The molecular mechanism(s) of N-methyl-D-aspartate (NMDA) neuroprotective properties were investigated in primary cultures of cerebellar granule cell neurons. Granule cells express the neurotrophin receptor TrkB but not TrkA or TrkC. In these cells, the TrkB ligand brain-derived neurotrophic factor (BDNF) prevents glutamate toxicity. Therefore, we have tested the hypothesis that NMDA activates synthesis and release of BDNF, which may prevent glutamate toxicity by an autocrine loop. Exposure of granule cells for 2 and 5 min to a subtoxic concentration of NMDA (100 M) evoked an accumulation of BDNF in the medium without concomitant changes in the intracellular levels of BDNF protein or mRNA. The increase in BDNF in the medium is followed by enhanced TrkB tyrosine phosphorylation, suggesting that NMDA increases the release of BDNF and therefore the activity of TrkB receptors. To examine whether BDNF and TrkB signaling play a role in the NMDA-mediated neuroprotective properties, neurons were exposed to soluble trkB receptor-IgG fusion protein, which is known to inhibit the activity of extracellular BDNF, and to K252a, a tyrosine kinase inhibitor. Both compounds blocked the NMDA-mediated TrkB tyrosine phosphorylation and subsequently its neuroprotective properties. We suggest that NMDA activates the TrkB receptor via a BDNF autocrine loop, resulting in neuronal survival.Glutamate, the endogenous neurotransmitter required for normal physiological excitation, is also involved in the pathophysiology of hypoxic/ischemic neuronal injury (1-3). Although this neuropathological process can be mediated by any of the excitatory amino acid receptors, the N-methyl-D-aspartate (NMDA) 1 glutamate receptor subtype plays a major role. In addition, NMDA receptors mediate adaptive responses important for synaptic plasticity during development (4, 5). The molecular mechanism(s) by which NMDA receptors mediate such opposing effects are not clear.The role of NMDA receptors in glutamate-mediated excitotoxicity has been studied in rat cerebellar granule cells in vitro, in which overactivation of NMDA receptors results in neuronal cell death (6 -8). Paradoxically, in these cells subtoxic concentrations of NMDA have been shown to protect vulnerable neurons against an excitotoxic concentration of glutamate (9). Moreover, recent studies have shown that the neuroprotective effect of NMDA is blocked by coincubation with either the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide (9). Thus, the neuroprotective effect of NMDA may involve a polypeptide(s) with neuroprotective properties.Cerebellar granule cells are responsive to various neurotrophic factors and in particular to the neurotrophins (10, 11), a family of trophic factors related by primary amino acid sequence homology, whose members include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and NT-4/5 (12-17). The biological activity of neurotrophins depends upon the activation of high-affinity receptors (Trk...
Neurotrophins are critical to the development and maintenance of the mammalian central nervous system. Among them is brain-derived neurotrophic factor (BDNF), whose synthesis and release is targeted by activation of glutamate receptors. Perturbation of this process probably underlies neurodegenerative and psychiatric disorders. A naturally occurring variation in humans, in the form of a common single-nucleotide polymorphism in the pro region of the polypeptide at codon 66 (Val66-->Met), affects processing of the pro-BDNF polypeptide and its activation-dependent release. This variant is associated with differences in the volume of the hippocampal formation and with anxiety and depression-related phenotypes. Convergent findings supporting a role for BDNF in alterations to hippocampal structure and behavior are found in a "humanized" BDNF transgenic mouse. Also, recent human genetic studies have supported a role of BDNF signaling in addictive behaviors by allele-, genotype-, and haplotype-based association of the TrkB gene, which encodes the cognate receptor for BDNF, with alcohol dependence. A better understanding of the influence of BDNF-mediated pathways in cell survival and plasticity will aid in developing new approaches to restoring normal function in disease states.
The brain-derived neurotrophic factor (BDNF) gene is critical for neuronal function and survival, and is likely to be important in psychiatric disorders. In this study, we used single-nucleotide polymorphism (SNP) discovery, functional analyses, and genetic association studies to better understand the potential role of BDNF sequence variation in behavior. Screening 480 unrelated individuals for SNPs and genotyping was performed in US Caucasian, American Indian, and African American populations. Lifetime DSM-III-R psychiatric diagnoses were assigned and the Tridimensional Personality Questionnaire (TPQ) was administered to measure anxious temperament (harm avoidance (HA)) and novelty seeking (NS). A novel SNP (À281 C4A) in promoter 1 was discovered that had decreased DNA binding in vitro and decreased basal reporter gene activity in transfected rat hippocampal neurons. The frequency of the À281 A allele was 0.03 in a Caucasian sample, but was virtually absent in other populations. Association analyses in a community-based sample showed that individuals with the À281 A allele (13 heterozygotes) had lower TPQ HA (F ¼ 4.8, po0.05). In contrast, the Met 66 allele was associated with increased HA (F ¼ 4.1, p ¼ 0.02) and was most abundant in individuals with both anxiety disorders and major depression (po0.05). Among the Val66Val homozygotes, individuals who were -281 CA heterozygotes had significantly lower HA than the -281 CC homozygotes (po0.01). Our results suggest that in this population, the low activity -281 A allele may be protective against anxiety and psychiatric morbidity, whereas Met 66 may be a risk allele.
Cerebellar granule cells are susceptible to the excitotoxin glutamate, which acts at N-methyl-D-aspartate (NMDA) receptors, as well as the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+), the active cytotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Paradoxically, preincubation of cultured cerebellar granule cells with low concentrations of NMDA or glutamate markedly antagonizes the neurotoxicity resulting from subsequent exposure to toxic concentrations of either MPP+ or glutamate. The neuroprotective effects of NMDA and glutamate against MPP+ toxicity are observed at agonist concentrations as low as 1 IAM, are blocked by specific NMDA receptor antagonists, and require at least 30 min to develop fully. Moreover, NMDA receptor-mediated neuroprotection is prevented by the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. Thus, in cerebellar granule cells activation of NMDA receptors by glutamate can result in either neurotoxicity or neuroprotection, depending on the apparent degree of receptor stimulation. NMDA receptor-mediated neuroprotection requires new RNA and protein synthesis and therefore appears to be mediated by the expression of a neuroprotective protein(s). These data demonstrate the presence of an active NMDA receptor-mediated and transcriptionally directed neuroprotective mechanism in cerebellar granule cells.Glutamate, the major excitatory neurotransmitter in brain, is also a potent excitotoxin to many neurons under a variety of experimental conditions (1-3). Each of the three major subtypes of glutamate receptor has been implicated in glutamate neurotoxicity (4-6), although it appears that the N-methyl-D-aspartate (NMDA) receptor plays an important, if not exclusive, role in mediating neuronal death observed in most primary cultured neurons after either brief (7) or prolonged (8) glutamate exposure. Given the postulated role of NMDA receptor-mediated neurotoxicity in the underlying pathogenesis of ischemic brain injury (e.g., following stroke or trauma) and in a variety of neurodegenerative disorders (e.g., Alzheimer and Huntington disease) (9, 10), there has been considerable effort at both delineating and modifying the cellular and molecular events underlying NMDA receptormediated neurotoxicity.Cultured cerebellar granule cells, the most abundant neuronal subtype in the mammalian brain (11), have been used extensively for studying the cellular mechanisms underlying neuronal death induced by various endogenous and exogenous neurotoxins. These neurons express functional NMDA, as well as non-NMDA, glutamate receptors (12) and are highly susceptible to the excitotoxic actions of glutamate (13). Cerebellar granule cells are also killed by 1-methyl-4-phenylpyridinium ion (MPP+) (14), the active neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a parkinsonian-like syndrome in animals and humans. Although the exact cellular mechanism(s) underlying the neurotoxic actions of MPP+ are unknow...
The role of a nuclear factor kB (NF-kB) in NMDA receptormediated neuroprotection is not known. A candidate sequence from the 5H¯a nking region of exon 3 of the rat brain-derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 mM NMDA activated a speci®c DNA binding activity that was blocked by the NMDA receptor antagonist MK-801. Anti-p65 antibody or anti-p50 antibody`supershifted' the DNA binding activity, suggesting that the DNA±protein complex was composed of p65 and p50 subunits. NMDA receptor-mediated neuroprotection was blocked when cerebellar neurons were transfected with a double-stranded oligonucleotide containing the BDNF gene NF-kB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double-stranded NF-kB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF-kB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quanti®ed the level of exon 3-speci®c BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3-speci®c BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF-kB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF-kB DNA binding activity. Taken together, these data support a mechanism where NF-kB plays a critical role in NMDA-mediated neuroprotection.
The transcription factor DEC1 is induced by CD28 ligation and is required for optimal CD4+ T cell responses and the development of EAE.
Astrocytes have many neuronal characteristics, such as neurotransmitter receptors, ion channels, and neurotransmitter uptake systems. Cultured astrocytes were shown to express certain neuropeptide genes, with specificity for both the gene expressed and the brain region from which the cells were prepared. Somatostatin messenger RNA and peptides were detected only in cerebellar astrocytes, whereas proenkephalin messenger RNA and enkephalin peptides were present in astrocytes of cortex, cerebellum, and striatum. Cholecystokinin was not expressed in any of the cells. These results support the hypothesis that peptides synthesized in astrocytes may play a role in the development of the central nervous system.
Alpha-linolenic acid (ALA) is plant-based essential omega-3 polyunsaturated fatty acids that must be obtained through the diet. This could explain in part why the severe deficiency in omega-3 intake pointed by numerous epidemiologic studies may increase the brain's vulnerability representing an important risk factor in the development and/or deterioration of certain cardio- and neuropathologies. The roles of ALA in neurological disorders remain unclear, especially in stroke that is a leading cause of death. We and others have identified ALA as a potential nutraceutical to protect the brain from stroke, characterized by its pleiotropic effects in neuroprotection, vasodilation of brain arteries, and neuroplasticity. This review highlights how chronic administration of ALA protects against rodent models of hypoxic-ischemic injury and exerts an anti-depressant-like activity, effects that likely involve multiple mechanisms in brain, and may be applied in stroke prevention. One major effect may be through an increase in mature brain-derived neurotrophic factor (BDNF), a widely expressed protein in brain that plays critical roles in neuronal maintenance, and learning and memory. Understanding the precise roles of ALA in neurological disorders will provide the underpinnings for the development of new therapies for patients and families who could be devastated by these disorders.
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