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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The Wiskott–Aldrich syndrome protein family Verprolin-homologous protein (WAVE) complex has been proposed to link Rho GTPase activity with actin polymerization but its role in neuronal plasticity has never been documented. We now examined the presence, distribution and dynamics of WAVE3 in cultured hippocampal neurons. WAVE3 was localized to dendritic spines via its N-terminal domain. Green fluorescent protein (GFP)-tagged WAVE3 clusters demonstrate an F-actin-dependent high rate of local motility. Constitutive Rac activation translocates WAVE3 (via the N-terminus), to the leading edge of lamellipodia. Also, spinogenesis is associated with actin-based motility of the WAVE3 protein. Brain specific WAVE1 showed similar localization and effects on spine density. Cytoplasmic fragile X mental retardation protein interacting protein (CYFIP) and non-catalytic region of tyrosine kinase adaptor protein 1 (NCK-1), proteins that are assumed to complex with WAVE, have a somewhat similar cellular distribution and motility. We propose that the WAVE complex is a downstream effector of the Rac signaling cascade, localized to sites of novel synaptic contacts by means of its N-terminal domain, to guide local actin polymerization needed for morphological plasticity of neurons.
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: While a β-sheet-rich form of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an α-helical-rich conformation similar to that of PrPC, resulted in a 1.9-fold increase in neurons with a differentiated axon, a 13.5-fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic-like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N-terminal nor C-terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3-kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP-mediated signals. The results predict that full-length PrPC functions as a growth factor involved in development of neuronal polarity.
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The ‘cross-talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP-dependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ɛ, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca2+-independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p 〈 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 95 (2005), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia–reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O2 can be lowered to a concentration of 1 µm and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co-cultured neurones that had been subjected to a 15-min ischaemic insult with either non-insulted astrocytes or astrocyte-conditioned medium during the reperfusion stage. Both astrocytes and astrocyte-conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte-sourced neuronal glutathione synthesis may play a role in preventing neuronal death.
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The search for effective treatments that prevent oxidative stress associated with premature ageing and neurodegenerative diseases is an important area of neurochemical research. As age- and disease-related oxidative stress is frequently associated with mitochondrial dysfunction, amphiphilic antioxidant agents of high stability and selectivity that target these organelles can provide on-site protection. Such an amphiphilic nitrone protected human neuroblastoma cells at low micromolar concentrations against oxidative damage and death induced by exposure to the β-amyloid peptide, hydrogen peroxide and 3-hydroxykynurenine. Daily administration of the antioxidant at a concentration of only 5 μm significantly increased the lifespan of the individually cultured rotifer Philodina acuticornis odiosa Milne. This compound is unique in its exceptional anti-ageing efficacy, being one order of magnitude more potent than any other compound previously tested on rotifers. The nitrone protected these aquatic animals against the lethal toxicity of hydrogen peroxide and doxorubicin and greatly enhanced their survival when co-administered with these oxidotoxins. These findings indicate that amphiphilic antioxidants have a great potential as neuroprotective agents in preventing the death of cells and organisms exposed to enhanced oxidative stress and damage.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.
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  • 9
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT–PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABAA-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Half of congenital muscular dystrophy cases arise from laminin α2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean ± SD 1.42 ± 0.28 µmol acetylthiocholine/h/mg protein, U/mg) was decreased by ∼50% in dystrophic thymus (0.77 ± 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT–PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (〈inlineGraphic alt="inline image" href="urn:x-wiley:00223042:JNC3433:JNC_3433_mu1" location="equation/JNC_3433_mu1.gif"/〉, 64%) and monomers (〈inlineGraphic alt="inline image" href="urn:x-wiley:00223042:JNC3433:JNC_3433_mu2" location="equation/JNC_3433_mu2.gif"/〉, 19%), as well as hydrophilic tetramers (〈inlineGraphic alt="inline image" href="urn:x-wiley:00223042:JNC3433:JNC_3433_mu3" location="equation/JNC_3433_mu3.gif"/〉, 9%) and monomers (〈inlineGraphic alt="inline image" href="urn:x-wiley:00223042:JNC3433:JNC_3433_mu4" location="equation/JNC_3433_mu4.gif"/〉, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.
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  • 11
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPα, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPα as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the α-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.
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  • 12
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Alzheimer's disease is associated with genetic risk factors, of which the allele E4 of apolipoprotein E (apoE4) is the most prevalent, and it is also affected by environmental factors such as early life education. We have recently shown, utilizing apoE-deficient and apoE transgenic mice, that synaptogenesis in the hippocampus following environmental stimulation is affected by apoE. In view of the pivotal role of cholesterol in synaptic plasticity, and of its suggested role in synaptogenesis, we presently examined the effects of apoE and environmental stimulation on brain cholesterol homeostasis. The hippocampal levels of cholesterol and its precursors and metabolites in control mice were not affected by exposure to environmental stimulation. In contrast, the hippocampal levels of cholesterol and its precursors lathosterol and desmosterol and metabolite 24S-hydroxycholesterol were lower in apoE-deficient mice that were maintained in a regular environmental than those of corresponding control mice, whereas they were markedly elevated following environmental stimulation. Histological and immunohistochemical experiments revealed that the combined stimulatory effects of apoE deficiency and environmental stimulation on cholesterol metabolism were associated with marked activation of hippocampal astrocytes and with the abnormal accumulation of cholesterol in neurons and astrocytes. These effects were rescued similarly in apoE3 and apoE4 transgenic mice. These findings suggest that apoE plays an important role in the translocation of cholesterol from astrocytes to neurons in vivo and in the regulation and homeostasis of this process.
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  • 13
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
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  • 14
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 ± 8.5%; lumbar cord = 62 ± 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 ± 3.9%; lumbar cord = 27 ±4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.
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  • 15
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Reduced activity of the mitochondrial respiratory chain – particularly complex I – may be implicated in the etiology of both Parkinson's disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral α-synuclein or tau protein pathology. To determine experimentally whether chronic generalized complex I inhibition has an effect on the distribution of α-synuclein or tau, we infused rats systemically with the plant-derived isoflavonoid rotenone. Rotenone-treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of α-synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau-immunoreactive material consisted of straight 15-nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavin S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspase 3. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of α-synuclein.
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  • 16
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-α release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.
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  • 17
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 95 (2005), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The hexadecapeptide cerebellin is present in the brains of many vertebrate species and is derived from a larger protein, Cbln1 (cerebellin 1 precursor protein). Although cerebellin has features of a neuropeptide, Cbln1 belongs to the C1q/tumor necrosis factor superfamily of secreted proteins, suggesting that it is the biologically active molecule and the proteolytic events that generate cerebellin serve another function. Therefore, we assessed whether Cbln1 undergoes proteolytic processing and determined what consequences the cleavage events necessary to produce cerebellin have on the structure of Cbln1. Substantial degradation of Cbln1 was evident in the synaptic compartment of cerebellum and lysates of cultured cerebellar neurons and cells transfected with Cbln1 expression vectors. However, only uncleaved Cbln1 containing the cerebellin motif was released and assembled into hexameric complexes. Using yeast two hybrid and mammalian expression systems we show that the cleavages required to produce cerebellin influence the subunit stoichiometry of Cbln1 complexes. Cleavage at the N-terminus of the cerebellin sequence in Cbln1 yields trimeric complexes by separating the trimer-mediating C-terminal C1q domain from conserved N-terminal cysteine residues that mediate higher order oligomerization. Cleavage at the C-terminus of the cerebellin motif disrupts the C1q domain and abolishes subunit interactions. Functional implications of these data are discussed.
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  • 18
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The pattern of catecholaminergic innervation of the dentate gyrus (DG) of the hippocampus, particularly the relatively dense and selective noradrenergic input, creates favourable conditions for real-time monitoring of noradrenaline (NA) release following stimulation of the locus coeruleus (LC) by in vivo voltammetry. Two electrochemically active species with different temporal characteristics were registered in the DG following electrical stimulation of the LC. Several approaches, including testing of anatomical and pharmacological specificity, coating of microelectrodes with Nafion and use of fast cyclic voltammetry, were used to verify the characteristics of electrochemical responses. The first sharp peak that appeared immediately during stimulation was definitely associated with NA overflow. The second late peak was possibly attributable to ascorbic acid. We examined the characteristics of α-2 adrenoceptor regulation of NA release in the DG, and showed for the first time that noradrenergic terminals resemble dopaminergic terminals in their mechanisms of increasing the refilling rate of the readily releasable pool following stimulation repeated at short intervals. Amperometric registration of NA in the DG was complicated by interference with electrical activity of hippocampus. This interference could be used, after appropriate filtration, for simultaneous recording from the same microelectrode of NA release and electrical activity of the hippocampus.
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  • 19
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABAA receptor labelling in the hippocampal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extracellular levels were studied in control and lesioned rats. In vivo effects of 100 mm KCl perfusion and adenosine A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABAA receptors and decreased glutamate neurotransmission. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.
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  • 20
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a low-density, sphingolipid- and cholesterol-enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immunoseparation procedure. Under conditions designed to preserve lipid-mediated membrane organization, several proteins were found in the prion protein-enriched membrane domains (i.e. the non-receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The prion protein-rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid-enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid-mediated non-covalent interactions.
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  • 21
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The later phases of neuronal plasticity are invariably dependent on gene transcription. Induction of the transcription factor Zif268 (Egr-1) in neurones is closely associated with many forms of functional plasticity, yet the neuronal target genes modulated by Zif268 have not been characterized. After transfection of a neuronal cell line with Zif268 we identified genes that show altered expression using high density microarrays. Although some of the genes identified have previously been associated with forms of neuronal plasticity, the majority have not been linked with neuronal plasticity or Zif268 action. Altered expression of a representative sample of the novel target genes was confirmed in Zif268-transfected PC12 neurones, and in in vitro and in vivo models of Zif268-associated neuronal plasticity. In particular, altered expression of the protease inhibitor Cystatin C and the chemokine Cxcl10 was observed in striatal tissue after haloperidol administration. Surprisingly, the group of identified genes is enriched for components of the proteasome and the major histocompatibility complex. Our findings suggest that altered expression of these genes following Zif268 induction may be a key component of long lasting plasticity in the CNS.
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  • 22
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca2+-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations 〈 2 µm produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca2+ chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca2+ and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca2+. Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca2+, and is involved in the dopamine release in concert with Ca2+.
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  • 23
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Galanin is a neuropeptide involved in a variety of biological functions, including having a strong anticonvulsant activity. To assess a possible role of galanin in modulation of glutamatergic synapses and excitotoxicity, we studied effects of a galanin receptor 2(3) agonist (AR-M1896) on several molecular events induced by glutamate administration in primary neural hippocampal cells. Exposure of cells, after 5 days in vitro, to glutamate 0.5 mm for 10 min caused morphological alterations, including disaggregation of β-tubulin and MAP-2 cytoskeletal protein assembly, loss of neurites and cell shrinkage. When present in culture medium together with glutamate, 1 and 10 nm of AR-M1896 reduced these alterations. Moreover, AR-M1896 counteracted glutamate-induced c-fos mRNA and c-Fos protein up-regulation after 30–150 min, and 24 h, respectively. Massive nuclear alterations (Hoechst 33258 staining), observed 24 h after glutamate exposure, were also antagonized by AR-M1896 (0.1–100 nm) in a dose-dependent manner. These findings indicate that galanin, probably mainly through its type 2 receptor, interferes with events associated with glutamate toxicity.
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  • 24
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Ethanol is a potent teratogenic agent that disrupts several aspects of neuronogenesis, including the proliferation rate of cortical precursors. With regard to corticogenesis, possible targets of ethanol toxicity include soluble factors, like transforming growth factor β1 (TGFβ1), that regulate cortical growth and cell cycle proteins that control the kinetics of the cell cycle. The effect of ethanol on normal cell proliferation and TGFβ1-regulated cell proliferation in the developing cortex was assessed using an organotypic slice culture model. Ethanol elongated the cell cycle, possibly through a decrease in the expression of G1 cell cycle protein cyclin D1. Further, ethanol exposure antagonized the anti-proliferative action of TGFβ1 and blocked TGFβ1-dependent increases in cell cycle inhibitor p21. Collectively, this evidence suggests that disruption of appropriate cell cycle protein expression and inhibition of TGFβ1 activity are potential mechanisms underlying the effect of ethanol on cortical development.
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  • 25
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Interleukin-12 (IL-12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1-type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL-23, composed of the same p40 subunit as IL-12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen-presenting cell (APC) function can present antigen to myelin basic protein (MBP)-reactive T cells, and whether this presentation is blocked with antibodies against IL-12/IL-23p40. Interferon (IFN)-γ-treated APC induced proliferation of MBP-reactive T cells. Anti-IL-12/IL-23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL-12/IL-23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL-12p70. Because IL-12 and IL-23 share p40, we wanted to determine whether astrocytes also express IL-12p35 and IL-23p19, as microglia were already shown to express them. Astrocytes expressed IL-12p35 mRNA constitutively, and IL-23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL-12/IL-23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti-IL-12/IL-23p40 antibodies.
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  • 26
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The ability of cells to control the balance between the generation and quenching of reactive oxygen species is important in combating potentially damaging effects of oxidative stress. One mechanism that cells use to maintain redox homeostasis is the antioxidant response pathway. Antioxidant response elements (AREs) are cis-acting elements located in regulatory regions of antioxidant and phase II detoxification genes. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a member of the Cap ‘n’ Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes such as NAD(P)H:quinone oxidoreductase 1, glutamylcysteine synthetase and heme oxygenase. Activation of Nrf2 results in release from its negative repressor, Kelch-like ECH-associated protein 1 (Keap1), and allows Nrf2 to translocate into the nucleus to induce gene expression. In this study, we demonstrate that increasing Nrf2 activity by various methods, including chemical induction, Nrf2 overexpression or Keap1 siRNA knockdown, protects cells against specific types of oxidative damage. Cells were protected against 6-hydroxydopamine- and 3-morpholinosydnonimine-mediated toxicity but not against 1-methyl-1-4-phenylpyridinium toxicity. As oxidative stress is a hallmark of several neurodegenerative disorders, including Parkinson's disease, pharmacological agents that selectively target the Keap1-Nrf2 pathway may provide a novel neuroprotective strategy for the treatment of these diseases.
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  • 27
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Huntington's disease is a neurodegenerative illness caused by expansion of CAG repeats at the N-terminal end of the protein huntingtin. We examined longitudinal changes in brain metabolite levels using in vivo magnetic resonance spectroscopy in five different mouse models. There was a large (〉50%) exponential decrease in N-acetyl aspartate (NAA) with time in both striatum and cortex in mice with 150 CAG repeats (R6/2 strain). There was a linear decrease restricted to striatum in N171-82Q mice with 82 CAG repeats. Both the exponential and linear decreases of NAA were paralleled in time by decreases in neuronal area measured histologically. Yeast artificial chromosome transgenic mice with 72 CAG repeats, but low expression levels, had less striatal NAA loss than the N171–82Q mice (15% vs. 43%). We evaluated the effect of gene context in mice with an approximate 146 CAG repeat on the hypoxanthine phosphoribosyltransferase gene (HPRT). HPRT mice developed an obese phenotype in contrast to weight loss in the R6/2 and N171–82Q mice. These mice showed a small striatal NAA loss (21%), and a possible increase in brain lipids detectable by magnetic resonance (MR) spectroscopy and decreased brain water T1. Our results indicate profound metabolic defects that are strongly affected by CAG repeat length, as well as gene expression levels and protein context.
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  • 28
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Myelomeningocele (MMC), the most severe form of spina bifida (SB), causes neurological deficit. Injury to the spinal cord is thought to begin in utero. We investigated whether brain-specific proteins (BSPs) would enable us to monitor the development of MMC-related tissue damage during pregnancy in an animal model with naturally occurring SB (curly tail/loop tail mouse; n = 256). Amniotic fluid levels of neurofilament heavy chain (NfH), glial acidic fibrillary protein (GFAP) and S100B were measured by standard ELISA techniques. The amniotic fluid levels of all BSPs were similar in SB and control mice on embryonic day (E) 12.5 and 14.5, whereas a significant increase was observed for GFAP in SB mice on E16.5. Levels of all BSPs were significantly increased in SB mice on E18.5. The rapid increase in GFAP, paralleled by a moderate increase in NfH and S100B, suggests that spinal cord damage starts to accelerate around E16.5. The macroscopic size of the MMC was related to NfH level on E16.5 and E18.5, suggesting that axonal degeneration is most severe in large MMC. Amniotic fluid BSP measurements may provide important information for balancing the risks and benefits to mother and child of in utero surgery for MMC.
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  • 29
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin–proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.
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  • 30
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by a prominent loss of GABA-ergic medium-sized spiny neurons in the caudate putamen. There is evidence that impaired energy metabolism contributes to neuronal death in HD. Creatine is an endogenous substrate for creatine kinases and thereby supports cellular ATP levels. This study investigated the effects of creatine supplementation (5 mm) on cell survival and neuronal differentiation in striatal cultures. Chronic creatine treatment resulted in significant increased densities of GABA-immunoreactive (-ir) neurons, although total neuronal cell number and general viability were not affected. Similar effects were seen after short-term treatment, suggesting that creatine acted as a differentiation factor. Inhibitors of transcription or translation did not abolish the creatine-mediated effects, nor did omission of extracellular calcium, whereas inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly attenuated the creatine induced increase in GABA-ir cell densities. Creatine exhibited significant neuroprotection against toxicity instigated either by glucose- and serum deprivation or addition of 3-nitropropionic acid. In sum, the neuroprotective properties in combination with promotion of neuronal differentiation suggest that creatine has potential as a therapeutic drug in the treatment of neurodegenerative diseases, like HD.
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  • 31
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5′-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras–MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.
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  • 32
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We investigated the expression, activation and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38 MAPKs) and extracellular signal-regulated kinases (ERKs), using western blotting and immunohistochemistry, in the brains of hamsters infected with 263K scrapie agent, to clarify the role of these kinases in the pathogenesis of prion disease. The immunoblot analysis demonstrated that activation of JNK, p38 MAPK and ERK in whole brain homogenates was increased in infected animals. Phosphorylation of cAMP/calcium responsive element binding protein (CREB), a downstream transcription factor of active ERK, was significantly increased in scrapie-infected hamsters. The immunohistochemical study showed that active ERK was enhanced in infected hamsters compared with controls. Active ERK immunoreactivity was observed within neurons in the dentate gyrus and in glial fibrillary acidic protein (GFAP)-positive reactive astrocytes of infected animals. The expression level of c-Jun mRNA as well as protein, a substrate of active JNK, was increased in infected animals. A significant increase in JNK activity upon glutathione S-transferase (GST)-c-Jun was observed in infected compared with control animals. Phospho-c-Jun immunoreactivity was observed only in neurons of the thalamus in infected groups. These findings indicated that the JNK pathway was activated in the scrapie-infected group. The chronological activation of MAPKs using immunoblot analysis indicates that the kinases are sequentially activated during the pathophysiology of prion disease. Taken together, these results lend credence to the notion that MAPK pathways are dysregulated in prion disease, and also indicate an active role for this pathway in disease pathogenesis.
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  • 33
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Stem cell factor (SCF) is a highly expressed cytokine in the central nervous system. In the present study, we demonstrate a neuroprotective role for SCF and its tyrosine kinase receptor, c-kit, against camptothecin-induced apoptosis and glutamate excitotoxicity in rat cortical neurons. This protection was blocked by pharmacological or molecular inhibition of either the MEK/ERK or PI3K/Akt signaling pathways. The importance of these pathways was further confirmed by the activation of both ERK, in a MEK-dependent manner, and Akt, via PI3K. Activation of Akt increased the binding of the p50 and p65 subunits of NFκB, which was also important for neuroprotection. Akt inhibition prevented NFκB binding, suggesting a role for Akt in SCF-induced NFκB. Pharmacological inhibition of NFκB or dominant negative IκB also prevented neuroprotection by SCF. SCF up-regulated the anti-apoptotic genes, bcl-2 and bcl-xL in an NFκB-dependent manner. Together, these findings demonstrate a neuroprotective role for SCF in cortical neurons, an effect that was mediated by Akt and ERK, as well as NFκB-mediated gene transcription. SCF represents a novel therapeutic target in the treatment of neurodegenerative disease.
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  • 34
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI–TOF (matrix-assisted laser desorption/ionization–time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.
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  • 35
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Huntington disease (HD) is an adult onset neurodegenerative disorder characterized by selective atrophy and cell loss within the striatum. There is currently no treatment that can prevent the striatal neuropathology. Transglutaminase (TG) activity is increased in HD patients, is associated with cell death, and has been suggested to contribute to striatal neuronal loss in HD. This work assesses the therapeutic potential of cystamine, an inhibitor of TG activity with additional potentially beneficial effects. Specifically, we examine the effect of cystamine on striatal neuronal loss in the YAC128 mouse model of HD. We demonstrate here for the first time that YAC128 mice show a forebrain-specific increase in TG activity compared with wild-type (WT) littermates which is decreased by oral delivery of cystamine. Treatment of symptomatic YAC128 mice with cystamine starting at 7 months prevented striatal neuronal loss. Cystamine treatment also ameliorated the striatal volume loss and striatal neuronal atrophy observed in these animals, but was unable to prevent motor dysfunction or the down-regulation of dopamine and cyclic adenosine monophsophate-regulated phosphoprotein (DARPP-32) expression in the striatum. While the exact mechanism responsible for the beneficial effects of cystamine in YAC128 mice is uncertain, our findings suggest that cystamine is neuroprotective and may be beneficial in the treatment of HD.
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  • 36
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Activation of neural pathways originating in the cerebellar fastigial nucleus (FN) protects the brain from the deleterious effects of cerebral ischemia and excitotoxicity, a phenomenon termed central neurogenic neuroprotection. The neuroprotection is, in part, mediated by suppression of apoptosis. We sought to determine whether FN stimulation exerts its anti-apoptotic effect through mitochondrial mechanisms. Mitochondria were isolated from the cerebral cortex of rats in which the FN was stimulated for 1 h (100 μA; 1 s on/1 s off), 72 h earlier. Stimulation of the dentate nucleus (DN), a brain region that does not confer neuroprotection, served as control. Mitochondria isolated from FN-stimulated rats exhibited a marked increase in their ability to sequester Ca2+ and an increased resistance to Ca2+-induced membrane depolarization and depression in respiration. FN stimulation also leads to reduction in the release in cytochrome c, induced either by Ca2+ or the mitochondrial toxin mastoparan. Furthermore, in brain slices, FN stimulation reduced the staurosporine-induced insertion of the pro-apoptotic protein Bax into the mitochondria, a critical step in the mitochondrial mechanisms of apoptosis. Collectively, these results provide evidence that FN stimulation protects the mitochondria from dysfunction induced by Ca2+ loading, and inhibits mitochondrial pathways initiating apoptosis. These mitochondrial mechanisms are likely to play a role in the neuroprotection exerted by FN stimulation.
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  • 37
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Brain abscess is associated with local vasogenic edema, which leads to increased intracranial pressure and significant morbidity. Aquaporin-4 (AQP4) is a water channel expressed in astroglia at the blood–brain and brain–CSF barriers. To investigate the role of AQP4 in brain abscess-associated edema, live Staphylococcus aureus (105 colony-forming units) was injected into the striatum to create a focal abscess. Wild-type and AQP4-deficient mice had comparable immune responses as measured by brain abscess volume (∼ 3.7 mm3 at 3 days), bacterial count and cytokine levels in brain homogenates. Blood–brain barrier permeability was increased comparably in both groups as assessed by extravasation of Evans blue dye. However, at 3 days the AQP4 null mice had significantly higher intracranial pressure (mean ± SEM 27 ± 2 vs. 17 ± 2 mmHg; p 〈 0.001) and brain water content (81.0 ± 0.3 vs. 79.3 ± 0.5 % water by weight in the abscess-containing hemisphere; p 〈 0.01) than wild-type mice. Reactive astrogliosis was found throughout the abscess-containing hemisphere; however, only a subset of astrocytes in the peri-abscess region of wild-type mice had increased AQP4 immunoreactivity. Our findings demonstrate a protective effect of AQP4 on brain swelling in bacterial abscess, suggesting that AQP4 induction may reduce vasogenic edema associated with cerebral infection.
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  • 38
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glycogen synthase kinase-3β (GSK-3β) is a multifunctional enzyme involved in a variety of biological events including development, glucose metabolism and cell death. Its activity is inhibited by phosphorylation of the Ser9 residue and up-regulated by Tyr216 phosphorylation. Activated GSK-3β increases phosphorylation of tau protein and induces cell death in a variety of cultured neurons, whereas phosphorylation of phosphatidylinositol-3 (PI-3) kinase-dependent protein kinase B (Akt), which inhibits GSK-3β activity, is one of the best characterized cell survival signaling pathways. In the present study, the cholinergic immunotoxin 192 IgG-saporin was used to address the potential role of GSK-3β in the degeneration of basal forebrain cholinergic neurons, which are preferentially vulnerable in Alzheimer's disease (AD) brain. GSK-3β co-localized with a subset of forebrain cholinergic neurons and loss of these neurons was accompanied by a transient decrease in PI-3 kinase, phospho-Ser473Akt and phospho-Ser9GSK-3β levels, as well as an increase in phospho-tau levels, in the basal forebrain and hippocampus. Total Akt, GSK-3β, tau and phospho-Tyr216GSK-3β levels were not significantly altered in these brain regions in animals treated with 192 IgG-saporin. Systemic administration of the GSK-3β inhibitor LiCl did not significantly affect cholinergic marker or phospho-Ser9GSK-3β levels in control rats but did preclude 192-IgG saporin-induced alterations in PI-3 kinase/phospho-Akt, phospho-Ser9GSK-3β and phospho-tau levels, and also partly protected cholinergic neurons against the immunotoxin. These results provide the first evidence that increased GSK-3β activity, via decreased Ser9 phosphorylation, can mediate, at least in part, 192-IgG saporin-induced in vivo degeneration of forebrain cholinergic neurons by enhancing tau phosphorylation. The partial protection of these neurons following inhibition of GSK-3β kinase activity suggests a possible therapeutic role for GSK-3β inhibitors in attenuating the loss of basal forebrain cholinergic neurons observed in AD.
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  • 39
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1flop displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.
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  • 40
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or ‘energy crisis’ of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage-sensor, poly(ADP-ribose) polymerase-1 (PARP-1). A 2-h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP-1 was much activated and autopoly(ADP-ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl-2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis-inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP-1 inhibitors, 1,5-dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP-1. These results indicated that PARP-1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.
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  • 41
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF-κB, p38, and JNK pathways, whereas the dsRNA-induced CCL5 production was dependent on JNK, but not on the other signal-transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH-dependent activation of NF-κB and MAPK signaling pathways.
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  • 42
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.
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  • 43
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Δloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Δloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Δloop occurs at 37°C but not at 4°C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Δloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.
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  • 44
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate-mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate-induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N-methyl-d-aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin-4 (NT-4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT-4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK-801 increased natural cell death of specific cell populations of the post-natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF-dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.
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  • 45
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Phenylethanolamine N-methyltransferase (PNMT, EC2.1.1.28) catalyzes the N-methylation of norepinephrine to form epinephrine. As a step toward understanding the possible contribution of inheritance to individual variation in PNMT-catalyzed epinephrine formation, we ‘re-sequenced’ the entire human PNMT gene, including the three exons, the introns and approximately 1 kb of the 5′-flanking region (5′-FR), using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Within the 3.5 kb re-sequenced, 18 single nucleotide polymorphisms (SNPs) were observed, including four non-synonymous coding SNPs (cSNPs) that resulted in the following alterations in encoded amino acid sequence: Asn9Ser, Thr98Ala, Arg112Cys and Ala175Thr. When constructs for the non-synonymous cSNPs were transiently expressed in COS-1 cells, the Ala98 allozyme displayed significantly lower levels of both activity and immunoreactive protein (p 〈 0.002) than did the wild-type (WT) enzyme due, at least in part, to accelerated protein degradation by a proteasome-mediated process. Luciferase reporter gene constructs were also created for the six common PNMT 5′-FR haplotypes observed. Significant differences were observed among haplotypes in their ability to drive transcription. These observations raise the possibility of inherited variation in the ability to form epinephrine from norepinephrine as a result of variant PNMT polymorphisms and haplotypes.
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  • 46
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration and synaptic plasticity. This study describes a novel function of NCAM140 in stimulating integrin-dependent cell migration. Expression of NCAM140 in rat B35 neuroblastoma cells resulted in increased migration toward the extracellular matrix proteins fibronectin, collagen IV, vitronectin, and laminin. NCAM-potentiated cell migration toward fibronectin was dependent on β1 integrins and required extracellular-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activity. NCAM140 in B35 neuroblastoma cells was subject to ectodomain cleavage resulting in a 115 kDa soluble fragment released into the media and a 30 kDa cytoplasmic domain fragment remaining in the cell membrane. NCAM140 ectodomain cleavage was stimulated by the tyrosine phosphatase inhibitor pervanadate and inhibited by the broad spectrum metalloprotease inhibitor GM6001, characteristic of a metalloprotease. Moreover, treatment of NCAM140-B35 cells with GM6001 reduced NCAM140-stimulated cell migration toward fibronectin and increased cellular attachment to fibronectin to a small but significant extent. These results suggested that metalloprotease-induced cleavage of NCAM140 from the membrane promotes integrin- and ERK1/2-dependent cell migration to extracellular matrix proteins.
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  • 47
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Experiments compared a series of phenethylamine hallucinogens with their phenylisopropylamine analogues for binding affinity and ability to stimulate serotonin 5-HT2A receptor-mediated hydrolysis of phosphatidyl inositol in cells expressing cloned rat and human 5-HT2A receptors. The (±)phenylisopropylamine analogues had significantly higher intrinsic activities for 5-HT2A receptor-mediated hydrolysis of phosphatidyl inositol compared to their phenethylamine analogues. With respect to the effects of the stereochemistry of the phenylisopropylamines, those with the (R) absolute configuration at the alpha carbon had higher intrinsic activities for hydrolysis of phosphatidyl inositol in a cell line expressing the human 5-HT2A receptor compared to those with the (S) absolute configuration. In virtual docking studies comparing the (R)- and (S)-phenylisopropylamines with their phenethylamine analogues, there were distinct differences in the orientations of key ligand binding domain residues that have been identified as important by previous mutagenesis studies. In conclusion, our data support the hypothesis that phenylisopropylamines have higher hallucinogenic potency than their phenethylamine analogues primarily because they have higher intrinsic activities at 5-HT2A receptors. Although virtual ligand binding led to significant perturbations of certain key residues, our results emphasize the conclusion reached by others that overall three-dimensional structural microdomains within the receptor must be considered.
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  • 48
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2β, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR α7 and α3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2α. Isoforms α and β are expressed in all tissues examined and have the same N-terminal and central regions but alternative C-terminal regions. Both isoforms interact with the α4 subunit. Within this subunit the interaction was localized to the N-terminal region of the large cytoplasmic loop. The CRELD2β protein is present at the endoplasmic reticulum where colocalized with α4β2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where α4β2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of α4β2 nAChRs in Xenopus leavis oocytes, without affecting α3β4 and α7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of α4β2 nAChR expression.
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  • 49
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Non-steroidal anti-inflammatory drugs (NSAIDs) and inhibitors of the cyclooxygenase (COX) pathways are currently recommended for the prevention and treatment of several inflammatory diseases, including neurodegenerative disorders. However non-selective blockade of COX was found to have pro-inflammatory properties, because they have the ability to alter the plasma glucocorticoid levels that play a critical role in the control of the innate immune response. The present study investigated the role of non-selective (ketorolac or indomethacin) or specific inhibitors of COX-1 (SC-560) and COX-2 (NS-398) in these effects. Mice challenged systemically with the endotoxin lipopolysaccharide (LPS) exhibited a robust hybridization signal for numerous inflammatory genes in vascular-associated cells of the brain and microglia across the cerebral tissue. Ketorolac, indomethacin and NS-398 significantly increased the ability of LPS to trigger such an innate immune response at time 3 h post challenge, whereas SC-560 failed to change gene expression in the brain of animals treated with the endotoxin. These data together with the crucial role of COX-2-derived prostaglandin E2 (PGE2) in the increase of glucocorticoids during systemic immune stimuli provide evidence that inhibition of this pathway results in an exacerbated early innate immune reaction. This may have a major impact on the use of these drugs in diseases where inflammation is believed to be a contributing and detrimental factor.
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  • 50
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Despite advances in our understanding of the basic biology of amyloid precursor protein (APP), the normal physiological function(s) of APP in learning and memory remains unclear. Here we show increased APP degradation in the hippocampus to be associated with the consolidation of a passive avoidance response. Neurone-specific APP695 expression became transiently reduced 2–4 h post-training through association with endosomal adaptin proteins and enhanced internalization. By contrast, internalization of glial-associated APP containing a Kunitz protease inhibitor-like domain (APP-KPI) was dependent on the low-density lipoprotein receptor-related protein (LRP). In addition, LRP expression and association with apolipoprotein E increased in the 2–4 h post-training period. The LRP antagonist receptor-associated protein prevented the APP-KPI internalization and LRP–apolipoprotein E association and this resulted in amnesia. Degradation of APP695 and APP-KPI did not appear to be related to α-secretase activity, as no learning-associated increase of secreted APP was observed in the CSF. Moreover, as internalization of APP isoforms was observed only in dentate gyrus, it probably relates to the learning-associated restructuring of the perforant path terminals. Memory-associated APP processing in both neuronal and glial compartments points to a role for glial unsheathing of synaptic connections, an event required for the synaptic restructuring that accompanies memory consolidation. These observations may have a direct relevance to understanding the pathophysiology of Alzheimer's disease as β/γ-secretase-derived β-amyloid is formed following internalization of cell surface APP into the endosomal compartment.
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  • 51
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-d-aspartate (NMDA), but not α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.
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  • 52
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 94 (2005), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
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  • 53
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: AML1/Runx1 (Runx1) is a mammalian transcription factor that plays critical roles in regulating the differentiation of a number of different cell types. In the present study, we have utilized mice expressing β-galactosidase (β-gal) under the control of the Runx1 promoter to characterize the spatiotemporal expression pattern of Runx1 during retinogenesis. Expression of β-gal was first detected at embryonic day 13.5 in post-mitotic cells located in the inner retina and overlapped with expression of the early amacrine and ganglion cell marker protein Islet1. During subsequent developmental stages, the number of β-gal-positive cells increased in a central-to-peripheral gradient until late embryogenesis but then decreased in the early post-natal retina. β-gal-positive cells were located primarily in the ganglion cell layer by late embryonic/early post-natal stages and were identified as a subpopulation of displaced amacrine cells by the continued expression of Islet1, as well as Pax6, and the coexpression of the amacrine cell subtype-specific markers choline acetyltransferase, calretinin and the 65-kDa isoform of glutamic acid decarboxylase. These findings identify Runx1 as a novel marker for a restricted amacrine cell subtype and suggest a role for this gene in regulating the post-mitotic development of these cells.
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  • 54
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Neurone glial-related cell adhesion molecule (NrCAM) is a member of the L1 family of transmembrane cell adhesion receptors which are involved in the development and function of the mammalian nervous system. How these receptors interact with intracellular signalling pathways is not understood. To date the only identified binding partner to the cytoplasmic terminus of NrCAM is ankyrin G. We screened a developing rat brain cDNA yeast two-hybrid library with the cytoplasmic domain of NrCAM to identify further intracellular binding partners. We identified synapse associated protein 102 (SAP102) as a new binding partner for NrCAM. The interaction was confirmed biochemically using glutathione S-transferase (GST)-pull-down and tandem affinity purification, and also immunocytochemically as NrCAM and SAP102 co-localized in COS-7 and cerebellar granule cells. Binding was specific to NrCAM as neither neurofascin nor L1 bound SAP102, and this interaction was reliant on the last three amino acids of NrCAM. Additionally, NrCAM constructs whose last three amino acids had been deleted appeared to have a dominant negative effect on neurite extension of cerebellar granule cells. This is the first interaction reported for NrCAM, and its association with SAP102 suggests that it is part of a larger complex which can interact with many different signalling pathways.
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  • 55
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN–) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN– cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN– cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.
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  • 56
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Ionizing radiation induced acute cell death in the dentate gyrus subgranular zone (SGZ) and the subventricular zone (SVZ). Hypomyelination was also observed. The effects of mild hypothermia and hyperthermia for 4 h after irradiation (IR) were studied in postnatal day 9 rats. One hemisphere was irradiated with a single dose of 8 Gy and animals were randomized to normothermia (rectal temperature 36°C for 4 h), hypothermia (32°C for 4 h) or hyperthermia (39°C for 4 h). Cellular injury, e.g. chromatin condensation and nitrotyrosine formation, appeared to proceed faster when the body temperature was higher. Caspase-3 activation was more pronounced in the hyperthermia group and nuclear translocation of p53 was less pronounced in the hypothermia group 6 h after IR. In the SVZ the loss of nestin-positive progenitors was more pronounced (48%) and the size was smaller (45%) in the hyperthermia group 7 days post-IR. Myelination was not different after hypo- or hyperthermia. This is the first report to demonstrate that hypothermia may be beneficial and that hyperthermia may aggravate the adverse side-effects after radiation therapy to the developing brain.
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  • 57
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the β-subunit gene (HEXB) associated with simultaneous deficiencies of β-hexosaminidase A (HexA; αβ) and B (HexB; ββ), and excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylglucosamine (GlcNAc) residues at their non-reducing termini. Recent studies have shown the involvement of microglial activation in neuroinflammation and neurodegeneration of this disease. We isolated primary microglial cells from the neonatal brains of Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex β-subunit gene allele (Hexb–/–). The cells expressed microglial cell-specific ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactivity (IR) and antigen recognized by Ricinus communis agglutinin lectin-120 (RCA120), but not glial fibrillary acidic protein (GFAP)-IR specific for astrocytes. They also demonstrated significant intracellular accumulation of GM2 and GlcNAc-oligosaccharides. We produced a lentiviral vector encoding for the murine Hex β-subunit and transduced it into the microglia from SD mice with the recombinant lentivirus, causing elimination of the intracellularly accumulated GM2 and GlcNAc-oligosaccharides and secretion of Hex isozyme activities from the transduced SD microglial cells. Recomibinant HexA isozyme isolated from the conditioned medium of a Chinese hamster ovary (CHO) cell line simultaneously expressing the human HEXA (α-subunit) and HEXB genes was also found to be incorporated into the SD microglia via cell surface cation-independent mannose 6-phosphate receptor and mannose receptor to degrade the intracellularly accumulated GM2 and GlcNAc-oligosaccharides. These results suggest the therapeutic potential of recombinant lentivirus encoding the murine Hex β-subunit and the human HexA isozyme (αβ heterodimer) for metabolic cross-correction in microglial cells involved in progressive neurodegeneration in SD mice.
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  • 58
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable ‘upstream’ targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia–reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.
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  • 59
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly and is a complex disorder that involves altered proteolysis, oxidative stress and disruption of ion homeostasis. Animal models have proven useful in studying the impact of mutant AD-related genes on other cellular signaling pathways, such as Ca2+ signaling. Along these lines, disturbances of intracellular Ca2+ ([Ca2+]i) homeostasis are an early event in the pathogenesis of AD. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate disturbances in Ca2+ homeostasis in primary cortical neurons from a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Application of caffeine to mutant presenilin-1 knock-in neurons (PS1KI) and 3xTg-AD neurons evoked a peak rise of [Ca2+]i that was significantly greater than those observed in non-transgenic neurons, although all groups had similar decay rates of their Ca2+ transient. This finding suggests that Ca2+ stores are greater in both PS1KI and 3xTg-AD neurons as calculated by the integral of the caffeine-induced Ca2+ transient signal. Western blot analysis failed to identify changes in the levels of several Ca2+ binding proteins (SERCA-2B, calbindin, calsenilin and calreticulin) implicated in the pathogenesis of AD. However, ryanodine receptor expression in both PS1KI and 3xTg-AD cortex was significantly increased. Our results suggest that the enhanced Ca2+ response to caffeine observed in both PS1KI and 3xTg-AD neurons may not be attributable to an alteration of endoplasmic reticulum store size, but to the increased steady-state levels of the ryanodine receptor.
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  • 60
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine-induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double-labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine-induced antinociception in mice.
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  • 61
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Mammalian cells often receive multiple extracellular stimuli under physiological conditions, and the various signaling inputs have to be integrated for the processing of complex biological responses. G protein-coupled receptors (GPCRs) are critical players in converting extracellular stimuli into intracellular signals. In this report, we examined the integration of different GPCR signals by mitogen-activated protein kinases (MAPKs) using the SK-N-MC human brain neuroepithelioma cells as a neuronal model. Stimulation of the Gi-coupled neuropeptide Y1 and Gq-coupled muscarinic M1 acetylcholine receptors, but not the Gs-coupled dopamine D1 receptor, led to the activation of extracellular signal-regulated kinase (ERK). All three receptors were also capable of stimulating c-Jun NH2-terminal kinases (JNK) and p38 MAPK. The Gi-mediated ERK activation was completely suppressed upon inhibition of Src tyrosine kinases by PP1, while the Gq-induced response was suppressed by both PP1 and the Ca2+ chelator, BAPTA-AM. In contrast, activations of JNK and p38 by Gs-, Gi-, and Gq-coupled receptors were sensitive to PP1 and BAPTA-AM pretreatments. Simultaneous stimulation of Gi- and Gq-coupled receptors resulted in the synergistic activation of ERK, but not JNK or p38 MAPK. The Gi/Gq-induced synergistic ERK activation was PTX-sensitive, and appeared to be a co-operative effect between Ca2+ and Src family tyrosine kinases. Enhanced ERK activation was associated with an increase in CREB phosphorylation, while the JNK and p38-responsive transcription factor ATF-2 was weakly enhanced upon Gi/Gq-induction. This report provides evidence that G protein signals can be integrated at the level of MAPK, resulting in differential effects on ERK, JNK and p38 MAPK in SK-N-MC cells.
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  • 62
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Large-conductance Ca2+-activated K+ (BKCa) channels are activated by membrane depolarization and modulated by intracellular Ca2+. Here, we report the direct interaction of cereblon (CRBN) with the cytosolic carboxy-terminus of the BKCa channel α subunit (Slo). Rat CRBN contained the N-terminal domain of the Lon protease, a ‘regulators of G protein-signaling’ (RGS)-like domain, a leucine zipper (LZ) motif, and four putative protein kinase C (PKC) phosphorylation sites. RNA messages of rat cereblon (rCRBN) were widely distributed in different tissues with especially high-levels of expression in the brain. Direct association of rCRBN with the BKCa channel was confirmed by immunoprecipitation in brain lysate, and the two proteins were co-localized in cultured rat hippocampal neurons. Ionic currents evoked by the rSlo channel were dramatically suppressed upon coexpression of rCRBN. rCRBN decreased the formation of the tetrameric rSlo complex thus reducing the surface expression of functional channels. Therefore, we suggest that CRBN may play an important role in assembly and surface expression of functional BKCa channels by direct interaction with the cytosolic C-terminus of its α-subunit.
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  • 63
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1–3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.
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  • 64
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Stimulation of hippocampal 5-HT1A receptors impairs memory retention. The highly selective 5-HT1A antagonist, WAY-100635, prevents the cognitive deficits induced not only by 5-HT1A stimulation but also by cholinergic or NMDA receptor blockade. On this basis, the effects of WAY-100635 on molecular events associated with memory storage were explored. In rat hippocampus, WAY-100635 produced a rapid increase in phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) and in Ca2+-independent CaMKII and protein kinase A (PKA) enzyme activity. This increase was followed a few hours later by an enhanced membrane expression of AMPA receptor subunits, especially of the GluR1 subunit phosphorylated at the CaMKII site, pGluR1(Ser831). The same qualitative effects were found with the weaker 5-HT1A antagonist NAN-190. The effects of both antagonists were no longer apparent in rats with a previous 5-HT depletion induced by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), suggesting that 5-HT1A receptor blockade removes the tonic inhibition of 5-HT through 5-HT1A receptor stimulation on excitatory hippocampal neurons, with the consequent increase in PKA activity. In addition, administration of WAY-100635 potentiated the learning-specific increase in the hippocampus of phospho-CaMKII, Ca2+-independent CaMKII activity, as well as the phosphorylation of either the CaMKII or the PKA site on the AMPA receptor GluR1 subunit. This study suggests that blockade of hippocampal 5-HT1A receptors favours molecular events critically involved in memory formation, and provides an in vivo molecular basis for the proposed utility of 5-HT1A receptor antagonists in the treatment of cognitive disorders.
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  • 65
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Long-term potentiation (LTP) is impaired in the CA1 area of hippocampal slices from rats with chronic moderate hyperammonemia. We studied the mechanisms by which hyperammonemia in vivo impairs LTP. This process requires sequential activation of soluble guanylate cyclase, cyclic GMP-dependent protein kinase (PKG) and cyclic GMP-degrading phosphodiesterase. Application of the tetanus induced a rapid increase of cyclic GMP in slices from control or hyperammonemic rats, which is followed in control slices by a sustained decrease in cyclic GMP due to sustained activation of cyclic GMP-degrading phosphodiesterase, which in turn is due to sustained activation of PKG. In slices from rats with chronic hyperammonemia tetanus-induced decrease in cyclic GMP was delayed and transient due to lower and transient activation of PKG and of the phosphodiesterase. Hyperammonemia-induced impairment of LTP may be involved in the alterations of cognitive function in patients with hepatic encephalopathy.
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  • 66
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: IN-1, the monoclonal antibody against the exon 3-encoded N-terminal domain of Nogo-A, and the Nogo-66 receptor (NgR) antagonist NEP1-40 have both shown efficacy in promoting regeneration in animal spinal cord injury models, the latter even when administered subcutaneously 1 week after injury. These results are supportive of the hypothesis that the Nogo–NgR axis is a major path for inhibition of spinal cord axonal regeneration and uphold the promises of these neutralizing agents in clinical applications. However, mice with targeted disruption of Nogo and NgR have, surprisingly, only modest regenerative capacity (if any) compared with treatment with IN-1 or NEP1-40. Disruption of the Nogo gene by various groups yielded results ranging from significant regenerative improvement in young mice to no improvement. Likewise, knockout of NgR produced some improvement in raphespinal and rubrospinal axonal regeneration, but not that of corticospinal neurons. Other than invoking possible differences in genetic background, we suggest here some possible and testable explanations for the above phenomena. These possibilities include effects of IN-1 and NEP1-40 on the CNS beyond neutralization of Nogo and NgR functions, and the latter's possible role in the CNS beyond that of neuronal growth inhibition.
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  • 67
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481–490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481–490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.
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  • 68
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: l-DOPA is the most effective treatment for Parkinson's disease but in isolated neuronal cultures it is neurotoxic for dopamine (DA) neurones. Experiments in vivo and clinical studies have failed to show toxicity of l-DOPA in animals or patients but that does not exclude the possibility of a toxic effect of l-DOPA on patients with certain genetic risk factors. Mutations of the parkin gene are the most frequent cause of hereditary parkinsonism. Parkin null mice have a mild phenotype that could be modified by different neurotoxins. The aim of this study was to investigate whether the toxic effects of l-DOPA on DA neurones are amplified in parkin null mice. We have measured the effects of l-DOPA on cell viability, tyrosine hydroxylase (TH) expression, DA metabolism and glutathione levels of parkin knockout (PK-KO) midbrain cultures. Neuronal-enriched cultures from PK-KO mice have similar proportions of the different cell types with the exception of a significant increment of microglial cells. l-DOPA (400 µm for 24 h) reduced the number of TH-immunoreactive cells to 50% of baseline and increased twofold the percentage of apoptotic cells in cultures of wild-type (WT) animals. The PK-KO mice, however, are not only resistant to the l-DOPA-induced pro-apoptotic effects but they have an increased number of TH-immunoreactive neurones after treatment with l-DOPA, suggesting that l-DOPA is toxic for neurones of WT mice but not those of parkin null mice. MAPK and phosphatidylinositol-3 kinase signalling pathways are not involved in the differential l-DOPA effects in WT and PK-KO cultures. Intracellular levels of l-DOPA were not different in WT and parkin null mice but the intracellular and extracellular levels of DA and 3-4-dihydroxyphenylacetic acid, however, were significantly increased in parkin null animals. Furthermore, monoamine oxidase activity was significantly increased in parkin null mice, suggesting that these animals have an increased metabolism of DA. The levels of glutathione were further increased in parkin null mice than in controls both with and without treatment with l-DOPA, suggesting that a compensatory mechanism may protect DA neurones from neuronal death. This study opens new avenues for understanding the mechanisms of action of l-DOPA on DA neurones in patients with Park-2 mutations.
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  • 69
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glioma cell-surface binding to hyaluronan (HA), a major constituent of the brain extracellular matrix (ECM) environment, is regulated through a complex membrane type-1 matrix metalloproteinase (MT1-MMP)/CD44/caveolin interaction that takes place at the leading edges of invading cells. In the present study, intracellular transduction pathways required for the HA-mediated recognition by infiltrating glioma cells in brain was investigated. We show that the overexpression of the GTPase RhoA up-regulated MT1-MMP expression and triggered CD44 shedding from the U-87 glioma cell surface. This potential implication in cerebral metastatic processes was also observed in cells overexpressing the full-length recombinant MT1-MMP, while the overexpression of a cytoplasmic domain truncated from of MT1-MMP failed to do so. This suggests that the cytoplasmic domain of MT1-MMP transduces intracellular signaling leading to RhoA-mediated CD44 shedding. Treatment of glioma cells with the Rho-kinase (ROK) inhibitor Y27632, or with EGCg, a green tea catechin with anti-MMP and anti-angiogenesis activities, antagonized both RhoA- and MT1-MMP-induced CD44 shedding. Conversely, overexpression of recombinant ROK stimulated CD44 release. Taken together, our results suggest that RhoA/ROK intracellular signaling regulates MT1-MMP-mediated CD44 recognition of HA. These molecular processes may partly explain the diffuse brain-infiltrating character of glioma cells within the surrounding parenchyma and thus be a target for new approaches to anti-tumor therapy.
    Type of Medium: Electronic Resource
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  • 70
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Gabapentin is an anticonvulsant that successfully treats many neuropathic pain syndromes, although the mechanism of its antihyperalgesic action remains elusive. This study aims to help delineate gabapentin's antihyperalgesic mechanisms. We assessed the effectiveness of gabapentin at decreasing mechanical and cold hypersensitivity induced in a rat model of neuropathic pain. Thus, we compared the effectiveness of pre- or post-treatment with systemic or intrathecal (i.t.) gabapentin at reducing the development and maintenance of the neuropathic pain symptoms. Gabapentin successfully decreased mechanical and cold hypersensitivity, both as a pretreatment and post-treatment. Furthermore, both i.t. and systemic administration of gabapentin were effective in reducing the behavioral hypersensitivity; however, the i.t. administration was superior to the systemic. We also examined gabapentin's effects at inhibiting hindpaw formalin-induced release of excitatory amino acids (EAAs) in the spinal cord dorsal horn (SCDH) both in naïve rats and in rats with neuropathic pain. We present the first evidence that gabapentin reduces the formalin-induced release of both glutamate and aspartate in SCDH. Furthermore, i.t. gabapentin reduces the enhanced noxious stimulus-induced spinal release of glutamate seen in neuropathic rats. These data suggest that gabapentin reduces neuropathic pain symptoms by inhibiting the release of glutamate in the SCDH.
    Type of Medium: Electronic Resource
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  • 71
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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