Journal of Neuroscience Research

Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth.

Publication Date: 2010 Mar 5 PMID: 20209634
Authors: Renton, J. P. - Xu, N. - Clark, J. J. - Hansen, M. R.
Journal: J Neurosci Res

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl-2 prevents SGN death but inhibits neurite growth. Here we assessed the consequences of Bcl-2 targeted to either the mitochondria (GFP-Bcl-2-Maob) or the endoplasmic reticulum (ER, GFP-Bcl-2-Cb5) on cultured SGN survival and neurite growth. Transfection of wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob increased SGN survival, with GFP-Bcl-2-Cb5 providing the most robust response. Paradoxically, expression of GFP-Bcl-2-Maob results in SGN death in the presence of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears to be specific for neurotrophin signaling, insofar as coexpression of constitutively active mitogen-activated kinase kinase (MEKDeltaEE) or phosphatidyl inositol-3 kinase (P110), but not other prosurvival stimuli (e.g., membrane depolarization), also results in the loss of SGNs expressing GFP-Bcl-2-Maob. MEKDeltaEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT-3 or MEKDeltaEE. However, wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, and GFP-Bcl-2-Maob inhibit neurite growth even in the presence of neurotrophins, MEKDeltaEE, or P110. Historically, Bcl-2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl-2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl-2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. (c) 2010 Wiley-Liss, Inc.

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Regulation of spontaneous Ca(2+) spikes by metabotropic glutamate receptors in primary cultures of rat cortical neurons.

Publication Date: 2010 Mar 5 PMID: 20209633
Authors: Koga, K. - Iwahori, Y. - Ozaki, S. - Ohta, H.
Journal: J Neurosci Res

Periodic and spontaneous Ca(2+) spikes are observed in neurons during development of the central nervous system, and spontaneous changes in intracellular Ca(2+) concentration in neurons play important roles in the development of neural circuits. To clarify the roles of metabotropic glutamate receptors (mGluRs) in the regulation of spontaneous Ca(2+) spikes, we investigated the effects of selective and nonselective mGluRs ligands on primary cultures of rat cortical neurons. Cultured cortical neurons expressed all eight mGluR subtypes on reverse transcription-PCR. The mGluR2 and mGluR3 agonists LY379268, LY354740, and (2R,4R)-APDC increased the amplitude but decreased the frequency of spontaneous Ca(2+) spikes in cultured cortical neurons. The effects of these mGluR2 and mGluR3 agonists were completely inhibited by the presence of a potent mGluR2 and mGluR3 antagonist, LY341495, and by pretreatment with pertussis toxin. No significant effect was observed with either activation or inhibition of mGluR1, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8 on the spontaneous Ca(2+) spikes in cultured cortical neurons. These findings indicate that, among mGluRs, the group II mGluR subtypes mGluR2 and mGluR3 play principal roles in modulation of spontaneous Ca(2+) spikes. (c) 2010 Wiley-Liss, Inc.

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Brain-derived neurotrophic factor inhibits cell cycle reentry but not endoplasmic reticulum stress in cultured neurons following oxidative or excitotoxic stress.

Publication Date: 2010 Mar 5 PMID: 20209632
Authors: Boutahar, N. - Reynaud, E. - Lassabliere, F. - Borg, J.
Journal: J Neurosci Res

Neurotrophins protect neurons against glutamate and oxidative stress, but the underlying mechanism remains unclear. We investigated the neuroprotective role of the neurotrophin brain-derived neurotrophic factor (BDNF) in neuronal cultures subjected to NMDA or H(2)O(2) toxicity and analyzed the molecular mechanisms involved, particularly those related to regulation of cell cycle or endoplasmic reticulum (ER) stress. Preincubation with BDNF of cortical neuron cultures prevented NMDA- or H(2)O(2)-induced neuronal death as well as MAPK-ERK1/2 activation. Inhibition of phosphatidylinositol 3-kinase (PI3-K) abolished the protective effect of BDNF. NMDA and H(2)O(2) induced activation of cell cycle reentry regulators such as retinoblastoma (Rb) protein and E2F1 transcription factor. However, BDNF abolished the activation of both factors. NMDA-induced expression of chaperone encoding gene BIP was slightly inhibited by BDNF, but it did not affect expression of ER stress protein CHOP. Our results suggest that BDNF neuroprotection may be mediated through inhibition of Ras-MAPK pathway and cell cycle reentry during oxidative or excitotoxic stress responses. However, BDNF did not modify expression of ER stress signal induced by NMDA. (c) 2010 Wiley-Liss, Inc.

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Rostral brain axonal injury in congenital central hypoventilation syndrome.

Publication Date: 2010 Mar 5 PMID: 20209631
Authors: Kumar, R. - Macey, P. M. - Woo, M. A. - Harper, R. M.
Journal: J Neurosci Res

Brain injury underlying the state-related loss of ventilatory drive, autonomic, cognitive, and affective deficits in congenital central hypoventilation syndrome (CCHS) patients appears throughout the brain, as demonstrated by magnetic resonance (MR) T2 relaxometry and mean diffusivity studies. However, neither MR measure is optimal to describe types of axonal injury essential for assessing neural interactions responsible for CCHS characteristics. To evaluate axonal integrity and partition the nature of tissue damage (axonal vs. myelin injury) in CCHS, we measured water diffusion parallel (axial diffusivity) and perpendicular (radial diffusivity) to rostral brain fibers, indicative of axonal and myelin changes, respectively, with diffusion tensor imaging (DTI). We performed DTI in 12 CCHS (age 18.5 +/- 4.9 years, 7 male) and 30 control (17.7 +/- 4.6 years, 18 male) subjects, using a 3.0-Tesla MR imaging scanner. Axial and radial diffusivity maps were calculated, spatially normalized, smoothed, and compared between groups (analysis of covariance; covariates, age and gender). Significantly increased radial diffusivity, primarily indicative of myelin injury, emerged in fibers of the corona radiata, internal capsule, corpus callosum, hippocampus through the fornix, cingulum bundle, and temporal and parietal lobes. Increased axial diffusivity, suggestive of axonal injury, appeared in fibers of the internal capsule, thalamus, corona radiata, and occipital and temporal lobes. Multiple brain regions showed both higher axial and radial diffusivity, indicative of loss of tissue integrity with a combination of myelin and axonal injury, including basal ganglia, bed nucleus, and limbic, occipital, and temporal areas. The processes underlying injury are unclear, but likely stem from both hypoxic and developmental processes. (c) 2010 Wiley-Liss, Inc.

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A peptide derived from a trans-homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival.

Publication Date: 2010 Mar 5 PMID: 20209630
Authors: Kohler, L. B. - Soroka, V. - Korshunova, I. - Berezin, V. - Bock, E.
Journal: J Neurosci Res

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis-interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans-interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding. (c) 2010 Wiley-Liss, Inc.

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mu-Opioid receptor knockout mice are insensitive to methamphetamine-induced behavioral sensitization.

Publication Date: 2010 Mar 5 PMID: 20209629
Authors: Shen, X. - Purser, C. - Tien, L. T. - Chiu, C. T. - Paul, I. A. - Baker, R. - Loh, H. H. - Ho, I. K. - Ma, T.
Journal: J Neurosci Res

Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using mu-OR knockout mice, we examined the role of mu-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the mu-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the mu-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the mu-opioid system is involved in modulating the development of behavioral sensitization to METH. (c) 2010 Wiley-Liss, Inc.

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Wide-dynamic-range neurons are heterogeneous in windup responsiveness to changes in stimulus intensity and isoflurane anesthesia level in mice.

Publication Date: 2010 Mar 5 PMID: 20209628
Authors: Guan, Y. - Raja, S. N.
Journal: J Neurosci Res

The windup phenomenon in wide-dynamic-range (WDR) neurons represents a short-term neuronal sensitization to repetitive noxious inputs that may share similar mechanisms with those that trigger the development of persistent pain and hyperalgesia. Some WDR cells are readily sensitized and express prominent windup (windup(+)), whereas others do not (windup(-)). We recorded extracellular single-unit activity of deep laminae WDR neurons (350-700 mum) in C57BL/6 mice to determine how changes in stimulus intensity (1x and >2x C-component threshold, n = 53) and concentrations of isoflurane anesthesia (2.0% and 1.0%, n = 30) might differently modulate windup responsiveness in windup(+) and windup(-) cells. Two principally different analysis methods [absolute windup (the number of action potentials) and relative windup (the percentage of action potentials evoked by the first stimulus of the train)] were used to interpret windup data. We observed that increasing the stimulus intensity and decreasing the isoflurane concentration: 1) facilitated windup generation at 0.2-Hz stimulation and significantly enhanced absolute windup at both 0.2-Hz and 0.5-Hz stimulation predominantly in windup(+) cells but did not confer windup capability on windup(-) cells and 2) significantly increased relative windup at 0.2-Hz, but not 0.5-Hz, stimulation in windup(+) cells. Our findings advance our understanding of the neurobiology of deep WDR neurons in mice and demonstrate that two populations of cells differ in their windup responsiveness to changes in experimental conditions. We also elucidate the usefulness and potential limitations of two widely used methods for calculating and presenting windup data. (c) 2010 Wiley-Liss, Inc.

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Impaired neurogenesis is an early event in the etiology of familial Alzheimer's disease in transgenic mice.

Publication Date: 2010 Mar 5 PMID: 20209626
Authors: Demars, M. - Hu, Y. S. - Gadadhar, A. - Lazarov, O.
Journal: J Neurosci Res

Formation of new neurons in the adult brain takes place in the subventricular zone and in the subgranule layer of the dentate gyrus throughout life. Neurogenesis is thought to play a role in hippocampus- and olfaction-dependent learning and memory. However, whether impairments in neurogenesis take place in learning and memory disorders, such as Alzheimer's disease, is yet to be established. Importantly, it remains to be elucidated whether neurogenic impairments play a role in the course of the disease or are the result of extensive neuropathology. We now report that transgenic mice harboring familial Alzheimer's disease-linked mutant APPswe/PS1DeltaE9 exhibit severe impairments in neurogenesis that are evident as early as 2 months of age. These mice exhibit a significant reduction in the proliferation of neural progenitor cells and their neuronal differentiation. Interestingly, levels of hyperphosphorylated tau, the cytotoxic precursor of the Alzheimer's disease hallmark neurofibrillary tangles, are particularly high in the neurogenic niches. Isolation of neural progenitor cells in culture reveals that APPswe/PS1DeltaE9-expressing neurospheres exhibit impaired proliferation and tau hyperphosphorylation compared with wildtype neurospheres isolated from nontransgenic littermates. This study suggests that impaired neurogenesis is an early critical event in the course of Alzheimer's disease that may underlie memory impairments, at least in part, and exacerbate neuronal vulnerability in the hippocampal formation and olfaction circuits. Furthermore, impaired neurogenesis is the result of both intrinsic pathology in neural progenitor cells and extrinsic neuropathology in the neurogenic niches. Finally, hyperphosphorylation of the microtubule-associated protein tau, a critical player in cell proliferation, neuronal maturation, and axonal transport, is a major contributor to impaired neurogenesis in Alzheimer's disease. (c) 2010 Wiley-Liss, Inc.

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Neuroprotective effect of L-serine against temporary cerebral ischemia in rats.

Publication Date: 2010 Feb 22 PMID: 20186763
Authors: Wang, G. H. - Jiang, Z. L. - Chen, Z. Q. - Li, X. - Peng, L. L.
Journal: J Neurosci Res

To investigate the neuroprotective effect of L-serine and its underlying mechanisms, focal cerebral ischemia was induced in rats by occlusion of middle cerebral artery (MCAO) with a suture, and reperfusion was given by filament withdrawal 2 hr later. Meanwhile, rat hippocampal neurons were primarily cultured, and incubated in serum-free medium in an incubator containing 1% O(2) for hypoxic exposure of 5 hr, or incubated in serum-free medium containing 1 mM glutamate for glutamate exposure of 2 hr. Brain tissue injury and cell damage were then measured. L-serine dose-dependently decreased the neurology deficit score and infarct volume, elevated the cell viability and inhibited the leakage of lactate dehydrogenase. These effects were blocked by strychnine in both MCAO rats and cultured hippocampal neurons. Furthermore, L-serine (168 mg.kg(-1)) reduced the brain water content, permeability of blood-brain barrier, neuronal loss and the expression of activated caspase-3 in the cortex. In addition, L-serine effectively protected the brain from damage when it was administered within 6 hr after the end of MCAO. It is suggested that L-serine could exert a neuroprotective effect on the ischemic-reperfused brain and on the hypoxia- or glutamate-exposed hippocampal neurons, which may be mediated by activating glycine receptors. (c) 2010 Wiley-Liss, Inc.

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A mutation in the saposin C domain of the sphingolipid activator protein (Prosaposin) gene causes neurodegenerative disease in mice.

Publication Date: 2010 Feb 19 PMID: 20175216
Authors: Yoneshige, A. - Suzuki, K. - Suzuki, K. - Matsuda, J.
Journal: J Neurosci Res

Saposins A, B, C, and D are small amphiphatic glycoproteins that are encoded in tandem within a precursor protein (prosaposin, PSAP), and are required for in vivo degradation of sphingolipids. Humans with saposin C deficiency exhibit the clinical presentation of Gaucher-like disease. We generated two types of saposin C mutant mice, one carrying a homozygous missense mutation (C384S) in the saposin C domain of prosaposin (Sap-C(-/-)) and the other carrying the compound heterozygous mutation with a second null Psap allele (Psap(-/C384S)). During early life stages, both Sap-C(-/-) and Psap(-/C384S) mice grew normally; however, they developed progressive motor and behavioral deficits after 3 months of age and the majority of affected mice could scarcely move by about 15 months. They showed no signs of hepatosplenomegaly throughout their lives. No accumulation of glucosylceramide and glucosylsphingosine was detected in the brain or liver of both Sap-C(-/-) and Psap(-/C384S) mice. Neuropathological analyses revealed patterned loss of cerebellar Purkinje cells, widespread axonal spheroids filled with membrane-derived concentric or lamellar electron-dense bodies, and lipofuscin-like deposition in the neurons. Soap-bubble-like inclusion bodies were detected in the trigeminal ganglion cells and the vascular endothelial cells. Compound heterozygous Psap(-/C384S) mice showed qualitatively identical but faster progression of the neurological phenotypes than Sap-C(-/-) mice. These results suggest the in vivo role of saposin C in axonal membrane homeostasis, the disruption of which leads to neurodegeneration in lysosomal storage disease. (c) 2010 Wiley-Liss, Inc.

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Tissue plasminogen activator and plasminogen are critical for osmotic homeostasis by regulating vasopressin secretion.

Publication Date: 2010 Feb 19 PMID: 20175210
Authors: Imamura, Y. - Morita, S. - Nakatani, Y. - Okada, K. - Ueshima, S. - Matsuo, O. - Miyata, S.
Journal: J Neurosci Res

Systemic osmotic homeostasis is regulated mainly by neuroendocrine system of arginine-vasopressin (AVP) in mammalians. In the present study, we demonstrated that the immunoreactivity of tissue plasminogen activator (tPA) was observed specifically at neurosecretory granules of AVP-positive magnocellular terminals and that of plasminogen was seen at astrocytes in the neurohypophysis (NH). Both tPA and plasminogen knockout (KO) mice revealed higher plasma osmolarity upon water deprivation, a chronic osmotic stimulation, as compared with their wild-type (WT) animals, indicating abnormal osmotic control in these KO mice. tPA KO mice but not plasminogen ones revealed lower ability in secreting AVP into the blood circulation upon an acute osmotic stimulation. Both tPA and plasminogen KO animals showed lower ability in secreting AVP into the blood circulation upon a chronic osmotic stimulation. The recombinant tPA was able to promote the release of AVP from isolated NH. Chronic osmotic stimulation decreased the laminin expression level of neurohypophysial microvessel in WT mice but not in plasminogen KO ones. We suggest that AVP secretion is critically regulated by tPA-dependent facilitation of AVP release from terminals and plasminogen-dependent increase of AVP permeability across microvessels possibly via laminin degradation. (c) 2010 Wiley-Liss, Inc.

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Mitogen-activated protein kinases support survival of activated microglia that mediate thrombin-induced striatal injury in organotypic slice culture.

Publication Date: 2010 Feb 19 PMID: 20175209
Authors: Ohnishi, M. - Katsuki, H. - Izumi, Y. - Kume, T. - Takada-Takatori, Y. - Akaike, A.
Journal: J Neurosci Res

Intracerebral hemorrhage-associated tissue damage is triggered by blood-derived serine proteases such as thrombin. In addition, our previous studies have suggested that mitogen-activated protein (MAP) kinases contribute to intracerebral hemorrhage- and thrombin-induced striatal tissue damage in vivo. Here we addressed the mechanisms of MAP kinase involvement in thrombin cytotoxicity in rat corticostriatal slice culture, focusing on striatal tissue damage. Thrombin induced apoptotic nuclear condensation and fragmentation in striatal cells, which was suppressed by DEVD-CHO, a caspase-3 inhibitor. DEVD-CHO also prevented shrinkage of the striatal tissue induced by thrombin. Phagocytotic activity may be involved in tissue deterioration, because a phagocytosis inhibitor (cytochalasin D) and an inhibitor of phagocytosis of apoptotic cells (O-phospho-L-serine) suppressed shrinkage of the striatal tissue. OX42 immunostaining revealed that apoptosis-like microglial cell death was induced only when thrombin treatment was combined with application of inhibitors of MAP kinase/extracellular signal-regulated kinase kinase (PD98059), p38 MAP kinase (SB203580), or c-Jun N-terminal kinase (SP600125). Thrombin-induced increase in the number of microglia was also prevented by these inhibitors of MAP kinase pathways. We also found that thrombin-induced production of tumor necrosis factor (TNF)-alpha was inhibited by PD98059, SB203580, and SP600125. Finally, thrombin-induced neuronal apoptosis and shrinkage of the striatal tissue were significantly inhibited by anti-TNF-alpha neutralizing antibody. These results suggest that MAP kinases contribute to thrombin-induced striatal damage by supporting survival of activated microglia, which induce neuron death by producing TNF-alpha and cause tissue shrinkage by phagocytosing apoptotic cells. (c) 2010 Wiley-Liss, Inc.

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Antiapoptotic and antiautophagic effects of glial cell line-derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in rats.

Publication Date: 2010 Feb 19 PMID: 20175208
Authors: Shang, J. - Deguchi, K. - Yamashita, T. - Ohta, Y. - Zhang, H. - Morimoto, N. - Liu, N. - Zhang, X. - Tian, F. - Matsuura, T. - Funakoshi, H. - Nakamura, T. - Abe, K.
Journal: J Neurosci Res

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways. (c) 2010 Wiley-Liss, Inc.

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The fibroblast growth factor receptor (FGFR) agonist FGF1 and the neural cell adhesion molecule-derived peptide FGL activate FGFR substrate 2alpha differently.

Publication Date: 2010 Feb 19 PMID: 20175207
Authors: Chen, Y. - Li, S. - Berezin, V. - Bock, E.
Journal: J Neurosci Res

Activation of fibroblast growth factor (FGF) receptors (FGFRs) both by FGFs and by the neural cell adhesion molecule (NCAM) is crucial in the development and function of the nervous system. We found that FGFR substrate 2alpha (FRS2alpha), Src homologous and collagen A (ShcA), and phospholipase-Cgamma (PLCgamma) were all required for neurite outgrowth from cerebellar granule neurons (CGNs) induced by FGF1 and FGL (an NCAM-derived peptide agonist of FGFR1). Like FGF1, FGL induced tyrosine phosphorylation of FGFR1, FRS2alpha, ShcA, and PLCgamma in a time- and dose-dependent manner. However, the activation of FRS2alpha by FGL was significantly lower than the activation by FGF1, indicating a differential signaling profile induced by NCAM compared with the cognate growth factor. (c) 2010 Wiley-Liss, Inc.

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Ionic mechanisms of action of prion protein fragment PrP(106-126) in rat basal forebrain neurons.

Publication Date: 2010 Feb 19 PMID: 20175205
Authors: Alier, K. - Li, Z. - Mactavish, D. - Westaway, D. - Jhamandas, J. H.
Journal: J Neurosci Res

Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion-induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K(+)) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106-126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106-126) peptide, but not the "scrambled" sequence of PrP(106-126), evoked a reduction of whole-cell outward currents in a voltage range between -30 and +30 mV. Reduction of whole-cell outward currents was significantly attenuated in Ca(2+)-free external media and also in the presence of iberiotoxin, a blocker of calcium-activated potassium conductance. PrP(106-126) application also evoked a depression of the delayed rectifier (I(K)) and transient outward (I(A)) potassium currents. By using single cell RT-PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K(v)4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106-126) peptide used at nanomolar concentrations, induces a reduction of specific K(+) channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K(+) channels, and PrP-mediated neurotoxicity. (c) 2010 Wiley-Liss, Inc.

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Rapid increase of Nurr1 mRNA expression in limbic and cortical brain structures related to coping with depression-like behavior in mice.

Publication Date: 2010 Feb 19 PMID: 20175204
Authors: Rojas, P. - Joodmardi, E. - Perlmann, T. - Ogren, S. O.
Journal: J Neurosci Res

The immediate-early gene Nurr1 is a member of the inducible orphan nuclear receptor family. Nurr1 is essential to the differentiation, maturation, and maintenance of midbrain dopaminergic neurons and is expressed in different brain regions. We have reported that adult mice with reduced Nurr1 expression displayed an increase in immobility response to acute stress. These mice were also deficient in the retention of emotional memory. Thus, Nurr1 expression seems to be relevant to normal cognitive processes. To investigate the response of Nurr1 to a stress stimulus, Nurr1 mRNA expression was examined by in situ hybridization in adult mice using a depression-like behavior paradigm, the forced swim test. The Nurr1 gene was rapidly and widely up-regulated throughout the brain, including cortical areas (i.e., prefrontal cortex, primary and secondary visual cortex, primary auditory cortex, and secondary somatosensory cortex), hippocampus (dentate gyrus, CA1, CA2, and CA3), and midbrain (substantia nigra pars compacta and ventral tegmental area) at 30 min and 3 hr after the forced swim test. Dopamine content was reduced in prefrontal cortex and midbrain following swim stress. These results suggest that the increase in Nurr1 expression might be a compensatory mechanism to counteract the changes in forebrain dopamine transmission in coping with acute stress. (c) 2010 Wiley-Liss, Inc.

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Modulation of rumpshaker phenotype with wild-type PLP/DM20 suggests several pathogenic mechanisms.

Publication Date: 2010 Feb 19 PMID: 20175203
Authors: Barrie, J. A. - Montague, P. - Karim, S. - Kirkham, D. - Nave, K. A. - Anderson, T. J. - Griffiths, I. R. - McLaughlin, M.
Journal: J Neurosci Res

The rumpshaker mutation of the murine myelin proteolipid protein 1 (Plp1) gene generates misfolded PLP/DM20 protein, resulting in dysmyelination, increased oligodendrocyte apoptosis, and death prior to P40 when expressed on the C57 BL/6 background. In this study, we used transgenic complementation to normalize the levels of PLP/DM20 in myelin with wild-type protein to determine whether loss of normal PLP function or gain of toxic function is responsible for dysmyelination in the rumpshaker. Restoring myelin PLP/DM20 levels extended the survival time to at least P60, significantly reduced the density of apoptotic cells, increased myelin volume, and restored normal periodicity of myelin. Biochemical analysis found that several myelin proteins that are reduced in rumpshaker, including MAG, CNP, and SirT2, are markedly elevated at peak myelination (P20) in the rumpshaker transgenic mouse. Myelin basic protein, however, remained low at peak myelination but was restored at P60 when myelin had matured and entered into a maintenance phase. Markers of the unfolded protein response (UPR), BiP and XBP1, remained activated with the introduction of wild-type PLP. These data demonstrate that restoring wild-type PLP/DM20 levels in rumpshaker improves the phenotype and the integrity of myelin, but hypomyelination persists and stress pathways remain activated. This suggests that both gain- and loss-of-function mechanisms are involved in the pathogenesis of the rumpshaker. (c) 2010 Wiley-Liss, Inc.

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Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia.

Publication Date: 2010 Feb 19 PMID: 20175201
Authors: Shin, H. K. - Lee, H. R. - Lee, D. H. - Hong, K. W. - Lee, J. H. - Park, S. Y. - Lee, S. J. - Lee, J. S. - Lee, W. S. - Rhim, B. Y. - Kim, C. D.
Journal: J Neurosci Res

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.

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Stimulation of glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) induces inflammatory activation of microglia in culture.

Publication Date: 2010 Feb 16 PMID: 20162721
Authors: Hwang, H. - Lee, S. - Lee, W. H. - Lee, H. J. - Suk, K.
Journal: J Neurosci Res

Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) is a member of the tumor necrosis factor superfamily (TNFSF) and is known to act as a costimulator in the immune system by binding to GITR. GITRL is expressed in endothelial cells, dendritic cells, macrophages, and B cells, but it is not known whether GITRL is expressed in brain microglia cells. Here, we investigated the expression of GITR and GITRL and their potential role in microglia cells. Using BV-2 mouse microglia cells and mouse primary microglia cultures, we have demonstrated that 1) both GITR and GITRL are expressed in microglia cells; 2) stimulation of GITRL induces inflammatory activation of microglia on the basis of production of nitric oxide (NO) and expression of inducible nitric oxide synthase, cyclooxygenase-2, CD40, and matrix metalloproteinase-9; 3) GITRL-mediated microglial NO production partially depends on p38 MAPK, JNK, and nuclear factor-kappaB pathways; and 4) GITRL stimulation also induces microglia cell death. These results indicate that GITR and GITRL are functionally expressed on brain microglia and that the stimulation of GITRL can induce inflammatory activation of microglia. The GITR/GITRL system may play an important role in neuroinflammation. (c) 2010 Wiley-Liss, Inc.

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Involvement of Src-suppressed C kinase substrate in experimental autoimmune encephalomyelitis: A link between release of astrocyte proinflammatory factor and oligodendrocyte apoptosis.

Publication Date: 2010 Feb 12 PMID: 20155814
Authors: Li, X. - Yan, M. - Hu, L. - Sun, L. - Zhang, F. - Ji, H. - Jiang, J. - Wang, P. - Liu, H. - Gao, Y. - Tao, T. - He, X. - Cheng, C. - Shen, A.
Journal: J Neurosci Res

Src-suppressed C kinase substrate (SSeCKS) is involved in inflammation in the central nervous system (CNS), and plays a role in control of cell signaling and cytoskeletal arrangement. However, the expression and function of SSeCKS and its function in multiple sclerosis (MS) and its common animal model, experimental autoimmune encephalomyelitis (EAE) remained to be elucidated. In the present study, we first reported that SSeCKS was remarkably increased in astrocytes of EAE rats in vivo. TNF-alpha and NO were significantly induced in astrocytes stimulated with LPS/IFN-gamma in vitro, which was blocked in astrocytes transfected with SSeCKS siRNA. These results indicated that SSeCKS played a role in the production of TNF-alpha and NO in astrocytes with inflammatory stimulation. As excessive release of TNF-alpha and NO were major mediators in autoimmune diseases and correlated with oligodendrocyte cell death, we further investigated whether SSeCKS participated in oligodendrocyte apoptosis. Conditioned media (CM) from astrocytes treated with LPS/IFN-gamma decreased oligodendrocyte cell viability, while siRNA targeted to SSeCKS in astrocytes inhibited oligodendrocyte cell death. The results from antibody neutralization and NO inhibition suggested that the oligodendrocyte apoptosis may be due to the production of astrocyte-derived proinflammatory factors (TNF-alpha and NO). These findings revealed that there was a pathogenic interaction between SSeCKS expression in astrocytes and oligodendrocyte apoptosis. Understanding the mechanism of SSeCKS in the pathogenesis of EAE may contribute to the development of new therapeutic strategies against EAE and MS. (c) 2010 Wiley-Liss, Inc.

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Regulation of cholesterol efflux by amyloid beta secretion.

Publication Date: 2010 Feb 12 PMID: 20155813
Authors: Umeda, T. - Mori, H. - Zheng, H. - Tomiyama, T.
Journal: J Neurosci Res

Amyloid beta (Abeta) is a key molecule in the pathogenesis of Alzheimer's disease, but its physiological function remains unclear. Abeta is produced from amyloid precursor protein (APP) by beta- and gamma-secretases, which is enhanced by high levels of cellular cholesterol, so cholesterol is a risk factor for Alzheimer's disease. This linkage led us to hypothesize that Abeta is produced to regulate cellular cholesterol levels in response to high-cholesterol stimulation. Here we show that Abeta production caused a reduction of cellular cholesterol levels in transfected HEK293 cells and neuronal IMR-32 and Neuro2a cells, which was accompanied by an increase in efflux of cholesterol from cells. Fractionation of the culture media by ultracentrifugation and subsequent immunoelectron microscopic observation revealed that Abeta assembled high-density lipoprotein-like particles with cellular cholesterol during its secretion. This assembly was mediated by the ATP-binding cassette transporter A1. APP transgenic and knockout mice exhibited lower and higher levels of cellular cholesterol in their brains, suggesting that Abeta-mediated regulation of cellular cholesterol is physiological. Furthermore, we found that, when injected into mouse cerebral ventricle, reconstituted lipoproteins with Abeta were excreted into the peripheral tissues more efficiently than those without Abeta. This result suggests that Abeta mediates cholesterol transport from the brain to the circulation. We propose, based on these findings, a novel, apolipoprotein-like function for Abeta that is involved in maintenance of cellular and cerebral cholesterol homeostasis. (c) 2010 Wiley-Liss, Inc.

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Suppression of hypoxia-inducible factor-1alpha and its downstream genes reduces acute hyperglycemia-enhanced hemorrhagic transformation in a rat model of cerebral ischemia.

Publication Date: 2010 Feb 12 PMID: 20155812
Authors: Chen, C. - Ostrowski, R. P. - Zhou, C. - Tang, J. - Zhang, J. H.
Journal: J Neurosci Res

We evaluated a role of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream genes in acute hyperglycemia-induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats weighing 280-300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF-1alpha inhibitors, 2-methoxyestradiol (2ME2) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF-1alpha inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF-1alpha was increased in ischemic brain tissues after MCAO and reduced by HIF-1alpha inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase-2 and -9 (MMP-2/MMP-9) in the ipsilateral hemisphere. Both 2ME2 and YC-1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF-1alpha and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia. (c) 2010 Wiley-Liss, Inc.

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Chronic foot-shock stress potentiates the influx of bone marrow-derived microglia into hippocampus.

Publication Date: 2010 Feb 12 PMID: 20155811
Authors: Brevet, M. - Kojima, H. - Asakawa, A. - Atsuchi, K. - Ushikai, M. - Ataka, K. - Inui, A. - Kimura, H. - Sevestre, H. - Fujimiya, M.
Journal: J Neurosci Res

For several years, a new population of microglia derived from bone marrow has been described in multiple settings such as infection, trauma, and neurodegenerative disease. The aim of this study was to investigate the migration of bone marrow-derived cells to the brain parenchyma after stress exposure. Stress exposure was performed in mice that had received bone marrow transplantation from GFP mice, allowing identification of blood-derived elements within the brain. Electric foot-shock exposure was chosen because of its ability to serve as fundamental and physical stress in mice. Bone marrow-derived GFP(+) cells migrated to the ventral part of the hippocampus and acquired a ramified microglia-like morphology. Microglia marker Iba1 was expressed by 100% of the ramified cells, whereas ramified cells were negative for the astrocyte marker GFAP. Compared with the case in the control group, ramified cells significantly increased after chronic exposure to stress (5 days). One month after 5 days of stress exposure, ramified cells significantly decreased in ventral hippocampus compared with the group examined immediately after the last stress exposure. We report for the first time the migration of bone marrow-derived cells to the ventral hippocampus after stress exposure. These cells have the characteristics of microglia. Mechanisms responsible for this migration and their roles in the brain remain to be determined. (c) 2010 Wiley-Liss, Inc.

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Secreted amyloid precursor protein and holo-APP bind amyloid beta through distinct domains eliciting different toxic responses on hippocampal neurons.

Publication Date: 2010 Feb 12 PMID: 20155808
Authors: Kedikian, G. - Heredia, F. - Salvador, V. R. - Raimunda, D. - Isoardi, N. - Heredia, L. - Lorenzo, A.
Journal: J Neurosci Res

Amyloid beta (Abeta) is a metabolic product of Abeta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor-like protein for Abeta fibrils (fAbeta). In this study, we present further experimental support for the direct interaction of APP with fAbeta and for its involvement in Abeta neurotoxicity. Using recombinant purified holo-APP (h-APP), we have shown that it directly binds fAbeta. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAbeta. One sequence in the n-terminus of APP is required for binding of fAbeta to secreted APP (s-APP) but not to h-APP. In addition, the extracellular juxtamembrane Abeta-sequence mediates binding of fAbeta to h-APP but not to s-APP. Deletion of the extracellular juxtamembrane Abeta sequence abolishes abnormal h-APP accumulation and toxicity induced by fAbeta deposition, whereas deletions in the n-terminus of APP do not affect Abeta toxicity. These experiments show that interaction of toxic Abeta species with its membrane-anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD. (c) 2010 Wiley-Liss, Inc.

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Elevation of heme oxygenase-1 by proteasome inhibition affords dopaminergic neuroprotection.

Publication Date: 2010 Feb 12 PMID: 20155807
Authors: Yamamoto, N. - Izumi, Y. - Matsuo, T. - Wakita, S. - Kume, T. - Takada-Takatori, Y. - Sawada, H. - Akaike, A.
Journal: J Neurosci Res

Postmortem studies have shown that heme oxygenase-1 (HO-1) immunoreactivity is increased in patients with Parkinson disease. HO-1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO-1 content. MG-132, a proteasome inhibitor, increased the amount of HO-1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT-PCR analysis revealed that lactacystin upregulated HO-1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter-driven expression of Flag-HO-1 protein and resulted in an accumulation of ubiquitinated Flag-HO-1 in Flag-HO-1-overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag-HO-1 protein was slowed by MG-132. Next, the function of HO-1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced toxicity and this neuroprotection was abrogated by co-treatment with zinc protoporphyrin IX, a HO-1 inhibitor. Furthermore, 6-OHDA-induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO-1-catalyzed degradation of heme. These results suggest that mesencephalic HO-1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection. (c) 2010 Wiley-Liss, Inc.

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Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice.

Publication Date: 2010 Feb 12 PMID: 20155805
Authors: Panas, H. N. - Lynch, L. J. - Vallender, E. J. - Xie, Z. - Chen, G. L. - Lynn, S. K. - Scanlan, T. S. - Miller, G. M.
Journal: J Neurosci Res

3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 muM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 +/- 0.5 muM; KO, 1.2 +/- 0.4 muM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 +/- 0.6 muM; KO, 4.7 +/- 1.1 muM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 +/- 0.4, KO -5.6 +/- 1.0; and 25 mg/kg at 30 min: WT -2.7 +/- 0.4, KO -3.0 +/- 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens. (c) 2010 Wiley-Liss, Inc.

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Tieg1/Klf10 is upregulated by NGF and attenuates cell cycle progression in the pheochromocytoma cell line PC12.

Publication Date: 2010 Feb 12 PMID: 20155803
Authors: Spittau, G. - Happel, N. - Behrendt, M. - Chao, T. I. - Krieglstein, K. - Spittau, B.
Journal: J Neurosci Res

The transcription factor Tieg1/Klf10 belongs to a family of Sp1/Klf proteins that have been shown to play important roles during development and maintenance of various tissues and cell types. Upregulation of Tieg1/Klf10 has been reported for TGF-beta, BMP2, BMP4, ActivinA and GDNF as members of the TGF-beta superfamily. Moreover, estrogen, the cytostatic drugs homoharringtonine and velcade as well as nitric oxide are also able to trigger Tieg1/Klf10 transcription. Recent studies suggest a role for members of the neurotrophin family in regulating Tieg1/Klf10 transcriptional upregulation. Using semi-quantitative RT-PCR and immunoblotting, we present data describing that nerve growth factor (NGF) regulates the expression of Tieg1/Klf10 in the pheochromocytoma cell line PC12 in a TrkA-dependent manner. Moreover, we provide evidence for the existence of NGF-responsive elements in the 5'-regulatory region of Tieg1/Klf10 that contain binding sites for the transcription factors Sp1 and CREB. After treatment with NGF PC12 cells exit the cell cycle and start to differentiate towards a neuron-like phenotype indicated by neurite outgrowth. Using flow cytometry and differentiation assays we demonstrate that Tieg1/Klf10 reduces cell cycle progression in PC12 cells but fails to promote their terminal differentiation. Together, our results identify Tieg1/Klf10 as a new NGF target gene and substantiate its anti-proliferative function in the NGF signaling pathway in PC12 cells. (c) 2010 Wiley-Liss, Inc.

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Inflammation-induced preterm birth alters neuronal morphology in the mouse fetal brain.

Publication Date: 2010 Feb 12 PMID: 20155801
Authors: Burd, I. - Bentz, A. I. - Chai, J. - Gonzalez, J. - Monnerie, H. - Le Roux, P. D. - Cohen, A. S. - Yudkoff, M. - Elovitz, M. A.
Journal: J Neurosci Res

Adverse neurological outcome is a major cause of long-term morbidity in ex-preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non-infectious model of preterm birth by administering RU486. Pro-inflammatory cytokines (IL-10, IL-1beta, IL-6 and TNF-alpha) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro-inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation-induced preterm birth, but not non-infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule-associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation-induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology. (c) 2010 Wiley-Liss, Inc.

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Transcriptional upregulation of nitric oxide synthase II by nuclear factor-kappaB promotes apoptotic neuronal cell death in the hippocampus following experimental status epilepticus.

Publication Date: 2010 Feb 12 PMID: 20155797
Authors: Chuang, Y. C. - Chen, S. D. - Lin, T. K. - Chang, W. N. - Lu, C. H. - Liou, C. W. - Chan, S. H. - Chang, A. Y.
Journal: J Neurosci Res

Whereas status epilepticus, or the condition of continuous epileptic seizures, produces a characteristic pattern of preferential neuronal cell loss in the hippocampus, the underlying mechanism is still unsettled. Based on an experimental model of temporal lobe status epilepticus, we demonstrated previously that prolonged seizures prompted an overproduction of nitric oxide (NO) by upregulation of NO synthase II (NOS II) in the hippocampal CA3 subfield, followed by the activation of mitochondrial apoptotic signaling cascade. Using the same animal model, the present study evaluated the hypothesis that transcriptional upregulation of NOS II gene by nuclear factor-kappaB (NF-kappaB) promotes apoptotic neuronal cell death in the hippocampus following status epilepticus. In Sprague-Dawley rats, significantly augmented nucleus-bound translocation of NF-kappaB p50 and p65 subunits and DNA binding activity of NF-kappaB were observed in hippocampal CA3 neurons as early as 30 min after elicitation of sustained seizure activity by microinjection of kainic acid into the CA3 subfield, followed by a progressive elevation that peaked at 90 min. In addition, application bilaterally into the hippocampal CA3 subfield of a selective NF-kappaB inhibitor, pyrrolidine dithiocarbamate or double-stranded kappaB decoy DNA significantly antagonized the activated NOS II-peroxynitrite signaling cascade (3 hr) and the associated manifestations of apoptotic cell death (7 days) in the hippocampus. We conclude that activation of NF-kappaB in hippocampal CA3 neurons upregulates NOS II gene expression following experimental temporal lobe status epilepticus, leading to apoptotic neuronal cell death in the hippocampus. (c) 2010 Wiley-Liss, Inc.

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Role of mast cell activation in inducing microglial cells to release neurotrophin.

Publication Date: 2010 May 1 PMID: 20025063
Authors: Yuan, H. - Zhu, X. - Zhou, S. - Chen, Q. - Zhu, X. - Ma, X. - He, X. - Tian, M. - Shi, X.
Journal: J Neurosci Res

The brain-derived neurotrophic factor (BDNF) plays a critical role in pain hypersensitivity. BDNF is the ligand of P2X4 receptors (P2X4R) in the microglia. The causative factors involving the P2X4R over expression in the microglia remains unclear. Mast cell activation has a close relation with pain hypersensitivity. However, the underlying mechanism between mast cell activation and pain hypersensitivity is unknown. The present study aimed to elucidate the mechanism by which mast cell activation promoted the expression of P2X4R in the microglia. The results of present study showed that mast cell activation markedly promoted the expression of P2X4R and BDNF in microglial cells, which significantly enhanced the release of BDNF from microglial cells upon exposure to adenosine triphosphate. Mast cell-derived tryptase activated PAR2 that resulted in promoting the expression of P2X4R in microglial cells. Pretreatment with antibodies against tryptase or PAR2, or using tryptase-deficient HMC-1 cells or PAR2-deficient microglial cells abolished the increase in P2X4R expression and BDNF release. Increase in mitogen activated protein kinase phosphorylation was observed in the processes of mast cell-induced BDNF release and P2X4R expression. We conclude that mast cell activation has the capacity to promote the expression of P2X4R and BDNF in microglial cells.

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Comparative structural and functional analysis of the GLT-1/EAAT-2 promoter from man and rat.

Publication Date: 2010 May 1 PMID: 19998491
Authors: Allritz, C. - Bette, S. - Figiel, M. - Engele, J.
Journal: J Neurosci Res

In the vertebrate CNS, glutamate transport predominantly occurs through the glutamate transporter subtype, GLT-1/EAAT-2, which prevails in astrocytes. GLT-1/EAAT-2 expression is impaired in many acute and chronic brain diseases, leading to increases in extracellular glutamate and subsequent excitotoxic neuronal cell death. An obvious therapeutical approach to prevent glutamate-induced brain damage would be targeting GLT-1/EAAT-2 expression. Since so far, insights into the mechanisms modulating GLT-1/EAAT-2 expression mostly originated from work with rat astrocytes, we now sought to determine whether this modulatory network would also apply to humans. To this end, we have cloned the previously unknown rat GLT-1/EAAT-2 promoter and compared it to the human promoter sequence. In reporter assays, the cloned 2.7-kb region immediately flanking the 5'-end of the rat GLT-1/EAAT-2 gene allowed for similar increases in constitutive gene expression as the human promoter sequence. Sequence analysis demonstrated the presence of highly conserved regions on the rat and human GLT-1/EAAT-2 promoters, which turned out to be likewise essential for constitutive GLT-1/EAAT-2 expression, stimulation of gene transcription by EGF, TGFalpha, and PACAP as well as inhibition of gene transcription by TNFalpha. Intriguingly, endothelin-1 which inhibits endogenous GLT-1/EAAT-2 expression, promoted activity of both rat and human reporter constructs, indicating the existence of (an) inhibitory mechanism(s) not operational in the reporter gene assay. Our findings establish close similarities in the regulation of GLT-1/EAAT-2 expression in rat and man and, hence, validate rat astrocytes as an assay system for studying the molecular mechanisms affecting glutamate homeostasis in the healthy and diseased human brain.

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Neuroprotective effects of PDGF against oxidative stress and the signaling pathway involved.

Publication Date: 2010 May 1 PMID: 19998489
Authors: Zheng, L. - Ishii, Y. - Tokunaga, A. - Hamashima, T. - Shen, J. - Zhao, Q. L. - Ishizawa, S. - Fujimori, T. - Nabeshima, Y. - Mori, H. - Kondo, T. - Sasahara, M.
Journal: J Neurosci Res

The neuroprotective effects of platelet-derived growth factor (PDGF) and the major signaling pathways involved in these were examined using primary cultured mouse cortical neurons subjected to H(2)O(2)-induced oxidative stress. The specific function of the PDGF beta-receptor (PDGFR-beta) was examined by the selective deletion of the corresponding gene using the Cre-loxP system in vitro. In wild-type neurons, PDGF-BB enhanced the survival of these neurons and suppressed H(2)O(2)-induced caspase-3 activation. The prosurvival effect of PDGF-AA was less than that of PDGF-BB. PDGF-BB highly activated Akt, extracellular signal-regulated kinase (ERK), c-jun amino-terminal kinase (JNK) and p38. PDGF-AA activated these molecules at lesser extent than PDGF-BB. In particular, PDGF-AA induced activation of Akt was at very low level. The neuroprotective effects of PDGF-BB were antagonized by inhibitors of phosphatidylinositol 3-kinase (PI3-K), mitogen-activated protein kinase kinase (MEK), JNK and p38. The PDGFR-beta-depleted neurons showed increased vulnerability to oxidative stress, and less responsiveness to PDGF-BB-induced cytoprotection and signal activation, in which Akt activation was most strongly suppressed. After all, these results demonstrated the neuroprotective effects of PDGF and the signaling pathways involved against oxidative stress. The effects of PDGF-BB were more potent than those of PDGF-AA. This might be due to the activation and additive effects of two PDGFRs after PDGF-BB stimulation. Furthermore, the PI3-K/Akt pathway that was deduced to be preferentially activated by PDGFR-beta may explain the potent effects of PDGF-BB.

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N-methyl-D-aspartate preconditioning improves short-term motor deficits outcome after mild traumatic brain injury in mice.

Publication Date: 2010 May 1 PMID: 19998488
Authors: Costa, T. - Constantino, L. C. - Mendonca, B. P. - Pereira, J. G. - Herculano, B. - Tasca, C. I. - Boeck, C. R.
Journal: J Neurosci Res

Traumatic brain injury (TBI) causes impairment of fine motor functions in humans and nonhuman mammals that often persists for months after the injury occurs. Neuroprotective strategies for prevention of the sequelae of TBI and understanding the molecular mechanisms and cellular pathways are related to the glutamatergic system. It has been suggested that cellular damage subsequent to TBI is mediated by the excitatory neurotransmitters, glutamate and aspartate, through the excessive activation of the N-methyl-D-aspartate (NMDA) receptors. Thus, preconditioning with a low dose of NMDA was used as a strategy for protection against locomotor deficits observed after TBI in mice. Male adult mice CF-1 were preconditioned with NMDA (75 mg/kg) 24 hr before the TBI induction. Under anesthesia with O(2)/N(2)O (33%: 66%) inhalation, the animals were subjected to the experimental model of trauma that occurs by the impact of a 25 g weight on the skull. Sensorimotor gating was evaluated at 1.5, 6, or 24 hr after TBI induction by using footprint and rotarod tests. Cellular damage also was assessed 24 hr after occurrence of cortical trauma. Mice preconditioned with NMDA were protected against all motor deficits revealed by footprint tests, but not those observed in rotarod tasks. Although mice showed motor deficits after TBI, no cellular damage was observed. These data corroborate the hypothesis that glutamatergic excitotoxicity, especially via NMDA receptors, contributes to severity of trauma. They also point to a putative neuroprotective mechanism induced by a sublethal dose of NMDA to improve motor behavioral deficits after TBI.

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Enhanced neurogenesis in the olfactory bulb in adult mice after injury induced by acute treatment with trimethyltin.

Publication Date: 2010 May 1 PMID: 19998485
Authors: Yoneyama, M. - Kawada, K. - Ogita, K.
Journal: J Neurosci Res

In adults, the subventricular zone is known to contain undifferentiated neural progenitor cells that proliferate and generate the olfactory bulb (OB) interneurons throughout life. We earlier showed that trimethyltin (TMT) causes neuronal damage in the granular cell layer of the OB in adult mice. In the current study, we examined neurogenesis in the OB in adult mice after injury induced by acute treatment with TMT. On day 2 post-TMT treatment, enhanced incorporation of 5-bromo-2'-deoxyuridine (BrdU) was seen in the granular cell layer of the OB. Many of the BrdU-labeled cells were undifferentiated cells on day 2 post-treatment. On day 30 post-TMT treatment, BrdU-labeled neuronal cells were dramatically increased in number in the granular cell layer of the OB. However, TMT treatment was ineffective in affecting the migration of BrdU-labeled cells from the subventricular zone to the OB. The results of a neurosphere assay revealed that the number of neurospheres derived from the OB was significantly increased on day 2 post-TMT treatment. The neurosphere-forming neural progenitor cells derived from the OB of TMT-treated animals were capable of differentiating into neuronal cells as well as into astrocytes. Taken together, our data suggest that the OB has the ability to undergo enhanced neurogenesis following TMT-induced neuronal injury in adult mice.

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Human umbilical cord blood cell therapy blocks the morphological change and recruitment of CD11b-expressing, isolectin-binding proinflammatory cells after middle cerebral artery occlusion.

Publication Date: 2010 May 1 PMID: 19998484
Authors: Leonardo, C. C. - Hall, A. A. - Collier, L. A. - Ajmo, C. T. Jr - Willing, A. E. - Pennypacker, K. R.
Journal: J Neurosci Res

Secondary neurodegeneration resulting from stroke is mediated by delayed proinflammatory signaling and immune cell activation. Although it remains unknown which cell surface markers signify a proinflammatory phenotype, increased isolectin binding occurs on CD11b-expressing immune cells within injured brain tissue. Several reports have confirmed the efficacy of human umbilical cord blood (HUCB) cell therapy in reducing ischemic injury in rat after middle cerebral artery occlusion (MCAO), and these effects were attributed in part to dampened neuroinflammation. The present study examined the time course of lectin binding to cells of microglia/macrophage lineage within 96 hr after MCAO and whether delayed HUCB cell treatment alters the migration and/or morphological characteristics of these cells throughout the period of infarct expansion. Isolectin binding was up-regulated in response to injury, was maximal at 96 hr, and colocalized with cells that expressed the putative proinflammatory markers MMP-9 and nitric oxide. Isolectin-tagged fluorescence was also significantly increased at 72 hr and localized to greater numbers of amoeboid, CD11b-expressing cells relative to 51 hr. Treatment with 1 x 10(6) HUCB cells significantly reduced total lectin binding at 72 hr, as well as the total area occupied by lectin-tagged fluorescence at both 51 and 72 hr, relative to vehicle-treated controls. This effect was accompanied by a shift in the morphology of CD11b-positive cells from amoeboid to ramified shape. These data indicate that HUCB cell therapy suppressed the recruitment of proinflammatory, isolectin-binding cells during the period of infarct expansion, thus offering a potential mechanism for the protective effects of HUCB cell therapy.

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Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation.

Publication Date: 2010 May 1 PMID: 19998483
Authors: Soane, L. - Li Dai, W. - Fiskum, G. - Bambrick, L. L.
Journal: J Neurosci Res

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O(2) and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre- and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate-cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up-regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.

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Development of integrins in the vasculature of germinal matrix, cerebral cortex, and white matter of fetuses and premature infants.

Publication Date: 2010 May 1 PMID: 19960540
Authors: Dummula, K. - Vinukonda, G. - Xu, H. - Hu, F. - Zia, M. T. - Braun, A. - Shi, Q. - Wolk, J. - Ballabh, P.
Journal: J Neurosci Res

Germinal matrix (GM) vasculature is selectively vulnerable to hemorrhage in premature infants during the first 48 hr of life. This is attributed to rapid angiogenesis of this brain region, resulting in formation of nascent vessels that show a paucity of pericytes and immaturity of extracellular matrix. Integrins are key regulators of angiogenesis and contribute to stabilization of cerebral vasculature by providing endothelial- and astrocyte-matrix adhesion. Therefore, we asked whether GM exhibited a distinct regional pattern of integrin expression that was dissimilar from that of the cerebral cortex and white matter in human fetuses and premature infants. To this end, we measured protein and gene expression of integrins in the GM, cortex, and white matter of human fetuses (15-22 weeks), premature infants (23-35 weeks), and mature infants (36-40 weeks). We found that protein levels of alpha5beta1 integrin were greater in the GM than in the cortex or white matter by 1.6-fold for both fetuses and premature infants. alpha5beta1 integrin mRNA expression was higher in the GM than in the cortex or white matter by 2-fold for fetuses but not for premature infants. alphaVbeta3, alphaVbeta5, alphaVbeta8, and alpha4beta1 integrin expression were comparable among GM, cortex, and white matter in fetuses and premature infants. Because alpha5beta1 integrin is a central regulator of angiogenesis, its elevation in the GM of fetuses and premature infants indicates that this might be a key activator of endothelial proliferation in this brain region. We speculate that selective alpha5beta1 integrin inhibition might suppress angiogenesis in the GM and thus prevent brain hemorrhage in premature infants.

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Acute hyperosmotic stimulus-induced Fos expression in neurons depends on activation of astrocytes in the supraoptic nucleus of rats.

Publication Date: 2010 May 1 PMID: 19938175
Authors: Yuan, H. - Gao, B. - Duan, L. - Jiang, S. - Cao, R. - Xiong, Y. F. - Rao, Z. R.
Journal: J Neurosci Res

Acute hyperosmolarity induced a time-dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expression occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons.

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Neuronal expression of two isoforms of mouse Septin 5.

Publication Date: 2010 May 1 PMID: 19937814
Authors: Asada, A. - Takahashi, J. - Taniguchi, M. - Yamamoto, H. - Kimura, T. - Saito, T. - Hisanaga, S.
Journal: J Neurosci Res

Septin 5 (Sept5) is a member of the Septin GTPase family and is thought to be involved in exocytosis through interactions with syntaxin 1 in postmitotic neurons. In rats, Sept5 is alternatively spliced to produce a short (Sept5_v2) and long (Sept5_v1) isoform. We recently identified Sept5 in rat brain as a substrate for Cdk5/p35, which phosphorylates Ser17 of Sept5_v1. To date, however, only the short Sept5_v2 isoform has been reported in the mouse. To determine the general expression of the Sept5_v1 isoform in mammals, we isolated Sept5_v1 cDNA by PCR using mouse brain total RNA. Mouse Sept5_v1 cDNA showed a high degree of nucleotide and amino acid sequence homology to the corresponding isoform of rat and human Sept5. Both isoforms were expressed mainly in brain and testis at the mRNA level, but expression was restricted to brain at the protein level. Whereas Sept5_v1 mRNA was highly expressed in the cortex and hippocampus, Sept5_v2 mRNA was expressed at the similar extent across in various brain regions. The protein ratio of Sept5_v1 to Sept5_v2 was high in the hippocampus, roughly equivalent in the cortex and low in the cerebellum and medulla. Sept5_v2 expression increased gradually from E17 to P30, but expression of Sept5_v1 was delayed until P10. The two isoforms were distinguished by their pattern of N-terminal phosphorylation. Thus, these different expression and phosphorylation patterns suggest isoform-specific functions for Sept5 and that a phosphorylation-specific antibody will be useful to study this idea.

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Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate.

Publication Date: 2010 May 1 PMID: 19937813
Authors: Brustovetsky, T. - Bolshakov, A. - Brustovetsky, N.
Journal: J Neurosci Res

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na(+)/Ca(2+) exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate-induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate-induced jump in cytosolic Ca(2+), completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate-treated hippocampal neurons, the initial increase in cytosolic Ca(2+) that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD.

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Glutamate transporters in the central nervous system of a pond snail.

Publication Date: 2010 May 1 PMID: 19937812
Authors: Hatakeyama, D. - Mita, K. - Kobayashi, S. - Sadamoto, H. - Fujito, Y. - Hiripi, L. - Elekes, K. - Ito, E.
Journal: J Neurosci Res

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm.

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A metallothionein mimetic peptide protects neurons against kainic acid-induced excitotoxicity.

Publication Date: 2010 Apr PMID: 19937811
Authors: Sonn, K. - Pankratova, S. - Korshunova, I. - Zharkovsky, A. - Bock, E. - Berezin, V. - Kiryushko, D.
Journal: J Neurosci Res

Metallothioneins I and II (MTI/II) are metal-binding proteins overexpressed in response to brain injury. Recently, we have designed a peptide, termed EmtinB, which is modeled after the beta-domain of MT-II and mimics the biological effects of MTI/II in vitro. Here, we demonstrate the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)-induced neurotoxicity. We show that EmtinB passes the blood-brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg/kg) and high (30 mg/kg) KA doses and tends to decrease mortality induced by the high KA dose. Histopathological evaluation of hippocampal (CA3 and CA1) and cortical areas of mice treated with 20 mg/kg KA shows that EmtinB treatment reduces KA-induced neurodegeneration in the CA1 region. These findings establish EmtinB as a promising target for therapeutic development.

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Selection of reference genes for real-time quantitative reverse transcription-polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures.

Publication Date: 2010 Apr PMID: 19937810
Authors: Pernot, F. - Dorandeu, F. - Beaup, C. - Peinnequin, A.
Journal: J Neurosci Res

Reference genes are often used to normalize expression of data from real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and only a validation of their stability during a given experimental paradigm leads to reliable interpretations. The present study was thus designed to validate potential reference genes in a mouse model of mesiotemporal lobe epilepsy (MTLE) with focal seizures after unilateral intrahippocampal injection of kainate (KA). Ipsilateral and contralateral hippocampi were removed during nonconvulsive status epilepticus (5 hr), epileptogenesis (7 days), and the chronic period of recurrent focal seizures (21 days). Naive animals were equally studied. The stability of eight potential reference genes (hypoxanthine phosphoribosyltransferase, Hprt1; peptidylprolyl isomerase A, Ppia; TATA box binding protein, Tbp; beta-actin, Actb; acidic ribosomal phosphoprotein P0, Arbp; glyceraldehyde-3-phosphate dehydrogenase, Gapdh; ribosomal RNA 18S, 18S rRNA; and glucuronidase beta, Gusb) were determined using geNorm and NormFinder software. The first five (Hprt1, Ppia, Tbp, Actb, and Arbp) were found to be stable across the different phases of the disease and appeared adequate for normalizing RT-qPCR data in this model. This was in contrast to the other three (18S rRNA, Gapdh, and Gusb), which showed unstable expressions and should be avoided. The analysis of KA-induced changes in the expression of glial fibrillary acidic protein (Gfap) gene resulted in various relative expressions or even a completely different pattern when unstable reference genes were used. These results highlight the absolute need to validate the reference genes for a correct interpretation of mRNA quantification.

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Genome wide profiling of altered gene expression in the neocortex of Alzheimer's disease.

Publication Date: 2010 May 1 PMID: 19937809
Authors: Tan, M. G. - Chua, W. T. - Esiri, M. M. - Smith, A. D. - Vinters, H. V. - Lai, M. K.
Journal: J Neurosci Res

Alzheimer's disease (AD) is characterized by a complex neurodegenerative process affecting multiple genes and proteins in the neocortex, many of which have not been well-studied. In this study, we investigated genome-wide gene alterations in the temporal cortex of a well-characterized cohort of AD patients using a recently developed microarray platform, and compared some of the transcript changes with immunoblotting. Of the 5485 genes found to be significantly altered in AD, there were consistent patterns of changes which show that the AD transcriptome in neocortex is characterized by changes indicative of synaptic dysfunction, perturbed neurotransmission and activation of neuroinflammation. We also highlighted several genes of potential pathogenic significance which have not been well studied in AD. The current study aims to add to the growing body of knowledge relating to gene changes in AD and provide further insights into pathogenic mechanisms and potential targets of pharmacotherapy.

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Nitric oxide regulates BDNF release from nodose ganglion neurons in a pattern-dependent and cGMP-independent manner.

Publication Date: 2010 May 1 PMID: 19937808
Authors: Hsieh, H. Y. - Robertson, C. L. - Vermehren-Schmaedick, A. - Balkowiec, A.
Journal: J Neurosci Res

Activity of arterial baroreceptors is modulated by neurohumoral factors, including nitric oxide (NO), released from endothelial cells. Baroreceptor reflex responses can also be modulated by NO signaling in the brainstem nucleus tractus solitarius (NTS), the primary central target of cardiovascular afferents. Our recent studies indicate that brain-derived neurotrophic factor (BDNF) is abundantly expressed by developing and adult baroreceptor afferents in vivo, and released from cultured nodose ganglion (NG) neurons by patterns of baroreceptor activity. Using electrical field stimulation and ELISA in situ, we show that exogenous NO nearly abolishes BDNF release from newborn rat NG neurons in vitro stimulated with single pulses delivered at 6 Hz, but not 2-pulse bursts delivered at the same 6-Hz frequency, that corresponds to a rat heart rate. Application of L-NAME, a specific inhibitor of endogenous NO synthases, does not have any significant effect on activity-dependent BDNF release, but leads to upregulation of BDNF expression in an activity-dependent manner. The latter effect suggests a novel mechanism of homeostatic regulation of activity-dependent BDNF expression with endogenous NO as a key player. The exogenous NO-mediated effect does not involve the cGMP-protein kinase G (PKG) pathway, but is largely inhibited by N-ethylmaleimide and TEMPOL that are known to prevent S-nitrosylation. Together, our current data identify previously unknown mechanisms regulating BDNF availability, and point to NO as a likely regulator of BDNF at baroafferent synapses in the NTS.

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The chemokine SDF-1/CXCL12 contributes to the 'homing' of umbilical cord blood cells to a hypoxic-ischemic lesion in the rat brain.

Publication Date: 2010 May 1 PMID: 19937807
Authors: Rosenkranz, K. - Kumbruch, S. - Lebermann, K. - Marschner, K. - Jensen, A. - Dermietzel, R. - Meier, C.
Journal: J Neurosci Res

Previous studies have shown that transplanted human umbilical cord blood (hUCB)-derived mononuclear cells exert therapeutic effects in various animal models of CNS impairments, including those of perinatal hypoxic-ischemic brain injury. However, the mechanisms of how transplanted cells exert their beneficial effects on the damaged tissue are still unclear. As detection of hUCB cells at the lesion site coincides with the therapeutic effects observed in our model, we investigated the role of the chemokine stromal derived factor (SDF)-1 (CXCL12) as a possible candidate for chemotaxis-mediated 'homing' of transplanted hUCB cells to a hypoxic-ischemic lesion in the perinatal rat brain. Following the hypoxic-ischemic insult expression of SDF-1 significantly increased in lesioned brain hemispheres and was mainly associated with astrocytes. Transplanted hUCB cells expressing the SDF-1 receptor CXCR4 migrated to the lesion site within one day. Inhibition of SDF-1 by application of neutralizing antibodies in vivo resulted in a significantly reduced number of hUCB cells at the lesioned area. The increase in glial SDF-1 expression shortly after induction of the lesion and hUCB cells expressing the corresponding receptor makes SDF-1 a potential chemotactic factor for hUCB cell migration. The reduction of hUCB cells present at the lesion site upon functional inhibition of SDF-1 strengthens the view that the SDF-1/CXCR4 axis is of major importance for cell 'homing'.

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The embryonic blood-CSF barrier has molecular elements to control E-CSF osmolarity during early CNS development.

Publication Date: 2010 May 1 PMID: 19937806
Authors: Parvas, M. - Bueno, D.
Journal: J Neurosci Res

In vertebrates, brain development takes place at the expanded anterior end of the neural tube. After closure of the anterior neuropore, the brain wall forms a physiologically sealed cavity that encloses embryonic cerebrospinal fluid (E-CSF), a complex and protein-rich fluid. E-CSF has several crucial roles in brain anlagen development. In this respect, during the initiation of neurogenesis, increases in the volume of brain cavities account for 70% of the total growth of the brain primordium, and are accompanied by a parallel increase in E-CSF volume. Recently, we reported the presence of several blood vessels located in the brain stem lateral to the ventral midline, at the mesencephalon and prosencephalon level, which have a transient blood-CSF barrier function in chick embryos by transporting proteins in a selective manner via transcellular routes. These blood vessels control E-CSF protein composition and homeostasis during this early stage of CNS development, just after closure of the neuropores. Here we report that in chick and rat embryos these same blood vessels, which lie close to the neuroectoderm, express several molecules related to water and ion transport, namely AQP1, AQP4 and Kir4.1. Our results confirm that a blood-CSF barrier controls E-CSF composition and homeostasis from early stages of brain development in chick embryos, including water and ion influx, thus regulating E-CSF osmolarity. On the basis of our findings, we also propose that a similar blood-CSF barrier is present in mammals at equivalent developmental stages of the brain.

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Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Publication Date: 2010 May 1 PMID: 19937805
Authors: Price, M. - Badaut, J. - Thevenet, J. - Hirt, L.
Journal: J Neurosci Res

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

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Conserved cellular function and stress-mediated regulation among members of the proteolipid protein family.

Publication Date: 2010 May 1 PMID: 19937804
Authors: Fernandez, M. E. - Alfonso, J. - Brocco, M. A. - Frasch, A. C.
Journal: J Neurosci Res

Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews, including atrophy of CA3 dendrites and loss of synapses. The molecular mechanisms underlying these structural changes remain largely unknown. We have previously identified M6a as a stress responsive gene and shown that M6a is involved in filopodium/spine outgrowth and, likely, synapse formation. M6a belongs to the proteolipid protein (PLP) family, all of their members having four transmembrane domains that allow their localization at the plasma membrane. In the present work, we analyzed other members of this family, the closely related M6b as well as PLP and its splice variant DM20. We found that chronic restraint stress in mice reduces M6b and DM20, but not PLP, mRNA levels in the hippocampus. In addition, M6b and DM20, but again not PLP, induce filopodium formation in primary cultures of hippocampal neurons. Several M6b protein isoforms were studied, all of them having similar effects except for the one lacking the transmembrane domains. Our results reveal a conserved cellular function and a stress-mediated regulation among members of the proteolipid protein family, suggesting an involvement of proteolipid proteins in the stress response.

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CCAAT/enhancer binding protein delta in microglial activation.

Publication Date: 2010 Apr PMID: 19908286
Authors: Ejarque-Ortiz, A. - Gresa-Arribas, N. - Straccia, M. - Mancera, P. - Sola, C. - Tusell, J. M. - Serratosa, J. - Saura, J.
Journal: J Neurosci Res

The transcription factor CCAAT/enhancer binding protein delta (C/EBP delta) regulates transcription of genes that play important roles in glial activation. Previous studies have shown the astroglial expression of C/EBP delta but the microglial expression of C/EBP delta remains virtually unexplored, with the exception of two microarray studies. In this report, using murine primary cultures and BV2 cells we clearly demonstrate that C/EBP delta is expressed by microglia and it is upregulated in microglial activation. Lipopolysaccharide upregulates C/EBP delta both in microglia and in astrocytes. This effect is time-dependent, with a maximum effect at 3 hr at mRNA level and at 4-8 hr at protein level, and concentration-dependent, with a maximum effect at 100 ng/mL. The lipopolysaccharide-induced C/EBP delta upregulation in BV2 microglia is mimicked by agonists of the toll-like receptors 2, 3 and 9 and can be prevented by an inhibitor of extracellular signal-regulated kinase activation. C/EBP delta from activated BV2 microglia binds to the cyclooxygenase-2 promoter and forms complexes with C/EBP beta isoforms. These results point to C/EBP delta as a putative key regulator of proinflammatory gene expression in microglial activation.

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Alterations in GluR2 AMPA receptor phosphorylation at serine 880 following group I metabotropic glutamate receptor stimulation in the rat dorsal striatum.

Publication Date: 2010 Apr PMID: 19908285
Authors: Ahn, S. M. - Choe, E. S.
Journal: J Neurosci Res

Phosphorylation of ionotropic glutamate receptors in the brain plays a crucial role in the regulation of synaptic plasticity. In this study, we investigated the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor phosphorylation by the stimulation of group I metabotropic glutamate receptors (mGluRs) in the dorsal striatum in vivo. The results showed that intrastriatal infusion of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG, 250 nmol), enhanced the sensitivity of GluR2 subunit in its phosphorylation at serine 880 (S880) in the dorsal striatum. This enhancement of the sensitivity of GluR2-S880 phosphorylation was reduced by blocking group I mGluRs and N-methyl-D-aspartate (NMDA) receptors. Similar reduction of the enhancement was also induced by inhibiting phospholipase C (PLC), calcium/calmodulin-dependent protein kinase (CaMK), c-Jun N-terminal kinase (JNK), and protein kinase C (PKC). Inhibition of protein phosphatase (PP) 1/2A and calcineurin (PP2B) alone enhanced GluR2-S880 phosphorylation in the dorsal striatum, whereas inhibition of these phosphatases did not further enhance the S880 phosphorylation by DHPG stimulation. In addition, inhibition of PP1/2A or PP2B also enhanced the phosphorylation of CaMKII, JNK and PKC. These data suggest that the phosphorylation of AMPA receptor GluR2 subunit at S880 is subject to the upregulation by the stimulation of group I mGluRs. Interactions among glutamate receptors, protein kinases, and PPs participate in this upregulation.

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Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters.

Publication Date: 2010 Apr PMID: 19908284
Authors: Contreras, L. - Urbieta, A. - Kobayashi, K. - Saheki, T. - Satrustegui, J.
Journal: J Neurosci Res

The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons.

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Description of distributed features of the nestin-containing cells in brains of adult mice: a potential source of neural precursor cells.

Publication Date: 2010 Apr PMID: 19908282
Authors: Xu, R. - Wu, C. - Tao, Y. - Yi, J. - Yang, Y. - Yang, R. - Zhang, X. - Zhang, Y. - Liu, R.
Journal: J Neurosci Res

The distribution of neural precursor cells (NPCs) in adult mice brain has so far not been described. Therefore, we investigated the distribution of NPCs by analyzing the nestin-containing cells (NCCs) in distinct brain regions of adult nestin second-intron enhancer-controlled LacZ reporter transgenic mice through LacZ staining. Results showed that NCCs existed in various regions of adult mouse brain. In cerebellum, the greatest number of NCCs existed in cortex of the simple lobule, followed by cortex of the cerebellar lobule. In olfactory bulb, NCCs were most numerous in the granular cell layer, followed by the mitral cell layer and the internal plexiform, glomerular, and external plexiform layers. In brain nuclei (nu), NCCs were most numerous in the marginal nu, followed by the brainstem and diencephalon nu. NCCs in sensory nu of brainstem were more numerous than in motor nu, and NCCs in the dorsal of sensory nu were more numerous than in the ventral part. In brain ventricle systems, NCCs were largely distributed in the center of and external to the lateral ventricle, the inferior part of the third ventricle, the dorsal and inferior parts of the fourth ventricle, and the gray matter around the cerebral aqueduct. NCCs in the left vs. right brain were not significantly different. These data collectively indicate that NCCs were extensively distributed in the cerebellum and olfactory bulb, the partial nu of the marginal system, the partial brain nu adjacent to brain ventricle systems, the subependymal zone, and the cerebral cortex around the marginal lobe and were a potential source of NPCs.

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Tau hyperphosphorylation and axonal damage induced by N,N-diethyldithiocarbamate (DEDTC) treatment along late postnatal development is followed by a rescue during adulthood.

Publication Date: 2010 Apr PMID: 19908281
Authors: Utrera, J. - Junyent, F. - de Lemos, L. - Pallas, M. - Camins, A. - Romero, R. - Auladell, C.
Journal: J Neurosci Res

Axonal degeneration has been described as the pathological hallmark of peripheral neuropathies induced by DEDTC. In addition, axonal damage has also been observed in the brain of mice treated daily with DEDTC along postnatal development, though with this experimental model there was observed to be axonal recovery after treatment, during the adulthood. To focus on this axonal dynamic activity, damage-recovery, a key axonal protein, the microtubule associated protein tau, was analyzed in this DEDTC model. Tau is a phosphoprotein and its dynamic site-specific phosphorylation is essential for its proper function; in fact, high levels are correlated with cell dysfunction. Furthermore, the levels of tau phosphorylation are associated with dynamic microtubules during periods of high plasticity. Thus, phosphorylated tau at two sites of phosphorylation, Ser(199) and Ser(396), were evaluated during the second week of postnatal development and throughout adulthood. The results obtained by Western blot made it evident that the levels of p-tau Ser(199) and p-tau Ser(396) were higher in treated mice than in controls. Interestingly, by immunohistochemistry there was shown to be an increase in p-tau-immunolabeling in neuronal soma together with axonal tract alterations in treated animals with respect to controls, and the analyses of GSK3 beta and cdk5 revealed an increase in its activity in DEDTC-treated animals. Nevertheless, in the adult a general decline in p-tau was observed together with a rescue of axonal tract. All these data support the idea that the axonal damage induced by DEDTC treatment along postnatal development is followed by an axonal rescue during adulthood.

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Differing in vitro survival dependency of mouse and rat NG2+ oligodendroglial progenitor cells.

Publication Date: 2010 Apr PMID: 19908280
Authors: Horiuchi, M. - Lindsten, T. - Pleasure, D. - Itoh, T.
Journal: J Neurosci Res

NG2 chondroitin sulfate proteoglycan is a surface marker of oligodendroglial progenitor cells (OPCs) in various species. In contrast to well-studied rat OPCs, however, we found that purified mouse NG2 surface positive cells (NG2(+) cells) require additional activation of cyclic AMP (cAMP) signaling for survival in a medium containing 30% B104 neuroblastoma conditioned medium supplemented with fibroblast growth factor-2 (B104CM+FGF2), whereas B104CM+FGF2 alone is sufficient for survival and selective proliferation of rat OPCs. After induction of in vitro differentiation, more than 90% of mouse NG2(+) cells became O4-positive, and a majority expressed myelin basic protein by 5 day of differentiation, which confirmed the identity of isolated mouse NG2(+) cells as OPCs. In comparison to rat OPCs, mouse OPCs in B104CM+FGF2 were less motile, and demonstrated lower basal phosphorylation levels of ERK1/2 and cAMP response element-binding protein (CREB) and a higher incidence of apoptosis mediated by the intrinsic pathway. Transient up-regulation of cAMP-CREB signaling partially inhibited apoptosis of mouse OPCs independently of the ERK pathway. This study demonstrates a difference in trophic requirements between mouse and rat OPCs, with an essential role for cAMP signaling to preserve viability of mouse OPCs.

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Peptides derived from the solvent-exposed loops 3 and 4 of BDNF bind TrkB and p75(NTR) receptors and stimulate neurite outgrowth and survival.

Publication Date: 2010 May 1 PMID: 19908279
Authors: Fobian, K. - Owczarek, S. - Budtz, C. - Bock, E. - Berezin, V. - Pedersen, M. V.
Journal: J Neurosci Res

Brain-derived neurotrophic factor (BDNF) is critically involved in modeling the developing nervous system and is an important regulator of a variety of crucial functions in the mature CNS. BDNF exerts its action through interactions with two transmembrane receptors, either separately or in concert. BDNF has been implicated in several neurological disorders, and irregularities in BDNF function may have severe consequences. Administration of BDNF as a drug has thus far yielded few practicable results, and the potential side effects when using a multifunctional protein are substantial. In an effort to produce more specific compounds without side effects, small peptides mimicking protein function have been developed. The present study characterized two mimetic peptides, Betrofin 3 and Betrofin 4, derived from the BDNF sequence. Both Betrofins bound the cognate BDNF receptors, TrkB and p75(NTR), and induced neurite outgrowth and enhanced neuronal survival, probably by inducing signaling through tha Akt and MAPK pathways. Distinct, charged residues within the Betrofin sequences were identified as important for generating the neuritogenic response, which was also inhibited when BDNF was added together with either Betrofin, indicating partial agonistic effects of the peptides. Thus, two peptides derived from BDNF induced neurite outgrowth and enhanced neuronal survival, probably through binding to BDNF receptors.

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Sonic hedgehog (SHH) promotes the differentiation of mouse cochlear neural progenitors via the Math1-Brn3.1 signaling pathway in vitro.

Publication Date: 2010 Apr PMID: 19908278
Authors: Hu, X. - Huang, J. - Feng, L. - Fukudome, S. - Hamajima, Y. - Lin, J.
Journal: J Neurosci Res

Sonic hedgehog (SHH) is essential for the development of the cochlear duct that harbors the organ of Corti. However, little is known about the molecular signaling pathway through which SHH promotes the development of the organ of Corti, especially cochlear sensory epithelial cells. In this study, we demonstrated that SHH contributes to the differentiation of cochlear neural progenitors (CNPs), which are derived from the postnatal day 1 organ of Corti in mice. Addition of SHH to CNPs increased the formation of epithelial cell islands, simultaneously activated the expression of Math1 that is a transcription factor for the initial differentiation of auditory hair cells. The increased expression of Math1 then regulated the promoter activity of Brn3.1, another transcription factor that controls the further differentiation and survival of auditory hair cells. Taken together, our data suggest that SHH plays an important role in the promotion of auditory hair cell differentiation via the Math1-Brn3.1 signaling pathway.

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Maternal caffeine intake during gestation and lactation down-regulates adenosine A1 receptor in rat brain from mothers and neonates.

Publication Date: 2010 May 1 PMID: 19908252
Authors: Lorenzo, A. M. - Leon, D. - Castillo, C. A. - Ruiz, M. A. - Albasanz, J. L. - Martin, M.
Journal: J Neurosci Res

Even though caffeine can be excreted in breast milk, few studies have analyzed the effect of maternal caffeine consumption during lactation on neonatal brain. In the present work pregnant rats were treated daily with 1 g/L of caffeine in their drinking water during pregnancy and/or lactation and the effect on adenosine A(1) receptor in brains from both lactating mothers and 15 days-old neonates was assayed using radioligand binding and real time PCR assays. Mothers receiving caffeine during gestational period developed motor activation in gestational days 8-10 which was associated with a significant decrease of total adenosine A(1) receptor number (84%). A similar decrease was detected in mothers treated with caffeine during lactation (76%) and throughout gestation and lactation (73%); this was accompanied by a significant decrease in mRNA level coding adenosine A(1) receptor (28%). In male neonates, adenosine A(1) receptor was also decreased after chronic caffeine exposure during gestation (80%), lactation (76%) and gestation plus lactation (80%). In female neonates, adenosine A(1) receptor tended to decrease in response to caffeine exposure although no significant variations were found. No variation in the level of mRNA coding adenosine A(1) receptor was detected in neonates in any case. Concerning adenosine A(2A) receptor, radioligand binding assays revealed that this receptor remains unaltered in maternal and neonatal brain in response to caffeine exposure. However, caffeine consumption during gestation and lactation evoked a significant decrease in mRNA level coding A(2A) receptor (32%) in mothers' brain.

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Promoting directional axon growth from neural progenitors grafted into the injured spinal cord.

Publication Date: 2010 May 1 PMID: 19908250
Authors: Bonner, J. F. - Blesch, A. - Neuhuber, B. - Fischer, I.
Journal: J Neurosci Res

Spinal cord injury (SCI) is a devastating condition characterized by disruption of axonal connections, failure of axonal regeneration, and loss of motor and sensory function. The therapeutic promise of neural stem cells has been focused on cell replacement, but many obstacles remain in obtaining neuronal integration following transplantation into the injured CNS. This study investigated the neurotransmitter identity and axonal growth potential of neural progenitors following grafting into adult rats with a dorsal column lesion. We found that using a combination of neuronal and glial restricted progenitors (NRP and GRP) produced graft-derived glutamatergic and GABAergic neurons within the injury site, with minimal axonal extension. Administration of brain-derived neurotrophic factor (BDNF) with the graft promoted modest axonal growth from grafted cells. In contrast, injecting a lentiviral vector expressing BDNF rostral into the injured area generated a neurotrophin gradient and promoted directional growth of axons for up to 9 mm. Animals injected with BDNF lentivirus (at 2.5 and 5.0 mm) showed significantly more axons and significantly longer axons than control animals injected with GFP lentivirus. However, only the 5.0-mm-BDNF group showed a preference for extension in the rostral direction. We concluded that NRP/GRP grafts can be used to produce excitatory and inhibitory neurons, and neurotrophin gradients can guide axonal growth from graft-derived neurons toward putative targets. Together they can serve as a building block for neuronal cell replacement of local circuits and formation of neuronal relays.

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Balance dysfunction resulting from acute inner ear energy failure is caused primarily by vestibular hair cell damage.

Publication Date: 2010 May 1 PMID: 19908248
Authors: Mizutari, K. - Fujioka, M. - Nakagawa, S. - Fujii, M. - Ogawa, K. - Matsunaga, T.
Journal: J Neurosci Res

Inner ear energy failure is associated with disorders such as inner ear ischemia. Recently, we used the mitochondrial toxin 3-nitropropionic acid (3-NP) to establish an animal model of inner ear energy failure that presents with auditory dysfunction. Here we investigated the mechanisms underlying balance disorders in the 3-NP animal model. Spontaneous nystagmus peaked 6 hr after treatment with either 300 mM or 500 mM 3-NP. The nystagmus attenuated gradually and disappeared 3 days after 3-NP treatment. A caloric test using ice water was performed to evaluate residual vestibular function 7 days after 3-NP treatment. The response to caloric stimulation was reduced to approximately 40% of the response of the untreated ear following 300 mM 3-NP and was undetectable following 500 mM 3-NP. Structural changes in the peripheral vestibular organs were analyzed by light and electron microscopy. Severe loss of stereocilia was observed following 500 mM 3-NP, whereas disorganized and mildly reduced stereocilia were observed following 300 mM 3-NP. There was severe loss and degeneration of vestibular hair cells following 500 mM 3-NP but only slight loss and degeneration of hair cells following 300 mM 3-NP. These results indicate that acute inner ear energy failure causes balance dysfunction mainly by damaging hair cells in the vestibule, which is distinct from the mechanism underlying auditory disorders.

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Caspase-11 mediates ischemia-induced astrocyte death: involvement of endoplasmic reticulum stress and C/EBP homologous protein.

Publication Date: 2010 Apr PMID: 19890920
Authors: Fradejas, N. - Pastor, M. D. - Burgos, M. - Beyaert, R. - Tranque, P. - Calvo, S.
Journal: J Neurosci Res

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.

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Correlations between peripheral blood mononuclear cell production of BDNF, TNF-alpha, IL-6, IL-10 and cognitive performances in multiple sclerosis patients.

Publication Date: 2010 Apr PMID: 19885866
Authors: Patanella, A. K. - Zinno, M. - Quaranta, D. - Nociti, V. - Frisullo, G. - Gainotti, G. - Tonali, P. A. - Batocchi, A. P. - Marra, C.
Journal: J Neurosci Res

The aim of this study was to investigate the role of Brain Derived Neurotrophic Factor (BDNF) and inflammatory factors in the development of cognitive dysfunctions in Multiple Sclerosis (MS). We correlated peripheral blood mononuclear cell (PBMC) production of BDNF, Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin (IL)-6 and IL-10 with performances on specific neuropsychological tasks in a selected series of MS patients. We studied a sample of 30 patients with relapsing-remitting (RR)MS, segregated by gender and matched for age, education, disease duration, type of immunomodulating therapy, degree of disability and overall cognitive status. We found that low BDNF levels were correlated with increased time of execution on a divided attention and visual scanning task whereas high levels of IL-6 were correlated with low Mini Mental State Examination scores. We did not observe any significant correlations between IL-10, TNF-alpha levels and cognitive performances in our patients. In conclusion our study shows a correlation between low BDNF and high IL-6 production by PBMCs and poorer performances in cognitive tasks in RRMS patients suggesting a possible role of these factors in cognitive impairment in MS.

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Conserved fate and function of ferumoxides-labeled neural precursor cells in vitro and in vivo.

Publication Date: 2010 Apr PMID: 19885865
Authors: Cohen, M. E. - Muja, N. - Fainstein, N. - Bulte, J. W. - Ben-Hur, T.
Journal: J Neurosci Res

Recent progress in cell therapy research for brain diseases has raised the need for non-invasive monitoring of transplanted cells. For therapeutic application in multiple sclerosis, transplanted cells need to be tracked both spatially and temporally, in order to assess their migration and survival in the host tissue. Magnetic resonance imaging (MRI) of superparamagnetic iron oxide-(SPIO)-labeled cells has been widely used for high resolution monitoring of the biodistribution of cells after transplantation into the central nervous system (CNS). Here we labeled mouse glial-committed neural precursor cells (NPCs) with the clinically approved SPIO contrast agent ferumoxides and examined their survival and differentiation in vitro, as well as their functional response to environmental signals present within the inflamed brain of experimental autoimmune encephalomyelitis (EAE) mice in vivo. We show that ferumoxides labeling does not affect NPC survival and pluripotency in vitro. Following intracerebroventricular (ICV) transplantation in EAE mice, ferumoxides-labeled NPCs responded to inflammatory cues in a similar fashion as unlabeled cells. Ferumoxides-labeled NPCs migrated over comparable distances in white matter tracts and differentiated equally into the glial lineages. Furthermore, ferumoxides-labeled NPCs inhibited lymph node cell proliferation in vitro, similarly to non-labeled cells, suggesting a preserved immunomodulatory function. These results demonstrate that ferumoxides-based MRI cell tracking is well suited for non-invasive monitoring of NPC transplantation.

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The galanin receptor 2/3 agonist Gal2-11 protects the SN56 cells against beta-amyloid 25-35 toxicity.

Publication Date: 2010 Apr PMID: 19885864
Authors: Pirondi, S. - Giuliani, A. - Del Vecchio, G. - Giardino, L. - Hokfelt, T. - Calza, L.
Journal: J Neurosci Res

The neuropeptide galanin is a modulator of cholinergic function and may play a role in A beta peptide-induced degeneration of cholinergic forebrain neurons. We have studied the effect of galanin and its galanin receptor subtype 2/3 agonist Gal2-11on toxicity induced by freshly-prepared beta-amyloid(25-35) in the cholinergic cell line SN56. Both nuclear fragmentation and caspase-3 expression were analysed. beta-amyloid(25-35)-exposure induced a significant increase in caspase-3 mRNA expression after 30, 60, 90 or 150 min of beta-amyloid(25-35) exposure. These effects were abolished in the presence of Gal2-11 (10 nM). Similarly, beta-amyloid(25-35)-induced nuclear fragmentation was prevented by the galanin agonist at all time points studied. These findings indicate that the galanin 2/3 agonist Gal2-11 protects SN56 cholinergic cells from beta-amyloid(25-35)-induced cell death and that this action is mediated by an early reduction of caspase-3 expression.

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Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model.

Publication Date: 2010 Apr PMID: 19885863
Authors: Wakabayashi, K. - Nagai, A. - Sheikh, A. M. - Shiota, Y. - Narantuya, D. - Watanabe, T. - Masuda, J. - Kobayashi, S. - Kim, S. U. - Yamaguchi, S.
Journal: J Neurosci Res

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia-induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 x 10(6) immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotrophic factors and cytokines was evaluated by quantitative real-time RT-PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC-transplanted brain, among many neurotrophic factors, only human insulin-like growth factor 1 (IGF-1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF-1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain.

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Periaqueductal gray afferents synapse onto dopamine and GABA neurons in the rat ventral tegmental area.

Publication Date: 2010 Apr PMID: 19885830
Authors: Omelchenko, N. - Sesack, S. R.
Journal: J Neurosci Res

The midbrain central gray (periaqueductal gray; PAG) mediates defensive behaviors and is implicated in the rewarding effects of opiate drugs. Projections from the PAG to the ventral tegmental area (VTA) suggest that this region might also regulate behaviors involving motivation and cognition. However, studies have not yet examined the morphological features of PAG axons in the VTA or whether they synapse onto dopamine (DA) or GABA neurons. In this study, we injected anterograde tracers into the rat PAG and used immunoperoxidase to visualize the projections to the VTA. Immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA was then used to identify the phenotype of innervated cells. Electron microscopic examination of the VTA revealed axons labeled anterogradely from the PAG, including myelinated and unmyelinated fibers and axon varicosities, some of which formed identifiable synapses. Approximately 55% of these synaptic contacts were of the symmetric (presumably inhibitory) type; the rest were asymmetric (presumably excitatory). These findings are consistent with the presence of both GABA and glutamate projection neurons in the PAG. Some PAG axons contained dense-cored vesicles indicating the presence of neuropeptides in addition to classical neurotransmitters. PAG projections synapsed onto both DA and GABA cells with no obvious selectivity, providing the first anatomical evidence for these direct connections. The results suggest a diverse nature of PAG physiological actions on midbrain neurons. Moreover, as both the VTA and PAG are implicated in the reinforcing actions of opiates, our findings provide a potential substrate for some of the rewarding effects of these drugs.

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Oxidative stress up-regulates presenilin 1 in lipid rafts in neuronal cells.

Publication Date: 2010 Apr PMID: 19885829
Authors: Oda, A. - Tamaoka, A. - Araki, W.
Journal: J Neurosci Res

Oxidative stress is associated with beta-amyloid peptide (A beta) accumulation in the brains of Alzheimer's disease patients. A beta is generated upon the sequential proteolytic cleavage of transmembrane amyloid precursor protein (APP) by two membrane-bound proteases, beta-secretase (BACE1) and the gamma-secretase complex comprising presenilin 1 (PS1), nicastrin, APH-1 and PEN-2. Recent evidence suggests that significant amounts of BACE1 and gamma-secretase components localize in the cholesterol-rich region of membranes known as lipid rafts, where A beta production occurs preferentially. In this study, we investigated the effects of oxidative stress on the BACE1 and gamma-secretase components in lipid rafts using human neuroblastoma SH-SY5Y cells exposed to ethacrynic acid (EA), a compound that induces cellular glutathione depletion. Following exposure of cells to EA, heme oxygenase-1, a marker protein of oxidative stress, was strongly induced. Moreover, treatment with EA resulted in a significant increase in PS1 protein levels, but not those of nicastrin, APH-1, PEN-2 or BACE1, in both cell lysates and the lipid raft fraction. This increase in PS1 protein expression was prevented by co-treatment with an antioxidant, N-acetylcysteine (NAC). EA additionally induced a significant increase in PS1 mRNA expression, which was inhibited by NAC. Finally, EA treatment was found to promote A beta secretion from cells expressing Swedish mutant APP. It appears that in our cell culture model, oxidative stress enhances PS1 protein levels in lipid rafts via up-regulation of PS1 transcription, which may constitute the mechanism underlying the oxidative stress-associated promotion of A beta production.

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Normobaric hyperoximia increases hypoxia-induced cerebral injury: DTI study in rats.

Publication Date: 2010 Apr PMID: 19885827
Authors: Bockhorst, K. H. - Narayana, P. A. - Dulin, J. - Liu, R. - Rea, H. C. - Hahn, K. - Wosik, J. - Perez-Polo, J. R.
Journal: J Neurosci Res

Perinatal hypoxia affects normal neurological development and can lead to motor, behavioral and cognitive deficits. A common acute treatment for perinatal hypoxia is oxygen resuscitation (hyperoximia), a controversial treatment. Magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), was performed in a P7 rat model of perinatal hypoxia to determine the effect of hyperoximia. These studies were performed on two groups of animals: 1) animals which were subjected to ischemia followed by hypoxia (HI), and 2) HI followed by hyperoximic treatment (HHI). Lesion volumes on high resolution MRI and DTI derived measures, fractional anisotropy (FA), mean diffusivity (MD), and axial and radial diffusivities (lambda(l) and lambda(t), respectively) were measured in vivo one day, one week, and three weeks after injury. Most significant differences in the MRI and DTI measures were found at three weeks after injury. Specifically, three weeks after HHI injury resulted in significantly larger hyperintense lesion volumes (95.26 +/- 50.42 mm(3)) compared to HI (22.25 +/- 17.62 mm(3)). The radial diffusivity lambda(t) of the genu of corpus callosum was significantly larger in HHI (681 +/- 330 x 10(-6) mm(2)/sec) than in HI (486 +/- 96 x 10(-6) mm(2)/sec). Over all, most significant differences in all the DTI metrics (FA, MD, lambda(t), lambda(l)) at all time points were found in the corpus callosum. Our results suggest that treatment of perinatal hypoxia with normobaric oxygen does not ameliorate, but exacerbates damage.

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Protective role of COMP-Ang1 in ischemic rat brain.

Publication Date: 2010 Apr PMID: 19885826
Authors: Shin, H. Y. - Lee, Y. J. - Kim, H. J. - Park, C. K. - Kim, J. H. - Wang, K. C. - Kim, D. G. - Koh, G. Y. - Paek, S. H.
Journal: J Neurosci Res

In cerebral ischemia, the induction of angiogenesis may represent a natural defense mechanism that enables the hypoxic brain to avoid progression into infarction. Angiopoietin-1 (Ang1) is known to produce non-leaky and stable blood vessel formation mainly by the Tie2 receptor. Therefore, we envisioned that the application of cartilage oligomeric matrix protein-Ang1 (COMP-Ang1), a soluble, stable, and potent form of Ang1, would promote angiogenesis and provide a protective effect following unilateral middle cerebral artery occlusion (MCAO) in rats. To this end, we employed a 2-hour-MCAO model, and treated rats with adenovirus encoding COMP-Ang1 (Ade-COMP-Ang1) or control virus encoding beta-gal (Ade-beta-gal). Time course magnetic resonance images (MRIs) revealed significantly reduced infarct volume in the rats treated with Ade-COMP-Ang1 with an improvement of post-ischemic neurological deficits compared with rats treated with Ade-beta-gal. Moreover, compared to the rats treated with Ade-beta-gal, the rats treated with Ade-COMP-Ang1 showed an increase in blood vessels, especially in the border zone adjacent to the infarction, increased number of endogenous neuronal progenitor cells in the ischemic brain, and decreased number of TUNEL-positive cells. Taken together, COMP-Ang1 reduced infarct volume and consequently attenuated post-ischemic neurological deficits through enhanced angiogenesis and increased viable cell mass of neuronal cells.

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Synaptic localization of neuroligin 2 in the rodent retina: comparative study with the dystroglycan-containing complex.

Publication Date: 2010 Mar PMID: 19859968
Authors: Lui, L. - Levinson, J. N. - Noel, G. - Handrigan, G. R. - Richman, J. M. - El-Husseini, A. - Moukhles, H.
Journal: J Neurosci Res

Several recent studies have shown that neuroligin 2 (NL2), a component of the cell adhesion neurexins-neuroligins complex, is localized postsynaptically at hippocampal and other inhibitory synapses throughout the brain. Other studies have shown that components of the dystroglycan complex are also localized at a subset of inhibitory synapses and are coexpressed with NL2 in brain. These data prompted us to undertake a comparative study between the localization of NL2 and the dystroglycan complex in the rodent retina. First, we determined that NL2 mRNA is expressed both in the inner and in the outer nuclear layers. Second, we found that NL2 is localized both in the inner and in the outer synaptic plexiform layers. In the latter, the horseshoe-shaped pattern of NL2 and its extensive colocalization with RIM2, a component of the presynaptic active zone at ribbon synapses, argue that NL2 is localized presynaptically at photoreceptor terminals. Third, comparison of NL2 and the dystroglycan complex distribution patterns reveals that, despite their coexpression in the outer plexiform layer, they are spatially segregated within distinct domains of the photoreceptor terminals, where NL2 is selectively associated with the active zone and the dystroglycan complex is distally distributed in the lateral regions. Finally, we report that the dystroglycan deficiency in the mdx(3cv) mouse does not alter NL2 localization in the outer plexiform layer. These data show that the NL2- and dystroglycan-containing complexes are differentially localized in the presynaptic photoreceptor terminals and suggest that they may serve distinct functions in retina.

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Cellular expression pattern of the protease-activated receptor 4 in the hippocampus in naive rats and after global ischaemia.

Publication Date: 2010 Mar PMID: 19859967
Authors: Henrich-Noack, P. - Riek-Burchardt, M. - Reymann, K. G. - Reiser, G.
Journal: J Neurosci Res

A pronounced hippocampal expression of the Protease-activated Receptor 4 (PAR4) has recently been shown. In the current study the authors define the PAR4-associated sub-cellular structures and the influence of global ischaemia on the expression of PAR4. For that purpose the authors performed double labelling with fluorescence immunohistochemistry on tissue from naive and post-ischaemic rats. In naive animals - apart from the expression in granular and pyramidal neurons - there was an intensive lamellar expression of PAR4 in the CA4 region. Further analysis revealed that PAR4 was localised exclusively on mossy fibre axons in CA4 as detected by double-labelling with calbindin D-28k, but there was no overlap with markers of the neuronal cell body, interneurons, and post-synaptic, pre-synaptic and dendritic structures. Three and 14 days post ischaemia, CA1 neurons were degenerated and, consequently, there was no PAR4 signal in the CA1 band. In most other hippocampal structures no change in the PAR4 expression was detectable, with the exception of the CA3 region. Here, the fibre-associated PAR4 signal was diminished and disintegrated post ischaemia. Additionally, a redistribution from the membrane-bound neuronal localisation of PAR4 in control animals to a diffuse localisation all over the cell soma was revealed in the CA3 area 14 days post ischaemia. In conclusion, the current study proves for the first time that PAR4 is localised in mossy fibre axons. The altered expression in CA3 neurons after ischaemia indicates that PAR4 may be involved in post-ischaemic adaptive mechanisms.

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Activity-dependent somatostatin gene expression is regulated by cAMP-dependent protein kinase and Ca2+-calmodulin kinase pathways.

Publication Date: 2010 Mar PMID: 19859966
Authors: Sanchez-Munoz, I. - Sanchez-Franco, F. - Vallejo, M. - Fernandez, A. - Palacios, N. - Fernandez, M. - Cacicedo, L.
Journal: J Neurosci Res

Ca(2+) influx through L-type voltage-gated Ca(2+) channels (L-VSCC) is required for K(+)-induced somatostatin (SS) mRNA. Increase in intracellular Ca(2+) concentration leads to the activation of cyclic AMP-responsive element binding protein (CREB), a key regulator of SS gene transcription. Several different protein kinases possess the capability of driving CREB upon membrane depolarization. We investigated which of the signalling pathways involved in CREB activation mediates SS gene induction in response to membrane depolarization in cerebrocortical cells exposed to 56 mM K(+). Activity dependent phosphorylation of CREB in Ser(133) was immunodetected. Activation of CREB was biphasic showing two peaks at 5 and 60 min. The selective inhibitors of extracellular signal related protein kinase/mitogen-activated protein kinase (ERK/MAPK) PD098059, cyclic-AMPdependent protein kinase (cAMP/PKA) H89 and RpcAMPS, and Ca(2+)/calmodulin-dependent protein kinases (CaMKs) pathways KN62 and KN93 were used to determine the signalling pathways involved in CREB activation. Here we show that the early activation of CREB was dependent on cAMP/PKA along with CaMKs pathways whereas the ERK/MAPK and CaMKs were implicated in the second peak. We observed that H89, RpcAMPS, KN62 and KN93 blocked K(+)-induced SS mRNA whereas PD098059 did not. These findings indicate that K(+)-induced SSmRNA is mediated by the activation of cAMP/PKA and CaMKs pathways, thus suggesting that the early activation of CREB is involved in the induction of SS by neuronal activity. We also demonstrated, using transient transfections of cerebrocortical cells, that K(+) induces the transcriptional regulation of the SS gene through the cAMP-responsive element (CRE) sequence located in the SS promoter.

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DHA diet reduces AD pathology in young APPswe/PS1 Delta E9 transgenic mice: possible gender effects.

Publication Date: 2010 Apr PMID: 19859965
Authors: Perez, S. E. - Berg, B. M. - Moore, K. A. - He, B. - Counts, S. E. - Fritz, J. J. - Hu, Y. S. - Lazarov, O. - Lah, J. J. - Mufson, E. J.
Journal: J Neurosci Res

Epidemiological and clinical trial findings suggest that consumption of docosahexaenoic acid (DHA) lowers the risk of Alzheimer's disease (AD). We examined the effects of short-term (3 months) DHA enriched diet on plaque deposition and synaptic defects in forebrain of young APPswe/PS1 Delta E9 transgenic (tg) and non-transgenic (ntg) mice. Gas chromatography revealed a significant increase in DHA concomitant with a decrease of arachidonic acid in both brain and liver in mice fed with DHA. Female tg mice consumed relatively more food daily than ntg female mice, independent of diet. Plaque load was significantly reduced in the cortex, ventral hippocampus and striatum of female APPswe/PS1 Delta E9 tg mice on DHA diet compared to female tg mice on control diet. Immunoblot quantitation of the APOE receptor, LR11, which is involved in APP trafficking and A beta production, were unchanged in mice on DHA or control diets. Moreover drebrin levels were significantly increased in the hippocampus of tg mice on the DHA diet. Finally, in vitro DHA treatment prevented amyloid toxicity in cell cultures. Our findings support the concept that increased DHA consumption may play and important role in reducing brain insults in female AD patients.

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Caspase inhibition by cardiotrophin-1 prevents neuronal death in vivo and in vitro.

Publication Date: 2010 Apr PMID: 19859964
Authors: Peng, H. - Sola, A. - Moore, J. - Wen, T.
Journal: J Neurosci Res

Our previous studies showed that cardiotrophin-1 (CT-1), a cytokine in the interleukin-6 family, protected the developing rat brain against focal cerebral ischemia (FCI) in vivo and prevented cortical neuron death in vitro. However, the mechanisms by which CT-1 prevents neuronal death are not clearly understood. This in vivo study focused on whether CT-1 treatment prevented FCI-induced brain injuries in the postnatal day 7 (P7) rat through modulating activation of the initiator caspase-8 (C-8) and the downstream effector caspase-3 (C-3). FCI caused a significant increase in expressions of cleaved C-8 and C-3 and, meanwhile, a significant decrease in expression of microtubule-associated protein-2 (MAP2) in the left ischemic cortex of the P7 rat brain after FCI. Exogenous treatment of CT-1 significantly reduced the expression of cleaved C-8 or C-3 and attenuated the decline in MAP2 expression in the ischemic cortex from 12 to 24 hr after FCI. Subsequent in vitro experiments demonstrated that CT-1 treatment inhibited sodium nitroprusside (SNP)-induced activation of C-8 and C-3 and loss of MAP2-positive neurons in cortical neuron cultures. More importantly, CT-1 activated several pathways, including Janus kinase 2, signal transducers and activators of transcription 3, nuclear factor kappa B, mitogen-activated protein kinase (MAPK), and MAPK kinase in the cultures exposed to SNP. This is the first suggestion that CT-1 prevents neuronal injury in the developing central nervous system possibly through mediating multiple signal pathways, inhibiting activation of C-8 and C-3.

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Axon responses of embryonic stem cell-derived dopaminergic neurons to semaphorins 3A and 3C.

Publication Date: 2010 Apr PMID: 19859963
Authors: Tamariz, E. - Diaz-Martinez, N. E. - Diaz, N. F. - Garcia-Pena, C. M. - Velasco, I. - Varela-Echavarria, A.
Journal: J Neurosci Res

Class 3 Semaphorins are a subfamily of chemotropic molecules implicated in the projection of dopaminergic neurons from the ventral mesencephalon and in the formation of the nigrostriatal pathway (NSP) during embryonic development. In humans, loss of mesencephalic dopaminergic neurons leads to Parkinson's disease (PD). Cell replacement therapy with dopaminergic neurons generated from embryonic stem cells (ES-TH(+)) is being actively explored in models of PD. Among several requisites for this approach to work are adequate reconstruction of the NSP and correct innervation of normal striatal targets by dopaminergic axons. In this work, we characterized the response of ES-TH(+) neurons to semaphorins 3A, 3C, and 3F and compared it with that of tyrosine hidroxylase-positive neurons (TH(+)) obtained from embryonic ventral mesencephalon (VM-TH(+)). We observed that similar proportions of ES-TH(+) and VM-TH(+) neurons express semaphorin receptors neuropilins 1 and 2. Furthermore, the axons of both populations responded very similarly to semaphorin exposure: semaphorin 3A increased axon length, and semaphorin 3C attracted axons and increased their length. These effects were mediated by neuropilins, insofar as addition of blocking antibodies against these proteins reduced the effects on axonal growth and attraction, and only TH(+) axons expressing neuropilins responded to the semaphorins analyzed. The observations reported here show phenotypic similarities between VM-TH(+) and ES-TH(+) neurons and suggest that semaphorins 3A and 3C could be employed to guide axons of grafted ES-TH(+) in therapeutic protocols for PD.

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Recurrent systemic infections with Streptococcus pneumoniae do not aggravate the course of experimental neurodegenerative diseases.

Publication Date: 2010 Apr PMID: 19859962
Authors: Ebert, S. - Goos, M. - Rollwagen, L. - Baake, D. - Zech, W. D. - Esselmann, H. - Wiltfang, J. - Mollenhauer, B. - Schliebs, R. - Gerber, J. - Nau, R.
Journal: J Neurosci Res

Neurological symptoms of patients suffering from neurodegenerative diseases such as Alzheimer's dementia (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS) often worsen during infections. We assessed the disease-modulating effects of recurrent systemic infections with the most frequent respiratory pathogen, Streptococcus pneumoniae, on the course of AD, PD, and ALS in mouse models of these neurodegenerative diseases [transgenic Tg2576 mice, (Thy1)-[A30P]alpha SYN mice, and Tg(SOD1-G93A) mice]. Mice were repeatedly challenged intraperitoneally with live S. pneumoniae type 3 and treated with ceftriaxone for 3 days. Infection caused an increase of interleukin-6 concentrations in brain homogenates. The clinical status of (Thy1)-[A30P]alpha SYN mice and Tg(SOD1-G93A) mice was monitored by repeated assessment with a clinical score. Motor performance was controlled by the tightrope test and the rotarod test. In Tg2576 mice, spatial memory and learning deficits were assessed in the Morris water maze. In none of the three mouse models onset or course of the disease as evaluated by the clinical tests was affected by the recurrent systemic infections performed. Levels of alpha-synuclein in brains of (Thy1)-[A30P]alpha SYN mice did not differ between infected animals and control animals. Plaque sizes and concentrations of A beta 1-40 and A beta 1-42 were not significantly different in brains of infected and uninfected Tg2576 mice. In conclusion, onset and course of disease in mouse models of three common neurodegenerative disorders were not influenced by repeated systemic infections with S. pneumoniae, indicating that the effect of moderately severe acute infections on the course of neurodegenerative diseases may be less pronounced than suspected.

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Ciliary neurotrophic factor enhances nicotinic synaptic transmission in sympathetic neurons.

Publication Date: 2010 Mar PMID: 19830843
Authors: Nai, Q. - Wang, X. - Jin, Y. - Sun, D. - Li, M. - Hu, B. - Zhang, X.
Journal: J Neurosci Res

Nicotinic acetylcholine receptors mediate fast synaptic transmission in both central and peripheral nervous systems. These receptors play important roles in various physiological functions and are involved in different neurological diseases. A disruption in nicotinic receptor-mediated synaptic transmission due to the loss of nAChRs was detected in the brains of patients with Parkinson's disease and Alzheimer's disease. Although ciliary neurotrophic factor (CNTF) has been reported to promote the cholinergic properties by increasing the production and storage of acetylcholine, it is still unclear whether CNTF can enhance nicotinic synaptic neurotransmission. In this study, we found that CNTF dramatically enhanced the frequency and amplitude of nicotinic excitatory post-synaptic currents in rat superior cervical ganglion neurons maintained in a medium supplemented with nerve growth factor. Moreover, the number of neurons displaying nicotinic synaptic currents was also significantly increased by CNTF. These results suggest that CNTF could enhance nicotinic synaptic transmission via both presynaptic and postsynaptic mechanisms. The findings of this study reinforce the rationale for the usage of combinations of different neurotrophic factors for the therapy of neurodegenerative diseases.

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Degenerative abnormalities in transgenic neocortical neuropeptide Y interneurons expressing tau-green fluorescent protein.

Publication Date: 2010 Feb 15 PMID: 19830842
Authors: Rancillac, A. - Laine, J. - Perrenoud, Q. - Geoffroy, H. - Ferezou, I. - Vitalis, T. - Rossier, J.
Journal: J Neurosci Res

The introduction of a reporter gene into bacterial artificial chromosome (BAC) constructs allows a rapid identification of the cell type expressing the gene of interest. Here we used BAC transgenic mice expressing a tau-sapphire green fluorescent protein (GFP) under the transcriptional control of the neuropeptide Y (NPY) genomic sequence to characterize morphological and electrophysiological properties of NPY-GFP interneurons of the mouse juvenile primary somatosensory cortex. Electrophysiological whole-cell recordings and biocytin injections were performed to allow the morphological reconstruction of the recorded neurons in three dimensions. Ninety-six recorded NPY-GFP interneurons were compared with 39 wild-type (WT) NPY interneurons, from which 23 and 19 were reconstructed, respectively. We observed that 91% of the reconstructed NPY-GFP interneurons had developed an atypical axonal swelling from which emerge numerous ramifications. These abnormalities were very heterogeneous in shape and size. They were immunoreactive for the microtubule-associated protein tau and the lysosomal-associated membrane protein 1 (LAMP1). Moreover, an electron microscopic analysis revealed the accumulation of numerous autophagic and lysosomal vacuoles in swollen axons. Morphological analyses of NPY-GFP interneurons also indicated that their somata were smaller, their entire dendritic tree was thickened and presented a restricted spatial distribution in comparison with WT NPY interneurons. Finally, the morphological defects observed in NPY-GFP interneurons appeared to be associated with alterations of their electrophysiological intrinsic properties. Altogether, these results demonstrate that NPY-GFP interneurons developed dystrophic axonal swellings and severe morphological and electrophysiological defects that could be due to the overexpression of tau-coupled reporter constructs.

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alpha-Synuclein abnormalities in mouse models of peroxisome biogenesis disorders.

Publication Date: 2010 Mar PMID: 19830841
Authors: Yakunin, E. - Moser, A. - Loeb, V. - Saada, A. - Faust, P. - Crane, D. I. - Baes, M. - Sharon, R.
Journal: J Neurosci Res

alpha-Synuclein (alphaS) is a presynaptic protein implicated in Parkinson's disease (PD). Growing evidence implicates mitochondrial dysfunction, oxidative stress, and alphaS-lipid interactions in the gradual accumulation of alphaS in pathogenic forms and its deposition in Lewy bodies, the pathological hallmark of PD and related synucleinopathies. The peroxisomal biogenesis disorders (PBD), with Zellweger syndrome serving as the prototype of this group, are characterized by malformed and functionally impaired peroxisomes. Here we utilized the PBD mouse models Pex2-/-, Pex5-/-, and Pex13-/- to study the potential effects of peroxisomal dysfunction on alphaS-related pathogenesis. We found increased alphaS oligomerization and phosphorylation and its increased deposition in cytoplasmic inclusions in these PBD mouse models. Furthermore, we show that alphaS abnormalities correlate with the altered lipid metabolism and, specifically, with accumulation of long chain, n-6 polyunsaturated fatty acids that occurs in the PBD models.

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Divergent role for MMP-2 in myelin breakdown and oligodendrocyte death following transient global ischemia.

Publication Date: 2010 Mar PMID: 19830840
Authors: Walker, E. J. - Rosenberg, G. A.
Journal: J Neurosci Res

Transient global ischemia causes delayed white matter injury to the brain with oligodendrocyte (OLG) death and myelin breakdown. There is increasing evidence that hypoxia may be involved in several diseases of the white matter, including multiple sclerosis, vascular dementia, and ischemia. Matrix metalloproteinases (MMPs) are increased in rat and mouse models of hypoxic hypoperfusion and have been associated with OLG death. However, whether the MMPs act on myelin or OLGs remains unresolved. We hypothesized that delayed expression of MMPs caused OLG death and myelin breakdown. To test the hypothesis, adult mice underwent hypoxic hypoperfusion with transient bilateral occlusion of the carotid arteries. After 3 days of reperfusion, ischemic white matter had increased reactivity of astrocytes and microglia, MMP-2 localization in astrocytes, and increased protein expression and activity of MMP-2. In addition, there was a significant loss of myelin basic protein (MBP) by Western blot and caspase-3- mediated OLG death. Treatment with the broad-spectrum MMP inhibitor, BB-94, significantly decreased astrocyte reactivity and MMP-2 activity. More importantly, it reduced MBP breakdown. However, MMP inhibition had no effect on OLG loss. Our results implicate MMPs released by reactive astrocytes in delayed myelin degradation, while OLG death occurs by an MMP-independent mechanism. We propose that MMP-mediated myelin loss is important in hypoxic injury to the white matter.

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Cocaine withdrawal-induced anxiety in females: impact of circulating estrogen and potential use of delta-opioid receptor agonists for treatment.

Publication Date: 2010 Mar PMID: 19830839
Authors: Ambrose-Lanci, L. M. - Sterling, R. C. - Van Bockstaele, E. J.
Journal: J Neurosci Res

Sex differences in cocaine addiction warrants further research focused on examining the growing population of female cocaine addicts. As demonstrated in both clinical and preclinical research, females are more susceptible to drug relapse with anxiety being a contributing factor. In support of this, a recent clinical study from our laboratory highlights the importance of menstrual cycle phase and anxiety at treatment admission for cocaine addiction on treatment retention. In support of these trends in the clinical population, the purpose of the present study was to design an animal model to directly test the role of circulating hormone levels during cocaine withdrawal. To directly measure the influence of estrogen on anxiety-like behavior during early stages of withdrawal, both ovariectomized and intact female rodent models were employed. The elevated-plus maze and elevated-zero maze were used to assess anxiety-like behavior. Recent evidence in male rodents highlights a potential role for the delta opioid-receptor (DOR) system in the modulation of cocaine withdrawal-induced anxiety. In addition to the evaluation of hormonal effects, a potential anxiolytic specific for DOR was tested for its efficacy in females withdrawn from cocaine. Our results support the use of DOR agonists as a potential anxiolytic in females and highlight the importance of estrogen and other circulating hormones during all phases of cocaine addiction.

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Microglial/macrophage cells in mammalian olfactory nerve fascicles.

Publication Date: 2010 Mar PMID: 19830837
Authors: Smithson, L. J. - Kawaja, M. D.
Journal: J Neurosci Res

This is the first description of a population of Iba1- and annexin A3-immunopositive cells residing in the peripheral olfactory nerves of adult rats and adult cats. Based on their ramified appearance, positive immunostaining for the monocytic markers Iba1 and annexin A3, and reactivity to bulbectomy (in adult rats), these cells found within the olfactory nerve fascicles of both mammalian species meet several important criteria for their designation as microglia/macrophages. These Iba1-/annexin A3-immunopositive cells may be uniquely positioned to protect against the potential spread of dangerous environmental xenobiotics (such as viruses and toxins) into the brain, where such pathogens may contribute to the development of neurological diseases, such Alzheimer's and Parkinson's diseases.

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Fear learning and extinction are linked to neuronal plasticity through Rin1 signaling.

Publication Date: 2010 Mar PMID: 19830836
Authors: Bliss, J. M. - Gray, E. E. - Dhaka, A. - O'Dell, T. J. - Colicelli, J.
Journal: J Neurosci Res

The amygdala is known to have a crucial role in both the acquisition and extinction of conditioned fear, but the physiological changes and biochemical mechanisms underlying these forms of learning are only partly understood. The Ras effector Rin1 activates Abl tyrosine kinases and Rab5 GTPases and is highly expressed in mature neurons of the telencephalon including the amygdala, where it inhibits the acquisition of fear memories (Rin1(-/-) mice show enhanced learning of conditioned fear). Here we report that Rin1(-/-) mice exhibit profound deficits in both latent inhibition and fear extinction, suggesting a critical role for Rin1 in gating the acquisition and persistence of cue-dependent fear conditioning. Surprisingly, we also find that depotentiation, a proposed cellular mechanism of extinction, is enhanced at lateral-basolateral (LA-BLA) amygdaloid synapses in Rin1(-/-) mice. Inhibition of a single Rin1 downstream effector pathway, the Abl tyrosine kinases, led to reduced amygdaloid depotentiation, arguing that proper coordination of Abl and Rab5 pathways is critical for Rin1-mediated effects on plasticity. While demonstrating a correlation between amygdala plasticity and fear learning, our findings argue against models proposing a direct causative relationship between amygdala depotentiation and fear extinction. Taken together, the behavior and physiology of Rin1(-/-) mice provide new insights into the regulation of memory acquisition and maintenance. In addition, Rin1(-/-) mice should prove useful as a model for pathologies marked by enhanced fear acquisition and retention, such as posttraumatic stress disorder.

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Fas activation increases neural progenitor cell survival.

Publication Date: 2010 Mar PMID: 19830835
Authors: Knight, J. C. - Scharf, E. L. - Mao-Draayer, Y.
Journal: J Neurosci Res

Although there is a sizable amount of research focusing on adult neural progenitor cells (NPCs) as a therapeutic approach for many neurodegenerative diseases, including multiple sclerosis, little is known about the pathways that govern NPC survival and apoptosis. Fas, a member of the death receptor superfamily, plays a well-characterized role in the immune system, but its function in neural stem cells remains uncertain. Our study focuses on the effects of Fas on NPC survival in vitro. Activation of Fas by recombinant Fas ligand (FasL) did not induce apoptosis in murine NPCs in culture. In fact, both an increase in the amount of viable cells and a decrease in apoptotic and dying cells were observed with FasL treatment. Our data indicate that FasL-mediated adult NPC neuroprotection is characterized by a reduction in apoptosis, but not increased proliferation. Further investigation of this effect revealed that the antiapoptotic effects of FasL are mediated by the up-regulation of Birc3, an inhibitor of apoptosis protein (IAP). Conversely, the observed effect is not the result of altered caspase activation or FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein) up-regulation, which is known to inhibit caspase-8-mediated cell death in T cells. Our data indicate that murine adult NPCs are resistant to FasL-induced cell death. Activation of Fas increased cell survival by decreasing apoptosis through Birc3 up-regulation. These results describe a novel pathway involved in NPC survival.

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Developmental and age-related changes of peptidylarginine deiminase 2 in the mouse brain.

Publication Date: 2010 Mar PMID: 19830834
Authors: Shimada, N. - Handa, S. - Uchida, Y. - Fukuda, M. - Maruyama, N. - Asaga, H. - Choi, E. K. - Lee, J. - Ishigami, A.
Journal: J Neurosci Res

Peptidylarginine deiminases (PADs) are a group of posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a Ca(2+)-dependent manner. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and functions. Among the five isoforms of PADs, PAD2 and PAD4 are proved occupants of the central nervous system (CNS), and especially PAD2 is a main PAD enzyme expressed in the CNS. We previously reported that abnormal protein citrullination by PAD2 has been closely associated with the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and prion disease. Protein citrullination in these patients is thought to play a role during the initiation and/or progression of disease. However, the contribution of changes in PAD2 levels, and consequent citrullination, during developmental and aging processes remained unclear. Therefore, we used quantitative real-time RT-PCR, Western blot analysis, and immunohistochemical methods to measure PAD2 expression and localization in the brain during those processes. PAD2 mRNA expression was detected in the brains of mice as early as embryonic day 15, and its expression in cerebral cortex, hippocampus, and cerebellum increased significantly as the animals aged from 3 to 30 months old. No citrullinated proteins were detected during that period. Moreover, we found here, for the first time, that PAD2 localized specifically in the neuronal cells of the cerebral cortex and Purkinje cells of the cerebellum. These findings indicate that, despite PAD2's normally inactive status, it becomes active and citrullinates cellular proteins, but only when the intracellular Ca(2+) balance is upset during neurodegenerative changes.

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Astrocytes protect oligodendrocyte precursor cells via MEK/ERK and PI3K/Akt signaling.

Publication Date: 2010 Mar PMID: 19830833
Authors: Arai, K. - Lo, E. H.
Journal: J Neurosci Res

Accumulating evidence suggest that trophic coupling among different cell types in the brain is required to maintain normal CNS function. Here we show that astrocytes secrete soluble factors that can be oligodendrocyte-supportive. Oligodendrocyte precursor cells (OPCs) and astrocytes were prepared from neonatal rat brain and cultured separately. We conducted cell culture medium-transfer experiments to examine whether astrocytes secrete OPC-protective factors. Conditioned media from astrocytes protected OPCs against H(2)O(2)-induced oxidative stress, starvation, and oxygen-glucose deprivation. This protective effect may be mediated in part via ERK and Akt signaling pathways. Astrocyte-conditioned media upregulated the phosphorylation levels of ERK and Akt in OPC cultures. Blockade of ERK or Akt signaling with U0126 or LY294002 cancelled the OPC-protective effects of astrocyte-conditioned media. Taken together, these data suggest that astrocytes are an important source for oligodendrocyte-supportive factors. Coupling between these two major glial components in brain may be vital for sustaining white matter homeostasis.

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Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells.

Publication Date: 2010 Mar PMID: 19813266
Authors: George, J. - Banik, N. L. - Ray, S. K.
Journal: J Neurosci Res

The potent antiapoptotic molecule Bcl-2 is markedly up-regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein-induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. The cells were transfected with Bcl-2 siRNA plasmid vector, treated with 10 microM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real-time RT-PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha), Fas-associated death domain (FADD), and TNFR-1-associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor- and mitochondria-mediated apoptotic pathways. Treatment with the combination of Bcl-2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria-mediated apoptotic pathway. Genistein triggered the receptor-mediated apoptotic pathway through upregulation of TNF-alpha, FasL, TRADD, and FADD and activation of caspase-8. Combination of Bcl-2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl-2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma.

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Effect of rosmarinic acid in motor dysfunction and life span in a mouse model of familial amyotrophic lateral sclerosis.

Publication Date: 2010 Mar PMID: 19798750
Authors: Shimojo, Y. - Kosaka, K. - Noda, Y. - Shimizu, T. - Shirasawa, T.
Journal: J Neurosci Res

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease affecting motor neurons. About 2% of patients with the disease are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). The purpose of this study is to assess the effect of rosemary extract and its major constituents, rosmarinic acid (RA) and carnosic acid (CA), in human SOD1 G93A transgenic mice, which are well-established mouse models for ALS. The present study demonstrates that intraperitoneal administration of rosemary extract or RA from the presymptomatic stage significantly delayed motor dysfunction in paw grip endurance tests, attenuated the degeneration of motor neurons, and extended the life span of ALS model mice. In addition, RA administration significantly improved the clinical score and suppressed body weight loss compared with a vehicle-treated group. In conclusion, this study provides the first report that rosemary extract and, especially, RA have preventive effects in the mouse model of ALS.

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Synergistic effect between proteasome and autophagosome in the clearance of polyubiquitinated TDP-43.

Publication Date: 2010 Mar PMID: 19798749
Authors: Urushitani, M. - Sato, T. - Bamba, H. - Hisa, Y. - Tooyama, I.
Journal: J Neurosci Res

Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.

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Enzymatic-nonenzymatic cellular antioxidant defense systems response and immunohistochemical detection of MDMA, VMAT2, HSP70, and apoptosis as biomarkers for MDMA (Ecstasy) neurotoxicity.

Publication Date: 2010 Mar PMID: 19798748
Authors: Riezzo, I. - Cerretani, D. - Fiore, C. - Bello, S. - Centini, F. - D'Errico, S. - Fiaschi, A. I. - Giorgi, G. - Neri, M. - Pomara, C. - Turillazzi, E. - Fineschi, V.
Journal: J Neurosci Res

3,4-Methylenedioxymethamphetamine (MDMA)-induced neurotoxicity leads to the formation of quinone metabolities and hydroxyl radicals and then to the production of reactive oxygen species (ROS). We evaluated the effect of a single dose of MDMA (20 mg/kg, i.p.) on the enzymatic and nonenzymatic cellular antioxidant defense system in different areas of rat brain in the early hours (<6 hr) of the administration itself, and we identified the morphological expressions of neurotoxicity induced by MDMA on the vulnerable brain areas in the first 24 hr. The acute administration of MDMA produces a decrease of reduced and oxidized glutathione ratio, and antioxidant enzyme activities were significantly reduced after 3 hr and after 6 hr in frontal cortex. Ascorbic acid levels strongly increased in striatum, hippocampus, and frontal cortex after 3 and 6 hr. High levels of malonaldehyde with respect to control were measured in striatum after 3 and 6 hr and in hippocampus and frontal cortex after 6 hr. An immunohistochemical investigation on the frontal, thalamic, hypothalamic, and striatal areas was performed. A strong positive reaction to the antivesicular monoamine transporter 2 was observed in the frontal section, in the basal ganglia and thalamus. Cortical positivity, located in the most superficial layer was revealed only for heat shock protein 70 after 24 hr.

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Reduced size of the dendritic tree does not protect Purkinje cells from excitotoxic death.

Publication Date: 2010 Mar PMID: 19798747
Authors: Gugger, O. S. - Kapfhammer, J. P.
Journal: J Neurosci Res

Purkinje cell loss by excitotoxic damage is a typical finding in many cerebellar diseases. One important aspect of this high sensitivity of Purkinje cells to excitotoxic death might be the enormous size of their dendritic tree, with a high load of excitatory glutamate receptors. We have studied whether reduction in the size of the dendritic tree might confer resistance against excitotoxic death to Purkinje cells. We have grown Purkinje cells in organotypic cerebellar slice cultures under chronic activation of metabotropic glutamate receptors or of protein kinase C. Both treatments strongly reduced dendritic tree size. After this treatment, cells were exposed to the glutamate receptor agonist AMPA, which has a strong excitotoxic effect on Purkinje cells. We found that Purkinje cells with small dendritic trees were as sensitive to AMPA exposure as untreated control cells with large dendritic trees. Immunostaining against vesicular glutamate transporter 1 revealed that the small dendritic trees were densely covered by glutamatergic terminals. Our results indicate that the expansion of the dendritic tree and the total number of AMPA receptors per neuron do not play a major role in determining the susceptibility of Purkinje cells to excitotoxic death.

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Calcium imaging in single neurons from brain slices using bioluminescent reporters.

Publication Date: 2010 Mar PMID: 19798746
Authors: Drobac, E. - Tricoire, L. - Chaffotte, A. F. - Guiot, E. - Lambolez, B.
Journal: J Neurosci Res

Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.

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Stromal cell-secreted factors promote the survival of embryonic stem cell-derived early neural stem/progenitor cells via the activation of MAPK and PI3K-Akt pathways.

Publication Date: 2010 Mar PMID: 19798745
Authors: Ishii, S. - Okada, Y. - Kadoya, T. - Matsuzaki, Y. - Shimazaki, T. - Okano, H.
Journal: J Neurosci Res

Neural stem/progenitor cells (NS/PCs) have been studied extensively with the hope of using them clinically to repair the damaged central nervous system. However, little is known about the signals that regulate the proliferation, survival, and differentiation of NS/PCs in early development. To clarify the underlying mechanisms, we took advantage of an in vitro ES cell differentiation system from which we can obtain neurospheres containing NS/PCs with characteristics of the early caudal neural tube, by treating embryoid bodies (EBs) with a low concentration of retinoic acid (RA). We found that conditioned medium from the PA6 stromal cell line (PA6CM) increased the efficiency of neurosphere formation by suppressing apoptosis and promoting the survival of the NS/PCs. PA6CM also induced the phosphorylation of Erk1/2 and Akt1 in cells derived from the EBs. Furthermore, inhibitors of the MAPK and PI3K-Akt signaling pathways, U0126 and LY294002, attenuated the effects of PA6CM, significantly increasing the number of apoptotic cells and decreasing the number of viable cells among the ES cell-derived NS/PCs. Thus, PA6CM appears to contain soluble factors that promote the survival of ES cell-derived early NS/PCs through the activation of the MAPK and PI3K-Akt pathways.

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Down-regulation of diacylglycerol lipase-alpha during neural stem cell differentiation: identification of elements that regulate transcription.

Publication Date: 2010 Mar PMID: 19798744
Authors: Walker, D. J. - Suetterlin, P. - Reisenberg, M. - Williams, G. - Doherty, P.
Journal: J Neurosci Res

The diacylglycerol lipases (DAGLalpha and DAGLbeta) synthesize 2-arachidonoylglycerol (2-AG), a full agonist at cannabinoid receptors. Dynamic regulation of DAGL expression underpins its role in axonal growth and guidance during development, retrograde synaptic signalling at mature synapses, and maintenance of adult neurogenesis. We show here that DAGLalpha expression is dramatically down-regulated when neural stem (NS) cells are differentiated toward a gamma-aminobutyric acidergic neuronal phenotype. To understand how DAGLalpha expression might be controlled, we sought to identify the core promoter region and regulatory elements within it. The core promoter was identified and shown to contain both an enhancer and a suppressor region. Deletion analysis identified two elements, including a GC-box, that specifically promote expression in NS cells. Bioinformatic analysis identified three candidate transcription factors that might regulate DAGLalpha expression in NS cells by binding to the GC box; these were specificity protein 1 (Sp1), early growth response element 1 (EGR1), and zinc finger DNA-binding protein 89 (ZBP-89). However, Sp1 was the only factor that could bind to the GC-box. A specific mutation within the GC-box that inhibited Sp1 binding reduced DAGLalpha promoter activity in NS cells. Likewise, a dominant negative Sp1 was shown to bind to the GC-box and to suppress DAGLalpha promoter activity specifically in NS cells. Finally, like DAGLalpha, Sp1 was down-regulated during neuronal differentiation. A full characterization of the DAGLalpha promoter will help to elucidate the upstream pathways that regulate DAGLalpha expression in NS cells and their progeny.

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Mutual effects of caveolin and nerve growth factor signaling in pig oligodendrocytes.

Publication Date: 2010 Feb 15 PMID: 19795378
Authors: Schmitz, M. - Kloppner, S. - Klopfleisch, S. - Mobius, W. - Schwartz, P. - Zerr, I. - Althaus, H. H.
Journal: J Neurosci Res

Signaling of growth factors may depend on the recruitment of their receptors to specialized microdomains. Previous reports on PC12 cells indicated an interaction of raft-organized caveolin and TrkA signaling. Because porcine oligodendrocytes (OLs) respond to nerve growth factor (NGF), we were interested to know whether caveolin also plays a role in oligodendroglial NGF/TrkA signaling. OLs expressed caveolin at the plasma membrane but also intracellularly. This was partially organized in the classically Omega-shaped invaginations, which may represent caveolae. We could show that caveolin and TrkA colocalize by using a discontinuous sucrose gradient (Song et al. [1996] J. Biol. Chem. 271:9690-9697), MACS technology, and immunoprecipitation. However, differential extraction of caveolin and TrkA with Triton X-100 at 4 degrees C indicated that caveolin and TrkA are probably not exclusively present in detergent-resistant, caveolin-containing rafts (CCRs). NGF treatment of OLs up-regulated the expression of caveolin-1 (cav-1) and stimulated tyrosine-14 phosphorylation of cav-1. Furthermore, OLs were transfected with cav-1-specific small interfering RNA (siRNA). A knockdown of cav-1 resulted in a reduced activation of downstream components of the NGF signaling cascade, such as p21Ras and mitogen-activated protein kinase (MAPK) after NGF exposure of OLs. Subsequently, increased oligodendroglial process formation via NGF was impaired. The present study indicates that CCRs/caveolin could play a modulating role during oligodendroglial differentiation and regeneration.

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Huperzine a improves chronic inflammation and cognitive decline in rats with cerebral hypoperfusion.

Publication Date: 2010 Mar PMID: 19795377
Authors: Wang, J. - Zhang, H. Y. - Tang, X. C.
Journal: J Neurosci Res

Chronic cerebral hypoperfusion has been suggested to contribute to the progression of dementia. Inflammation and white matter lesion (WML) are involved in the pathologic process. This study investigated whether huperzine A, a natural acetylcholinesterase (AChE) inhibitor, has beneficial effects on long-lasting inflammation as well as cognitive impairment in a rat model of cerebral hypoperfusion and how it plays these roles. Chronic cerebral hypoperfusion was induced by occlusion of bilateral common carotid arteries (two-vessel occlusion; 2VO). Huperzine A was initially given 150 min after 2VO and daily for 3, 7, 14, and 28 days. Learning and memory dysfunction as tested by Morris water maze performance was observed in 2VO-operated rats and was significantly improved by huperzine A treatment. WML and activation staining of immune cells were evaluated by Kluver-Barrera (KB) and immunohistochemistry, respectively. Myelin damage and increased immunostains were found in optic tract at all indicated days. Huperzine A treatment significantly ameliorated all these phenomena. Moreover, huperzine A also suppressed overexpression of the inflammatory factor tumor necrosis factor-alpha (TNF-alpha) and overphosphorylation of JNK and p38 mitogen-activated protein kinases (MAPKs) in a cell model of chronic hypoxia. Preincubation with mecamylamine (MEC), a nicotinic acetylcholine receptor (nAChR) antagonist, for 30 min before hypoxia notably reversed the effects of huperzine A on TNF-alpha production and MAPKs phosphorylation. In conclusion, delayed and chronic administration of huperzine A could protect against 2VO-induced cognitive impairment, which might be related to its beneficial effects on WML, and the nAChR-dependent cholinergic anti-inflammation pathway plays an important role.

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Trimethyltin intoxication up-regulates nitric oxide synthase in neurons and purinergic ionotropic receptor 2 in astrocytes in the hippocampus.

Publication Date: 2010 Feb 15 PMID: 19795376
Authors: Latini, L. - Geloso, M. C. - Corvino, V. - Giannetti, S. - Florenzano, F. - Viscomi, M. T. - Michetti, F. - Molinari, M.
Journal: J Neurosci Res

Nitric oxide (NO) and purinergic ionotropic receptors (P2X) mediate cellular events in the central nervous system (CNS) under physiological conditions as well as during pathological events, and they have been recently proposed to interact in mediating CNS response to injury (Viscomi et al. [2004] Neuroscience 123:393-404; Florenzano et al. [2008] Pflugers Arch. 452:622-644). Trimethyltin (TMT) is an organotin compound that generates neurotoxic effects, and it has been used in a model of neurodegenerative disease and memory dysfunction. TMT causes neuronal death and reactive gliosis primarily in the hippocampus and other limbic regions. In the present study, we examined the degenerative events and the expression of nitric oxide synthase (NOS) and P2X receptor subtypes (P2X(1,2,4,7)Rs) that were induced by TMT administration at different time points (3, 7, 14, and 21 days) by conventional and confocal microscopy and Western blotting. Massive glial activation and neuronal death in the CA1 and CA3 regions were observed after TMT treatment. In these areas, astrocytic P2X(2)R and neuronal NOS were temporarily enhanced in association with the progression of neuronal death. In the hippocampus, the physiological expression of P2X(1)R, P2X(4)R, and P2X(7)R was not modified by TMT. The present data demonstrate that, as in other neurodegenerative models, TMT-induced hippocampal degeneration is associated with nitrergic and purinergic activations. Nevertheless, at odds with previous data, in this model the two systems are active in segregated cell populations, namely, P2XR in astrocytes and NOS in neurons. Finally, the temporal relations between P2XR and NOS expression and neuronal degeneration suggest interactions between P2XR/NO signaling and cell survival.

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Interaction of Rab31 and OCRL-1 in oligodendrocytes: its role in transport of mannose 6-phosphate receptors.

Publication Date: 2010 Feb 15 PMID: 19795375
Authors: Rodriguez-Gabin, A. G. - Ortiz, E. - Demoliner, K. - Si, Q. - Almazan, G. - Larocca, J. N.
Journal: J Neurosci Res

Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6-phosphate receptors (MPRs) from the trans-Golgi network (TGN) to endosomes and for Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL-1, a phosphatidylinositol 4,5-diphosphate 5-phosphatase (PI(4,5)P(2) 5-phosphatase) that regulates the levels of PI(4,5)P(2) and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL-1 in a yeast two-hybrid system, GST-Rab31 pull-down experiments, and coimmunoprecipitation of OCRL-1 using oligodendrocyte culture lysates. Rab31 and OCRL-1 colocalize in the TGN, post-TGN carriers, and endosomes. Cation-dependent MPR (CD-MPR) is sorted to OCRL-1-containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA-mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL-1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL-1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL-1 causes demyelination in humans.

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NAD(P)H oxidase contributes to neurotoxicity in an excitotoxic/prooxidant model of Huntington's disease in rats: protective role of apocynin.

Publication Date: 2010 Feb 15 PMID: 19795371
Authors: Maldonado, P. D. - Molina-Jijon, E. - Villeda-Hernandez, J. - Galvan-Arzate, S. - Santamaria, A. - Pedraza-Chaverri, J.
Journal: J Neurosci Res

Intrastriatal injection of quinolinic acid (QUIN) to rodents reproduces some biochemical, morphological, and behavioral characteristics of Huntington's disease. NAD(P)H oxidase is an enzymatic complex that catalyzes superoxide anion (O(2).(-)) production from O(2) and NADPH. The present study evaluated the role of NAD(P)H oxidase in the striatal damage induced by QUIN (240 nmol/microl) in adult male Wistar rats by means of apocynin (APO; 5 mg/kg i.p.), a specific NAD(P)H oxidase inhibitor. Rats were given APO 30 min before and 1 hr after QUIN injection or only 30 min after QUIN injection. NAD(P)H oxidase activity was measured in striatal homogenates by O2(*)(-) production. QUIN infusion to rats significantly increased striatal NAD(P)H oxidase activity (2 hr postlesion), whereas APO treatments decreased the QUIN-induced enzyme activity (2 hr postlesion), lipid peroxidation (3 hr postlesion), circling behavior (6 days postlesion), and histological damage (7 days postlesion). The addition of NADH to striatal homogenates increased NAD(P)H oxidase activity in striata from QUIN-treated animals but not from sham rats. Interestingly, O2(*)(-) production in QUIN-lesioned striata was unaffected by the addition of substrates for intramitochondrial O2(*)(-) production, xanthine oxidase and nitric oxide synthase, suggesting that NAD(P)H oxidase may be the main source of O2(*)(-) in QUIN-treated rats. Moreover, the administration of MK-801 to rats as a pretreatment resulted in a complete prevention of the QUIN-induced NAD(P)H activation, suggesting that this toxic event is completely dependent on N-methyl-D-aspartate receptor overactivation. Our results also suggest that NAD(P)H oxidase is involved in the pathogenic events linked to excitotoxic/prooxidant conditions.

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Rapid assessment of internodal myelin integrity in central nervous system tissue.

Publication Date: 2010 Mar PMID: 19795370
Authors: Kirschner, D. A. - Avila, R. L. - Gamez Sazo, R. E. - Luoma, A. - Enzmann, G. U. - Agrawal, D. - Inouye, H. - Bunge, M. B. - Kocsis, J. - Peters, A. - Whittemore, S. R.
Journal: J Neurosci Res

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

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3-nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions.

Publication Date: 2010 Feb 15 PMID: 19795369
Authors: Mirandola, S. R. - Melo, D. R. - Saito, A. - Castilho, R. F.
Journal: J Neurosci Res

The adult rat striatum is particularly vulnerable to systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid (3NP), which is known to induce degeneration of the caudate-putamen, as occurs in Huntington's disease. The aim of the present study was to compare the susceptibility of isolated mitochondria from different rat brain regions (striatum, cortex, and cerebellum) as well as from the liver, kidney, and heart to mitochondrial permeability transition (MPT) induced by 3NP and Ca(2+). In the presence of micromolar Ca(2+) concentrations, 3NP induces MPT in a dose-dependent manner, as estimated by mitochondrial swelling and a decrease in the transmembrane electrical potential. A 3NP concentration capable of promoting a 10% inhibition of ADP-stimulated, succinate-supported respiration was sufficient to stimulate Ca(2+)-induced MPT. Brain and heart mitochondria were generally more sensitive to 3NP and Ca(2+)-induced MPT than mitochondria from liver and kidney. In addition, a partial inhibition of mitochondrial respiration by 3NP resulted in more pronounced MPT in striatal mitochondria than in cortical or cerebellar organelles. A similar inhibition of succinate dehydrogenase activity was observed in rat tissue homogenates obtained from various brain regions as well as from liver, kidney, and heart 24 hr after a single i.p. 3NP dose. Mitochondria isolated from forebrains of 3NP-treated rats were also more susceptible to Ca(2+)-induced MPT than those of control rats. We propose that the increased susceptibility of the striatum to 3NP-induced neurodegeneration may be partially explained by its susceptibility to MPT, together with the greater vulnerability of this brain region to glutamate receptor-mediated Ca(2+) influx.

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Decreased levels of PSD95 and two associated proteins and increased levels of BCl2 and caspase 3 in hippocampus from subjects with amnestic mild cognitive impairment: Insights into their potential roles for loss of synapses and memory, accumulation of Abeta, and neurodegeneration in a prodromal stage of Alzheimer's disease.

Publication Date: 2010 Feb 15 PMID: 19774677
Authors: Sultana, R. - Banks, W. A. - Butterfield, D. A.
Journal: J Neurosci Res

Alzheimer's disease (AD) is the most common form of dementia and is pathologically characterized by senile plaques, neurofibrillary tangles, synaptic disruption and loss, and progressive neuronal deficits. The exact mechanism(s) of AD pathogenesis largely remain unknown. With advances in technology diagnosis of a pre-AD stage referred to as amnestic mild cognitive impairment (MCI) has become possible. Amnestic MCI is characterized clinically by memory deficit, but normal activities of daily living and no dementia. In the present study, compared to controls, we observed in hippocampus from subjects with MCI a significantly decreased level of PSD95, a key synaptic protein, and also decreased levels of two proteins associated with PSD95, the N-methyl-D-aspartate receptor, subunit 2A (NR2A) and the low-density lipoprotein receptor-1 (LRP1). PSD95 and NR2A are involved in long-term potentiation, a key component of memory formation, and LRP1 is involved in efflux of amyloid beta-peptide (1-42). Abeta (1-42) conceivably is critical to the pathogenesis of MCI and AD, including the oxidative stress under which brain in both conditions exist. The data obtained from the current study suggest a possible involvement of these proteins in synaptic alterations, apoptosis and consequent decrements in learning and memory associated with the progression of MCI to AD.

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Decreased level and defective function of circulating endothelial progenitor cells in children with moyamoya disease.

Publication Date: 2010 Feb 15 PMID: 19774676
Authors: Kim, J. H. - Jung, J. H. - Phi, J. H. - Kang, H. S. - Kim, J. E. - Chae, J. H. - Kim, S. J. - Kim, Y. H. - Kim, Y. Y. - Cho, B. K. - Wang, K. C. - Kim, S. K.
Journal: J Neurosci Res

Circulating endothelial progenitor cells (EPCs) play an important role in physiological and pathological neovascularization and may be involved in attenuating ischemic diseases. This study aimed to characterize circulating EPCs in moyamoya disease (MMD), one of the most common pediatric cerebrovascular diseases. Twenty-eight children with MMD prior to any surgical treatment and 12 healthy volunteers were recruited. Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured in endothelial cell growth medium. Temporal change of phenotype of cells was analyzed on days 0 and 7. The formation of EPC clusters was evaluated on day 7. The CD34(+), CD133(+), and KDR(+) cells, and the number of EPC clusters was significantly reduced in children with MMD. In controls, CD34(+) cells were significantly decreased on day 7 compared with day 0, but in MMD they were only slightly decreased. The change in KDR(+) cells on day 7 compared with day 0 was the reverse of that for CD34(+) cells. Functional assay of EPC demonstrated less tube formation and increased senescent-like phenotype in children with MMD. Analysis of the circulating EPCs of MMD children reveals decreased level and defective function. This study suggests that circulating EPCs may be associated with MMD pathogenesis.

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Overexpression of aldehyde dehydrogenase 1A1 reduces oxidation-induced toxicity in SH-SY5Y neuroblastoma cells.

Publication Date: 2010 Feb 15 PMID: 19774675
Authors: Zhang, M. - Shoeb, M. - Goswamy, J. - Liu, P. - Xiao, T. L. - Hogan, D. - Campbell, G. A. - Ansari, N. H.
Journal: J Neurosci Res

Oxidative stress leading to lipid peroxidation is a problem in neurodegenerative diseases, because the brain is rich in polyunsaturated fatty acids and low in endogenous antioxidants. One of the most toxic byproducts of lipid peroxidation, 4-hydroxynonenal (HNE), is implicated in oxidative stress-induced damage in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). In this study, the human neuroblastoma cell line SH-SY5Y was used to test the protective effects of increasing the detoxification of HNE by overexpressing the HNE-detoxifying enzyme aldehyde dehydrogenase 1A1 (ALDH1). Overexpression of ALDH1 in the SH-SY5Y cells acts to reduce production of protein-HNE adducts and activation of caspase-3. Our data suggest that detoxification of HNE could be therapeutic in preventing some of the toxic disruptions of the brain's redox systems found in many neurodegenerative diseases.

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Protective effects of peroxisome proliferator-activated receptors gamma coactivator-1alpha against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia.

Publication Date: 2010 Feb 15 PMID: 19774674
Authors: Chen, S. D. - Lin, T. K. - Yang, D. I. - Lee, S. Y. - Shaw, F. Z. - Liou, C. W. - Chuang, Y. C.
Journal: J Neurosci Res

Peroxisome proliferator-activated receptors gamma coactivator-1alpha (PGC-1alpha) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC-1alpha in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC-1alpha signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague-Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC-1alpha expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4-48 hr after TGI. In addition, knock-down of PGC-1alpha expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC-1alpha is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress.

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Different antipsychotics elicit different effects on magnocellular oxytocinergic and vasopressinergic neurons as revealed by Fos immunohistochemistry.

Publication Date: 2010 Feb 15 PMID: 19774673
Authors: Kiss, A. - Bundzikova, J. - Pirnik, Z. - Mikkelsen, J. D.
Journal: J Neurosci Res

Acute administration of antipsychotics elicits regionally distinct patterns of Fos expression in the rat brain. Stimulation of oxytocin (OXY) and vasopressin (AVP) release in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei indicates that antipsychotics may play a role in autonomic, neuroendocrine, and behavioral processes. This study was focused to reveal the responsiveness of hypothalamic OXY- and AVP- producing magnocellular neurons, in terms of quantitative and topographical distinctions, to antipsychotics displaying different pharmacological profiles. Naive male Wistar rats were injected intraperitoneally with haloperidol (1 mg/kg), clozapine (30 mg/kg), olanzapine (30 mg/kg), risperidone (2mg/kg), and vehicle (5% chremophor) and were sacrificed 60 min later by a fixative. Fos, Fos/OXY, and Fos/AVP labelings were visualized by immunohistochemistry in the SON, 5 accessory (ACS) cell groups, and 4 distinct PVN subdivisions using a computerized light microscope. Most apparent activation of single Fos, Fos/OXY, and Fos/AVP cells was induced by clozapine and olanzapine; effects of risperidone and haloperidol were substantially lower; no colocalizations were revealed in naive or vehicle treated control rats. The data indicate the existence of a substantial diversity in the stimulatory effect of the selected antipsychotics on quantity of Fos, Fos/OXY, and Fos/AVP immunostainings with the preferential action of the atypicals clozapine over olanzapine and little effects of risperidone and haloperidol. Variabilities in Fos distribution in the PVN, SON, and ACS induced by antipsychotics may be helpful to understand more precisely the extent of their extra-forebrain actions with possible presumption of their functional impact and side effect consequences.

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Achyranthes bidentata polypeptides confer neuroprotection through inhibition of reactive oxygen species production, Bax expression, and mitochondrial dysfunction induced by overstimulation of N-methyl-D-aspartate receptors.

Publication Date: 2010 Feb 15 PMID: 19774671
Authors: Shen, H. - Yuan, Y. - Ding, F. - Hu, N. - Liu, J. - Gu, X.
Journal: J Neurosci Res

Achyranthes bidentata polypeptides (ABPP), the important constituents separated from the aqueous extract of Achyranthes bidentata, have been shown to attenuate N-methyl-D-aspartate (NMDA)-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors. The present study sought to investigate the possible mechanism underlying the neuroprotective effect of ABPP on NMDA-induced cell death. Western blot analysis and colorimetric enzymatic assay demonstrated that ABPP pretreatment inhibited NMDA-induced increase of Bax protein expression or caspase-3 activity in cultured hippocampal neurons. Fluorescence measurements after staining with 2,7-dichlorofluorescin diacetate and rhodamine 123 showed that ABPP treatment also reversed NMDA-induced intracellular radical oxygen species (ROS) elevation and mitochondrial membrane potential depression in cultured hippocampal neurons. Furthermore, the in vivo effects of ABPP on cerebral neuronal damage during focal ischemia-reperfusion were also investigated. In rat middle cerebral artery occlusion (MCAO) model, ABPP attenuated the increase in the neurological deficit and cerebral infarction induced by focal ischemia-reperfusion, showing in vivo neuroprotective effects. The results collectively suggest that ABPP might exert neuroprotective actions through inhibiting Bax protein expression, caspase-3 activity, ROS production, and mitochondrial dysfunction that are all caused by overstimulation of NMDA receptors.

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p38 mitogen-activated protein kinase and calcium channels mediate signaling in depolarization-induced activation of peroxisome proliferator-activated receptor gamma coactivator-1alpha in neurons.

Publication Date: 2010 Feb 15 PMID: 19774670
Authors: Liang, H. L. - Dhar, S. S. - Wong-Riley, M. T.
Journal: J Neurosci Res

Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) coactivates a number of transcription factors critical for mitochondrial biogenesis. Previously, we found that the expression of PGC-1alpha is governed by neuronal activity, but the signaling mechanism is poorly understood. The present study aimed at testing our hypothesis that depolarizing activation of PGC-1alpha in neurons is mediated by p38 mitogen-activated protein kinase (MAPK) and calcium channels. Cultured primary neurons and N2a cells were depolarized with 20 mM KCl for varying times, and increases in PGC-1alpha mRNA and protein levels were found after 0.5 and 1 hr of stimulation, respectively. These levels returned to those of controls after the withdrawal of KCl. Significantly, 15 min of KCl stimulation induced an up-regulation of both p38 MAPK and phosphorylated p38 MAPK that were suppressed by 30 min of pretreatment with SB203580, a blocker of p38 MAPK that also blocked the up-regulation of PGC-1alpha by KCl. Likewise, 30 min of pretreatment with nifedipine, a calcium channel blocker, also prevented the up-regulation of PGC-1alpha mRNA and proteins by KCl. Furthermore, a knockdown of p38 MAPK with small interference hairpin RNA significantly suppressed PGC-1alpha mRNA and protein levels. Our results indicate that both p38 MAPK and calcium play important roles in mediating signaling in depolarization-induced activation of PGC-1alpha at the protein and message levels in neurons.

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Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

Publication Date: 2010 Feb 15 PMID: 19774669
Authors: Xu, J. - Yu, L. - Zhang, J. - Cai, R. - Sun, X.
Journal: J Neurosci Res

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing.

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Matrigel supports survival and neuronal differentiation of grafted embryonic stem cell-derived neural precursor cells.

Publication Date: 2010 Feb 15 PMID: 19774667
Authors: Uemura, M. - Refaat, M. M. - Shinoyama, M. - Hayashi, H. - Hashimoto, N. - Takahashi, J.
Journal: J Neurosci Res

Cell replacement therapy holds great promise as a means of treating neurological disorders, including Parkinson's disease. However, one of the major obstacles to the success of this treatment is the low survival rate of grafted cells, which probably results from mechanical damage, acute inflammation, and immunological rejection. To overcome this problem, we investigated the effect of different types of extracellular matrix (ECM) on the survival and differentiation of embryonic stem (ES) cell-derived neural precursor cells (NPCs). We tested materials from natural sources, including collagen, ornithine/laminin, and growth factor-reduced Matrigel (gfrMG), as well as the synthetic biomaterial PuraMatrix, which consists of self-assembling polypeptides. GfrMG efficiently supported cell survival, migration, and neurite outgrowth in vitro and promoted proliferation of grafted cells in vivo, resulting in larger graft volume and an increase in the number of TH-positive dopaminergic neurons in the graft. GfrMG did not induce dopaminergic differentiation directly; rather, it reduced the invasion of pan-leukocytic CD45-positive cells into the graft. Insofar as the inflammatory or immune response in the host brain inhibits neuronal differentiation of grafted NPCs, gfrMG may increase the number of TH-positive cells by suppressing this effect. Thus, gfrMG appears to provide a suitable scaffold that supports survival and differentiation of NPCs. However, because it is derived from mouse sarcomas, a human-derived matrix or synthetic biomaterial must be developed for clinical applications.

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Roles of astrocytes and microglia in seizure-induced aberrant neurogenesis in the hippocampus of adult rats.

Publication Date: 2010 Feb 15 PMID: 19774666
Authors: Yang, F. - Liu, Z. R. - Chen, J. - Zhang, S. J. - Quan, Q. Y. - Huang, Y. G. - Jiang, W.
Journal: J Neurosci Res

Recent evidence showed that epileptic seizures increase hippocampal neurogenesis in the adult rat, but prolonged seizures result in the aberrant hippocampal neurogenesis that often leads to a recurrent excitatory circuitry and thus contributes to epileptogenesis. However, the mechanism underlying the aberrant neurogenesis after prolonged seizures remains largely unclear. In this study, we examined the role of activated astrocytes and microglia in the aberrant hippocampal neurogenesis induced by status epilepticus. Using a lithium-pilocarpine model to mimic human temporal lobe epilepsy, we found that status epilepticus induced a prominent activation of astrocytes and microglia in the dentate gyrus 3, 7, 14, and 20 days after the initial seizures. Then, we injected fluorocitrate stereotaxicly into the dentate hilus to inhibit astrocytic metabolism and found that fluorocitrate failed to prevent the seizure-induced formation of ectopic hilar basal dendrites but instead promoted the degeneration of dentate granule cells after seizures. In contrast, a selective inhibitor of microglia activation, minocycline, inhibited the aberrant migration of newborn neurons at 14 days after status epilepticus. Furthermore, with stereotaxic injection of lipopolysaccharide into the intact dentate hilus to activate local microglia, we found that lipopolysaccharide promoted the development of ectopic hilar basal dendrites in the hippocampus. These results indicate that the activated microglia in the epileptic hilus may guide the aberrant migration of newborn neurons and that minocycline could be a potential drug to impede seizure-induced aberrant migration of newborn neurons.

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Notch signaling: key role in intrauterine infection/inflammation, embryonic development, and white matter damage?

Publication Date: 2010 Feb 15 PMID: 19768798
Authors: Yuan, T. M. - Yu, H. M.
Journal: J Neurosci Res

The mechanisms or pathophysiologies that lead to cerebral white matter damage during development are complex and not fully understood. It is postulated that exposure of the preterm brain to inflammatory cytokines during intrauterine infection/inflammation contributes to brain white matter damage, and this damage may affect the function and differentiation of progenitor oligodendrocyte cells under physiological conditions. The Notch pathway, an important signaling pathway controlling various cells' differentiation, functions in the timing of oligodendrocyte differentiation, and Notch signaling may contribute to white matter damage and may mediate neurogenesis in a pathophysiological phase. Recent studies have led to recognition of the role of the Notch pathway in neurogenesis in cerebral ischemic damage and in myelination and axonal damage of neurodegenerative diseases. Moreover, Notch plays a critical role in steering an immune response toward inflammation by regulating expression of various cytokines and proinflammatory cytokines resulting in the activation of Notch signaling. Thus, the Notch signaling pathway likely plays a key role in intrauterine infection/inflammation, brain development, and white matter damage, and future research directed toward understanding its role will be important. Insofar as Notch signaling could have an important effect on neurogenesis, mobilization of progenitor cells is one strategy for compensating for the neuronal losses seen in white matter damage after intrauterine infection/inflammation.

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Altered AMPA receptor expression with treadmill exercise in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse model of basal ganglia injury.

Publication Date: 2010 Feb 15 PMID: 19746427
Authors: VanLeeuwen, J. E. - Petzinger, G. M. - Walsh, J. P. - Akopian, G. K. - Vuckovic, M. - Jakowec, M. W.
Journal: J Neurosci Res

Dopamine depletion leads to impaired motor performance and increased glutamatergic-mediated hyperexcitability of medium spiny neurons in the basal ganglia. Intensive treadmill exercise improves motor performance in both saline treatment and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. In the present study, we investigated the effect of high-intensity treadmill exercise on changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit expression, because these receptor channels confer the majority of fast excitatory neurotransmission in the brain, and their subunit composition provides a key mechanism for regulating synaptic strength and synaptic neuroplasticity and is important in modulating glutamatergic neurotransmission. Within the dorsolateral striatum of MPTP mice, treadmill exercise increased GluR2 subunit expression, with no significant effect on GluR1. Furthermore, neurophysiological studies demonstrated a reduction in the size of excitatory postsynaptic currents (EPSCs) in striatal medium spiny neurons (as determined by the input-output relationship), reduced amplitude of spontaneous EPSCs, and a loss of polyamine-sensitive inward rectification, all supportive of an increase in heteromeric AMPAR channels containing the GluR2 subunit. Phosphorylation of GluR2 at serine 880 in both saline-treated and MPTP mice suggests that exercise may also influence AMPAR trafficking and thus synaptic strength within the striatum. Finally, treadmill exercise also altered flip isoforms of GluR2 and GluR1 mRNA transcripts. These findings suggest a role for AMPARs in mediating the beneficial effects of exercise and support the idea that adaptive changes in GluR2 subunit expression may be important in modulating experience-dependent neuroplasticity of the injured basal ganglia.

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Uptake of ferrous iron by cultured rat astrocytes.

Publication Date: 2010 Feb 15 PMID: 19746426
Authors: Tulpule, K. - Robinson, S. R. - Bishop, G. M. - Dringen, R.
Journal: J Neurosci Res

Astrocytes are considered to play an important role in iron homeostasis of the brain, yet the mechanisms involved in the uptake of iron into astrocytes remain elusive. To investigate the uptake of iron into astrocytes, we have applied ferric ammonium citrate (FAC) to rat astrocyte-rich primary cultures. These cultures express the mRNAs of two membrane-bound ferric reductases, Dcytb and SDR2, and reduce extracellular ferric iron (100 muM) with a rate of 3.2 +/- 0.4 nmol/(hr x mg). This reduction rate is substantially lower than the rate of cellular iron accumulation from 100 muM FAC [24.7 +/- 8.9 nmol/(hr x mg)], which suggests that iron accumulation from FAC does at best partially depend on extracellular ferric reduction. Nonetheless, when the iron in FAC was almost completely reduced by an excess of exogenous ascorbate, astrocytes accumulated iron in a time- and concentration-dependent manner with specific iron accumulation rates that increased linearly for concentrations of up to 100 muM ferrous iron. This accumulation was attenuated by lowering the incubation temperature, by the presence of ferrous iron chelators, or by lowering the pH from 7.4 to 6.8. These data indicate that, in addition to the DMT1-mediated uptake of ferrous iron, astrocytes can accumulate ferric and ferrous iron by mechanisms that are independent of DMT1 or transferrin.

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Effects of the flavonoid casticin from Brazilian Croton betulaster in cerebral cortical progenitors in vitro: direct and indirect action through astrocytes.

Publication Date: 2010 Feb 15 PMID: 19746423
Authors: de Sampaio e Spohr, T. C. - Stipursky, J. - Sasaki, A. C. - Barbosa, P. R. - Martins, V. - Benjamim, C. F. - Roque, N. F. - Costa, S. L. - Gomes, F. C.
Journal: J Neurosci Res

Neurodegenerative diseases are a major constraint on the social and economic development of many countries. Evidence has suggested that phytochemicals have an impact on brain pathology; however, both their mechanisms of action and their cell targets are incompletely known. Here, we investigated the effects of the flavonoid casticin, extracted from Croton betulaster, a common plant in the state of Bahia in Brazil, on rat cerebral cortex neurons in vitro. Treatment of neural progenitors with 10 microM casticin increased the neuronal population positive for the neuronal marker beta-tubulin III and the neuronal transcriptional factor Tbr2 by approximately 20%. This event was followed by a 50% decrease in neuronal death. Pools of astrocyte (GFAP and S100beta), neural (nestin), and oligodendrocyte (Olig2 and NG2) progenitors were not affected by casticin. Neither neuronal commitment nor proliferation of progenitors was affected by casticin, suggesting a neuroprotective effect of this compound. Culture of neural progenitors on casticin-treated astrocyte monolayers increased the neuronal population by 40%. This effect was reproduced by conditioned medium derived from casticin-treated astrocytes, suggesting the involvement of a soluble factor. ELISA assays of the conditioned medium revealed a 20% increase in interleukin-6 level in response to casticin. In contrast to the direct effect, neuronal death was unaffected, but a 52% decrease in the death of nestin-positive progenitors was observed. Together our data suggest that casticin influences the neuronal population by two mechanisms: 1) directly, by decreasing neuronal death, and 2) indirectly, via astrocytes, by modulating the pool of neuronal progenitors.

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Features of sequential learning in hemicerebellectomized rats.

Publication Date: 2010 Feb 15 PMID: 19746422
Authors: Mandolesi, L. - Foti, F. - Cutuli, D. - Laricchiuta, D. - Gelfo, F. - De Bartolo, P. - Petrosini, L.
Journal: J Neurosci Res

Because the sequencing property is one of the functions in which cerebellar circuits are involved, it is important to analyze the features of sequential learning in the presence of cerebellar damage. Hemicerebellectomized and control rats were tested in a four-choice visuomotor learning task that required both the detection of a specific sequence of correct choices and the acquisition of procedural rules about how to perform the task. The findings indicate that the presence of the hemicerebellectomy did not affect the first phases of detection and acquisition of the sequential visuomotor task, delayed but did not prevent the learning of the sequential task, slowed down speed-up and proceduralization phases, and loosened the reward-response associative structure. The performances of hemicerebellectomized animals in the serial learning task as well as in the open field task demonstrated that the delayed sequential learning task could not be ascribed to impairment of motor functions or discriminative abilities or to low levels of motivation. The delay in sequential learning observed in the presence of a cerebellar lesion appeared to be related mainly to a delay of the automatization of the response. In conclusion, it may be advanced that, through cortical and subcortical connections, the cerebellum provides the acquisition of rapid and accurate sensory-guided sequence of responses.

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Microglia of rat ventral midbrain recovers its resting state over time in vitro: let microglia rest before work.

Publication Date: 2010 Feb 15 PMID: 19739250
Authors: Cristovao, A. C. - Saavedra, A. - Fonseca, C. P. - Campos, F. - Duarte, E. P. - Baltazar, G.
Journal: J Neurosci Res

Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinson's disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68(+) cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 microg/ml) increased the total cell number, the percentage of CD68(+) cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 microg/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent.

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