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Par-4(1) (prostate apoptosis response 4) is known to function at an early stage in apoptosis in several different cell types, including neurons. On the other hand, activation of the transcription factor NF-kappaB can prevent apoptosis in various cancer cells and neurons. We now report that overexpression of full-length Par-4 in cultured PC12 cells results in a suppression of basal NF-kappaB DNA-binding activity and NF-kappaB activation following trophic factor withdrawal (TFW). The decreased NF-kappaB activity is correlated with enhanced apoptosis. Conversely, NF-kappaB activity is increased and vulnerability to apoptosis reduced in cells overexpressing a dominant-negative form of Par-4. Par-4 overexpression or functional blockade had no effect on AP-1 DNA-binding activity. Expression of the antiapoptotic protein Bcl-2 was dramatically reduced in PC12 cells overexpressing Par-4. Our data suggest that suppression of NF-kappaB activation plays a major role in the proapoptotic function of Par-4., (Copyright 2000 Wiley-Liss, Inc.)

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.

Activity-dependent neurotrophic factor (ADNF) is produced by astrocytes in response to neuronal depolarization and, in turn, promotes neuronal survival. A nineamino acid ADNF peptide (ADNF9) exhibits full neurotrophic activity and potently protects cultured embryonic rat hippocampal neurons from oxidative injury and apoptosis. Picomolar concentrations of ADNF9 induced an increase in nuclear factor-kappaB (NF-kappaB) DNA-binding activity within 1 h of exposure, with a maximum increase of approximately 10-fold by 6 h. Activation of NF-kappaB was correlated with increased resistance of neurons to apoptosis induced by exposure to Fe(2+). The antiapoptotic action of ADNF9 was abolished when NF-kappaB activation was specifically blocked with kappaB decoy DNA. Oxidative stress was attenuated in neurons pretreated with ADNF9, and this effect of ADNF9 was blocked by kappaB decoy DNA, suggesting that ADNF9 suppresses apoptosis by reducing oxidative stress. ADNF9 also prevented neuronal apoptosis following trophic factor withdrawal via an NF-kappaB-mediated mechanism. Thus, NF-kappaB mediates the neuron survival-promoting effects of ADNF9 in experimental models relevant to developmental neuronal death and neurodegenerative disorders.

The catalytic subunit of telomerase (TERT) is a specialized reverse transcriptase that has been associated with cell immortalization and cancer. It was reported recently that TERT is expressed in neurons throughout the brain in embryonic and early postnatal development, but is absent from neurons in the adult brain. We now report that suppression of TERT levels and function in embryonic mouse hippocampal neurons in culture using antisense technology and the telomerase inhibitor 3' -azido-2' 3' -dideoxythymidine significantly increases their vulnerability to cell death induced by amyloid beta-peptide, a neurotoxic protein believed to promote neuronal degeneration in Alzheimer's disease. Neurons in which TERT levels were reduced exhibited increased levels of oxidative stress and mitochondrial dysfunction following exposure to amyloid beta-peptide. Overexpression of TERT in pheochromocytoma cells resulted in decreased vulnerability to amyloid beta-peptide-induced apoptosis. Our findings demonstrate a neuroprotective function of TERT in an experimental model relevant to Alzheimer's disease, and suggest the possibility that restoration of TERT expression in neurons in the adult brain may protect against age-related neurodegeneration.

Apoptosis is a form of programmed cell death that occurs in neurons during development of the nervous system and may also be a prominent form of neuronal death in chronic neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Recent findings also implicate apoptosis in neuronal degeneration after ischemic brain injury in animal models of stroke. Activation of both apoptotic and antiapoptotic signaling cascades occurs in neurons in animal and cell culture models of stroke. Apoptotic cascades involve: increased levels of intracellular oxyradicals and calcium; induction of expression of proteins such as Par-4 (prostate apoptosis response-4), which act by promoting mitochondrial dysfunction and suppressing antiapoptotic mechanisms; mitochondrial membrane depolarization, calcium uptake, and release of factors (e.g., cytochrome c) that ultimately induce nuclear DNA condensation and fragmentation; activation of cysteine proteases of the caspase family; activation of transcription factors such as AP-1 that may induce expression of "killer genes." Antiapoptotic signaling pathways are activated by neurotrophic factors, certain cytokines, and increases in oxidative and metabolic stress. Such protective pathways include: activation of the transcription factors (e.g., nuclear factor-kappa B, NF-kappa B) that induce expression of stress proteins, antioxidant enzymes, and calcium-regulating proteins; phosphorylation-mediated modulation of ion channels and membrane transporters; cytoskeletal alterations that modulate calcium homeostasis; and modulation of proteins that stabilize mitochondrial function (e.g., Bcl-2). Intervention studies in experimental stroke models have identified a battery of approaches of potential benefit in reducing neuronal death in stroke patients, including administration of antioxidants, calcium-stabilizing agents, caspase inhibitors, and agents that activate NF-kappa B. Interestingly, recent studies suggest novel dietary approaches (e.g., food restriction and supplementation with antioxidants) that may reduce brain damage following stroke.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. PS1 mutations may perturb cellular Ca(2+) homeostasis and thereby render neurons vulnerable to excitotoxicity and apoptosis. We now report that PC12 cells expressing PS1 mutations and primary hippocampal neurons from PS1 mutant knockin mice exhibit greatly increased levels of ryanodine receptors (RyR) and enhanced Ca(2+) release following stimulation with caffeine. Double-labeling immunostaining and co-immunoprecipitation analyses indicate that PS1 and RyR are colocalized and interact physically. Caffeine treatment sensitizes neurons expressing mutant PS1 to apoptosis induced by amyloid beta-peptide, a neurotic peptide linked to the pathogenesis of AD. When taken together with recent evidence for alterations in RyR in brains of AD patients, our data suggest that PS1 mutations may promote neuronal degeneration in AD by increasing transcription and translation of RyR and altering functional properties of ryanodine-sensitive Ca(2+) pools.

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.

Carbon nanotubes are strong, flexible, conduct electrical current, and can be functionalized with different molecules, properties that may be useful in basic and applied neuroscience research. We report the first application of carbon nanotube technology to neuroscience research. Methods were developed for growing embryonic rat-brain neurons on multiwalled carbon nanotubes. On unmodified nanotubes, neurons extend only one or two neurites, which exhibit very few branches. In contrast, neurons grown on nanotubes coated with the bioactive molecule 4-hydroxynonenal elaborate multiple neurites, which exhibit extensive branching. These findings establish the feasability of using nanotubes as substrates for nerve cell growth and as probes of neuronal function at the nanometer scale.

Activation of ionotropic glutamate receptors of the AMPA and NMDA subtypes likely contributes to neuronal injury and death in various neurodegenerative disorders. Excitotoxicity can manifest as either apoptosis or necrosis, but the mechanisms that determine the mode of cell death are not known. We now report that levels of AMPA receptor subunits GluR-1 and GluR-4 are rapidly decreased in cultured rat hippocampal neurons undergoing apoptosis in response to withdrawal of trophic support (WTS), whereas levels of NMDA receptor subunits NR1, NR2A, and NR2B are unchanged. Exposure of isolated synaptosomal membranes to "apoptotic" cytosolic extracts resulted in rapid degradation of AMPA receptor subunits. Treatment of cells and synaptosomal membranes with the caspase inhibitors prevented degradation of AMPA receptor subunits, demonstrating a requirement for caspases in the process. Calcium responses to AMPA receptor activation were reduced after withdrawal of trophic support and enhanced after treatment with caspase inhibitors. Vulnerability of neurons to excitotoxic necrosis was decreased after withdrawal of trophic support and potentiated by treatment with caspase inhibitors. Our data indicate that caspase-mediated degradation of AMPA receptor subunits occurs during early periods of cell stress and may serve to ensure apoptosis by preventing excitotoxic necrosis.

Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations.

Endoplasmic reticulum (ER) is a multifaceted organelle that regulates protein synthesis and trafficking, cellular responses to stress, and intracellular Ca2+ levels. In neurons, it is distributed between the cellular compartments that regulate plasticity and survival, which include axons, dendrites, growth cones and synaptic terminals. Intriguing communication networks between ER, mitochondria and plasma membrane are being revealed that provide mechanisms for the precise regulation of temporal and spatial aspects of Ca2+ signaling. Alterations in Ca2+ homeostasis in ER contribute to neuronal apoptosis and excitotoxicity, and are being linked to the pathogenesis of several different neurodegenerative disorders, including Alzheimer's disease and stroke.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.

Although several prominent morphological features of apoptosis are evident in the cell body (e.g., cell shrinkage, membrane blebbing, and nuclear DNA condensation and fragmentation) the biochemical and molecular cascades that constitute the cell death machinery can be engaged in synaptic terminals and neurites. Initiating events such as oxyradical production and calcium influx, and effector processes such as Par-4 production, mitochondrial alterations and caspase activation, can be induced in synapses and neurites. Several prominent signal transduction pathways in synaptic terminals play important roles in either promoting or preventing neuronal death in physiological and pathological conditions. For example, activation of glutamate receptors in postsynaptic spines can induce neuronal apoptosis, whereas local activation of neurotrophic factor receptors in presynaptic terminals can prevent neuronal death. Factors capable of inducing nuclear chromatin condensation and fragmentation can be produced locally in synaptic terminals and neurites, and may propogate to the cell body. Recent findings suggest that, beyond their roles in inducing or preventing cell death, apoptotic and anti-apoptotic cascades play roles in synaptic plasticity (structural remodelling and long-term functional changes). For example, caspase activation results in proteolysis of glutamate receptor (AMPA) subunits, which results in altered neuronal responsivity to glutamate. Activation of neurotrophic factor receptors in synaptic terminals can result in local changes in energy metabolism and calcium homeostasis, and can induce long-term changes in synaptic transmission. The emerging data therefore suggest that synapses can be considered as autonomous compartments in which both pro- and anti-apoptotic signaling pathways are activated resulting in structural and functional changes in neuronal circuits. A better understanding of such synaptic signaling mechanisms may reveal novel approaches for preventing and treating an array of neurodegenerative conditions that are initiated by perturbed synaptic homeostasis.

Inheritance of the apolipoprotein E (apoE) epsilon4 allele increases the risk for Alzheimer's disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE-modified apoE in lumbar spinal cord samples from mice expressing an ALS-linked mutation in Cu/Zn-superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid beta-peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases.

Lysophosphatidic acid (LPA) elicits a unique response in primary hippocampal neurons and sympathetic neuron-like cells, PC12 cells differentiated with nerve growth factor; LPA is cytotoxic. Treatment of rat hippocampal neurons with 50 microM LPA resulted in necrosis, as determined morphologically and by release of lactate dehydrogenase. Lower concentrations of LPA, 0.1, and 1 microM, induced neuronal apoptosis, as assessed by chromatin condensation, annexin V binding, TUNEL staining, and the caspase sensitivity of these events. In addition, 10 and 25 microM LPA induced apoptosis of PC12 cells. In order to define intracellular events associated with this neuronal apoptosis, protective agents were identified. Neurons and PC12 cells were protected against LPA-induced apoptosis by pretreatment with the antioxidant, propyl gallate, or with nitric oxide synthase inhibitors. PC12 cells were protected by insulin and insulin-like growth-factor-1 treatment. There is also evidence for mitochondrial participation in LPA-mediated apoptosis, including cyclosporin A-mediated protection. Thus, LPA-induced neuronal apoptosis is associated with mitochondrial alterations, the generation of reactive oxygen species and nitric oxide, and protection by pretreatment with a serum constituent, insulin-like growth factor 1.

Although traumatic brain injury (TBI) often results in impaired learning and memory functions, the underlying mechanisms are unknown and there are currently no treatments that can preserve such functions. We studied plasticity at CA3-CA1 synapses in hippocampal slices from rats subjected to controlled cortical impact TBI. Long-term potentiation (LTP) of synaptic transmission was markedly impaired, whereas long-term depression (LTD) was enhanced, 48 h following TBI when compared to unoperated and sham control rats. Post-TBI administration of cyclosporin A, a compound that stabilizes mitochondrial function, resulted in a highly significant amelioration of the impairment of LTP and completely prevented the enhancement of LTD. Our data suggest that alterations in hippocampal synaptic plasticity may be responsible for learning and memory deficits resulting from TBI and that agents such as cyclosporin A that stabilize mitochondrial function may be effective treatments for TBI., (Copyright 2000 Academic Press.)

Although degeneration of lower motor neurons is the most striking abnormality in amyotrophic lateral sclerosis (ALS), more subtle alterations may occur in the brain. Mutations in copper/zinc superoxide dismutase (Cu/Zn-SOD) are responsible for some cases of inherited ALS, and expression of mutant Cu/Zn-SOD in transgenic mice results in progressive motor neuron loss and a clinical phenotype similar to that of ALS patients. It is now reported that Cu/Zn-SOD mutant mice exhibit increased vulnerability to focal ischemic brain injury after transient occlusion of the middle cerebral artery. Levels of glucose and glutamate transport in cerebral cortex synaptic terminals were markedly decreased, and levels of membrane lipid peroxidation were increased in Cu/Zn-SOD mutant mice compared to nontransgenic mice. These findings demonstrate that mutant Cu/Zn-SOD may endanger brain neurons by a mechanism involving impairment of glucose and glutamate transporters. Moreover, our data demonstrate a direct adverse effect of the mutant enzyme on synaptic function.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Studies of cultured neural cells suggest that PS1 mutations result in perturbed cellular calcium homeostasis and may thereby render neurons vulnerable to apoptosis. In light of evidence that metabolic impairment plays a role in AD, that cerebral ischemia may be a risk factor for AD, and that individuals with AD have increased morbidity and mortality after stroke, we examined the impact of a PS1 mutation on neuronal vulnerability to ischemic injury. We report that the extent of brain injury after focal cerebral ischemia reperfusion is increased, and behavioral outcome is worsened, in PS1 mutant knock-in mice compared to wild-type mice. Cultured cortical neurons from PS1 mutant mice exhibit increased vulnerability to glucose deprivation and chemical hypoxia compared to their wild-type counterparts. Calcium imaging studies demonstrated enhanced elevation of intracellular calcium levels after glucose deprivation and chemical hypoxia in neurons from PS1 mutant mice. Agents that block calcium release from IP(3)- and ryanodine-sensitive stores (xestospongin and dantrolene, respectively) protected against the endangering action of the PS1 mutation. Our data suggest that presenilin mutations may promote neuronal degeneration in AD by increasing the sensitivity of neurons to age-related ischemia-like conditions. The data further suggest that drugs that stabilize endoplasmic reticulum calcium homeostasis may prove effective in suppressing the neurodegenerative process in AD patients.

Calcium influx through N-methyl-d-aspartate (NMDA) receptors can result in neuronal apoptosis or necrosis and may play a pivotal role in neuronal death in many different neurodegenerative diseases. In the present study we employed primary neuronal cultures and three different excitoprotective factors, brain-derived neurotrophic factor (BDNF), activity-dependent neurotrophic factor (ADNF9), and tumor necrosis factor alpha (TNFalpha), to elucidate the mechanisms whereby trophic factors modify the excitotoxic process. Neurons pretreated with BDNF exhibited increased levels of the NMDA receptor subunits NR1 and NR2A, which was associated with increased calcium responses to NMDA and vulnerability to excitotoxic necrosis and reduced vulnerability to apoptosis. ADNF9 and TNFalpha suppressed calcium responses to glutamate and protected neurons against both excitotoxic necrosis and apoptosis, but had no effect on levels of NMDA receptor subunits. Inhibition of phosphorylation and DNA binding of NF-kappaB, by H7 and kappaB decoy DNA, respectively, suggest that the excitotoxic-modulating actions of BDNF are mediated by kinases, while those of ADNF9 and TNFalpha are mediated by both kinases and the transcription factor NF-kappaB. Our data show that, whereas BDNF increases neuronal responses to glutamate while ADNF9 and TNFalpha decrease the same, all three protect against excitotoxic apoptosis., (Copyright 2000 Academic Press.)

Primary spinal cord trauma can initiate a cascade of pathophysiologic events which markedly contribute to the expansion and amplification of the primary insult. The detailed mechanisms of these secondary neurochemical reactions are largely unknown; however, they involve membrane lipid derangements with the release of free fatty acids, in particular, arachidonic acid (AA). AA can induce several injury effects on spinal cord neurons. We hypothesize that upregulation of nitric oxide synthase (NOS) is among the most important mechanisms of arachidonic-acid-induced neuronal dysfunction and that nicotine can attenuate this effect. To study these hypotheses, spinal cord neurons were exposed to AA and/or nicotine, and several markers of neuronal nitric oxide synthase (nNOS) metabolism were measured. In addition, cotreatments with either inhibitors of nicotinic receptors or inhibitors of specific NOS isoforms were employed. Treatment with AA markedly increased activity of nNOS, as well as mRNA and protein levels of this enzyme. Changes in nNOS expression were accompanied by an increase in cellular cGMP and medium nitrite levels. Pretreatment with nicotine decreased AA-induced overexpression of nNOS and elevation of nitrite levels. In addition, it appeared that these nicotine effects could be partially modulated both by the alpha7 nicotinic receptors or by nonreceptor mechanisms. Alternatively, the observed changes could also be mediated by an alternate nicotinic receptor mechanism which is not blocked by alpha-bungarotoxin or mecamylamine. Results of the present study indicate that exposure to AA can lead to induction of nNOS in cultured spinal cord neurons. In addition, nicotine can exert a neuroprotective effect by attenuation of AA-induced upregulation of nNOS metabolism. These data may have therapeutic implications for the treatment of acute spinal cord trauma., (Copyright 2000 Academic Press.)

The cytokine tumor necrosis factor-alpha (TNF), well-known for its roles in cellular responses to tissue injury, has recently been shown to be produced in response to physiological activity in neuronal circuits. TNF stimulates receptors in neurons linked to the activation of the transcription factor NF-kappaB, and recent findings suggest that this signaling pathway can modulate neuronal excitability and vulnerability of neurons to excitotoxicity. Because data indicate that TNF is produced, and NF-kappaB activated, under conditions associated with learning and memory, we performed experiments in the hippocampal slice preparation aimed at elucidating roles for TNF and NF-kappaB in modulating synaptic plasticity. Whereas stimulation of Schaffer collateral axons at a frequency of 1 Hz induced long-term depression (LTD) of synaptic transmission in region CA1 of wild-type mice, LTD did not occur in slices from TNF receptor knockout mice. Stimulation at 100 Hz induced long-term potentiation (LTP) in slices from both wild-type mice and mice lacking TNF receptors. Basal transmission was unaltered in mice lacking TNF receptors. Pretreatment of slices from wild-type mice with kappaB decoy DNA prevented induction of LTD and significantly reduced the magnitude of LTP. Collectively, these data suggest important roles for TNF and signaling pathways that modulate NF-kappaB activity in regulation of hippocampal synaptic plasticity., (Copyright 2000 Wiley-Liss, Inc.)

Telomerase, a specialized reverse transcriptase (RT) linked to cell immortalization and cancer, has been thought not to be expressed in postmitotic cells. We now report that telomerase activity and its essential catalytic subunit, telomerase reverse transcriptase (TERT), are expressed in neurons in the brains of rodents during embryonic and early postnatal development, and are subsequently downregulated. Suppression of TERT expression in cultured embryonic hippocampal neurons increases their vulnerability to apoptosis and excitotoxicity. Overexpression of TERT in PC12 cells suppresses apoptosis induced by trophic factor withdrawal. TERT exerts its anti-apoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation. TERT may serve a neuron survival-promoting function in the developing brain, and downregulation of TERT in the adult brain may contribute to increased neuronal vulnerability in various age-related neurodegenerative disorders.

The transcription factor nuclear factor kappaB (NF-kappaB) is moving to the forefront of the fields of apoptosis and neuronal plasticity because of recent findings showing that activation of NF-kappaB prevents neuronal apoptosis in various cell culture and in vivo models and because NF-kappaB is activated in association with synaptic plasticity. Activation of NF-kappaB was first shown to mediate antiapoptotic actions of tumor necrosis factor in cultured neurons and was subsequently shown to prevent death of various nonneuronal cells. NF-kappaB is activated by several cytokines and neurotrophic factors and in response to various cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF-kappaB activation. Activation of NF-kappaB can interrupt apoptotic biochemical cascades at relatively early steps, before mitochondrial dysfunction and oxyradical production. Gene targets for NF-kappaB that may mediate its antiapoptotic actions include the antioxidant enzyme manganese superoxide dismutase, members of the inhibitor of apoptosis family of proteins, and the calcium-binding protein calbindin D28k. NF-kappaB is activated by synaptic activity and may play important roles in the process of learning and memory. The available data identify NF-kappaB as an important regulator of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death and promote neuronal plasticity. Because NF-kappaB may play roles in a range of neurological disorders that involve neuronal degeneration and/or perturbed synaptic function, pharmacological and genetic manipulations of NF-kappaB signaling are being developed that may prove valuable in treating disorders ranging from Alzheimer's disease to schizophrenia.

The calcium-binding protein calbindin D28k (CB) is expressed in limited subpopulations of neurons in the brain. In the hippocampus, CB is expressed in all dentate granule cells and a subpopulation of CA1 pyramidal neurons, but is absent from CA3 neurons. This pattern of CB expression is inversely correlated with neuronal vulnerability to seizure-induced damage suggesting the possibility that expression of CB confers resistance to excitotoxicity. While data from cell culture studies support an excitoprotective role for calbindin, it is not known whether CB is a key determinant of neuronal vulnerability in vivo. We therefore examined the pattern of damage to hippocampal neurons following intrahippocampal injection of the seizure-inducing excitotoxin kainate in CB homozygous (CB-/-) and CB heterozygous (CB+/-) knockout mice in comparison with wild-type mice (CB+/+). Whereas the extent of damage to CA1 neurons was similar in CB-/- and CB+/+ mice, damage to CA1 neurons was significantly reduced in CB+/- mice. Dentate granule neurons were not damaged following kainate-induced seizures in CB+/+, CB+/- or CB-/- mice. These findings suggest that CB can modify vulnerability of hippocampal CA1 neurons to seizure-induced injury, and that either CB is not a critical determinant of resistance of dentate granule neurons, or compensatory changes occur and lack of CB is not the only difference between CB-/- and CB+/+ mice.

The ultimate goal of Alzheimer's disease (AD) research is to prevent the onset of the neurodegenerative process and thereby allow successful aging without cognitive decline. Herein I argue that a simple and effective preventative approach for AD may be in hand. AD is a disorder associated with the aging process and is, accordingly, characterized by cellular and molecular changes that occur in age-related diseases in other organ systems. Such changes include increased levels of oxidative stress, perturbed energy metabolism, and accumulation of insoluble (oxidatively modified) proteins (prominent among which are amyloid beta-peptide and tau). The risk of several other prominent age-related disorders, including cardiovascular disease, cancer, and diabetes, is known to be influenced by the level of food intake--high food intake increases risk, and low food intake reduces risk. An overwhelming body of data from studies of rodents and monkeys has documented the profound beneficial effects of dietary restriction (DR) in extending life span and reducing the incidence of age-related diseases. Reduced levels of cellular oxidative stress and enhancement of energy homeostasis contribute to the beneficial effects of DR. Recent findings suggest that DR may enhance resistance of neurons in the brain to metabolic, excitotoxic, and oxidative insults relevant to the pathogenesis of AD and other neurodegenerative disorders. While further studies will be required to establish the extent to which DR will reduce the incidence of AD, it would seem prudent (based on existing data) to recommend DR as widely applicable preventative approach for age-related disorders including neurodegenerative disorders.

Previous studies have shown that several different neurotrophic factors can prevent death of cortical and hippocampal neurons induced by excitotoxic and oxidative insults in cell culture and in vivo. Because neuronal degeneration may be initiated by alterations occurring in synaptic compartments in disorders ranging from Alzheimer's disease to stroke, we tested the hypothesis that neurotrophic factors can exert direct protective actions at the level of the synapse. We now report that a nine amino acid bioactive fragment of activity-dependent neurotrophic factor (ADNF-9) enhances basal glucose and glutamate transport, and attenuates oxidative impairment of glucose and glutamate transport induced by amyloid beta-peptide and Fe(2+), in neocortical synaptosomes. Preservation of transporter function required only short-term (1-2 h) pretreatments. Basic fibroblast growth factor (bFGF) was also effective in suppressing oxidative impairment of synaptic transporter functions, while nerve growth factor (NGF) was less effective. Additional analyses showed that ADNF-9, bFGF and NGF suppress oxidative stress and mitochondrial dysfunction induced by amyloid beta-peptide and Fe(2+) in synaptosomes. Our data suggest that ADNF-9 can act locally in synaptic compartments to suppress oxidative stress and preserve function of glucose and glutamate transporters. Such synapto-protective actions suggest roles for activity-dependent trophic signaling in preventing degeneration of neuronal circuits, and indicate possible therapeutic applications of agents that stimulate local synaptic (transcription-independent) neurotrophic factor signaling pathways.

The transcription factor activator protein-1 (AP-1) is activated in response to physiological activity in neuronal circuits and in response to neuronal injury associated with various acute and chronic neurodegenerative conditions. The membrane lipid peroxidation product 4-hydroxy-2,3-nonenal (HNE) is increasingly implicated in the disruption of neuronal calcium homeostasis that occurs in various paradigms of neuronal excitotoxicity and apoptosis. The possible mechanistic links between lipid peroxidation and alterations in gene transcription during neuronal apoptosis have not previously been examined. We now report that exposure of cultured rat cortical neurons to an apoptotic concentration of HNE results in a large increase in AP-1 DNA-binding activity. The protein synthesis inhibitor cycloheximide blocked the induction of AP-1, consistent with a requirement for induction of expression of AP-1 family members. The broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde blocked HNE-induced increases in AP-1 DNA-binding activity, demonstrating a requirement for caspase activation in the activation of AP-1. HNE induced phosphorylation of c-Jun N-terminal kinase (JNK), which was prevented by caspase inhibitors, indicating that HNE was acting at or upstream of JNK phosphorylation. The intracellular calcium chelator BAPTA-acetoxymethyl ester completely prevented stimulation of AP-1 DNA-binding by HNE, indicating a requirement for calcium. Moreover, agents that suppress mitochondrial calcium uptake (ruthenium red) and membrane permeability transition (cyclosporin A) attenuated AP-1 activation by HNE, suggesting a contribution of mitochondrial alterations to AP-1 activation. Collectively, our data suggest a scenario in which HNE disrupts neuronal calcium homeostasis and perturbs mitochondrial function, resulting in caspase activation. Activated caspases, in turn, induce activation of JNK, resulting in stimulation of AP-1 DNA-binding protein production. This transcriptional pathway induced by HNE may modulate the cell death process.

Alzheimer's disease (AD) has a substantial inflammatory component, and activated microglia may play a central role in neuronal degeneration. CD40 expression was increased on cultured microglia treated with freshly solublized amyloid-beta (Abeta, 500 nanomolar) and on microglia from a transgenic murine model of AD (Tg APPsw). Increased tumor necrosis factor alpha production and induction of neuronal injury occurred when Abeta-stimulated microglia were treated with CD40 ligand (CD40L). Microglia from Tg APPsw mice deficient for CD40L demonstrated reduction in activation, suggesting that the CD40-CD40L interaction is necessary for Abeta-induced microglial activation. Finally, abnormal tau phosphorylation was reduced in Tg APPsw animals deficient for CD40L, suggesting that the CD40-CD40L interaction is an early event in AD pathogenesis.

Activity-dependent neurotrophic factor (ADNF) and a 14-amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid beta-peptide, N-methyl-D-aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine-amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Lipolytic products of triglyceride-rich lipoproteins, i.e., free fatty acids, may cause activation and dysfunction of the vascular endothelium. Mechanisms of these effects may include lipid peroxidation. One of the major and biologically active products of peroxidation of n-6 fatty acids, such as linoleic acid or arachidonic acid, is the aldehyde 4-hydroxynonenal (HNE). To study the hypothesis that HNE may be a critical factor in endothelial cell dysfunction caused by free fatty acids, human umbilical endothelial cells (HUVEC) were treated with up to160 microM of linoleic or arachidonic acid. HNE formation was detected by immunocytochemistry in cells treated for 24 h with either fatty acid, but more markedly with arachidonic acid. To study the cellulareffects of HNE, HUVEC were treated with different concentrations of this aldehyde, and several markers of endothelial cell dysfunction were determined. Exposure to HNE for 6 and 9 h resulted in increased cellular oxidative stress. However, short time treatment with HNE did not cause activation of nuclear factor-kappaB (NF-kappaB). In addition, HUVEC exposure to HNE caused a dose-dependent decrease in production of both interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). On the other hand, HNE exerted prominent cytotoxic effects in cultured HUVEC, manifested by morphological changes, diminished cellular viability, and impaired endothelial barrier function. Furthermore, HNE treatment induced apoptosis of HUVEC. These data provide evidence that HNE does not contribute to NF-kappaB-related mechanisms of the inflammatory response in HUVEC, but rather to endothelial dysfunction, cytotoxicity, and apoptotic cell death., (Copyright 1999 Wiley-Liss, Inc.)

Nuclear factor-kappaB (NF-kappaB) is activated in brain cells after various insults, including cerebral ischemia and epileptic seizures. Although cell culture studies have suggested that the activation of NF-kappaB can prevent neuronal apoptosis, the role of this transcription factor in neuronal injury in vivo is unclear, and the specific kappaB subunits involved are unknown. We now report that mice lacking the p50 subunit of NF-kappaB exhibit increased damage to hippocampal pyramidal neurons after administration of the excitotoxin kainate. Gel-shift analyses showed that p50 is required for the majority of kappaB DNA-binding activity in hippocampus. Intraventricular administration of kappaB decoy DNA before kainate administration in wild-type mice resulted in an enhancement of damage to hippocampal pyramidal neurons, indicating that reduced NF-kappaB activity was sufficient to account for the enhanced excitotoxic neuronal injury in p50(-/-) mice. Cultured hippocampal neurons from p50(-/-) mice exhibited enhanced elevations of intracellular calcium levels and increased levels of oxidative stress after exposure to glutamate and were more vulnerable to excitotoxicity than were neurons from p50(+/+) and p50(+/-) mice. Collectively, our data demonstrate an important role for the p50 subunit of NF-kappaB in protecting neurons against excitotoxic cell death.

Apoptosis is a form of cell death historically defined by morphological and biochemical changes that occur in the cell body and nucleus. However, in contrast to nonneuronal cells in which apoptosis has been most intensively studied, neurons exhibit elaborate morphologies with synaptic connections often located at sites a great distance from the cell body. Signaling events occurring in synaptic terminals are believed to play important roles in either promoting (e.g., activation of glutamate receptors in postsynaptic spines) or preventing (e.g., activation of neurotrophic factors in presynaptic terminals) neuronal cell death in various physiological and pathological settings. We have found that apoptotic biochemical cascades can be activated locally in synaptic terminals and neurites and have shown that such cascades can result in local functional and morphological alterations and can also propagate to the cell body resulting in neuronal death. Prostate apoptosis response-4 production, caspase activation, loss of plasma membrane phospholipid asymmetry, mitochondrial dysfunction, and production of factors capable of inducing nuclear chromatin condensation and fragmentation can all occur locally in synaptic terminals in response to various stimuli. Activation of receptors for neurotrophic factors (e.g., basic fibroblast growth factor, secreted form of amyloid precursor protein alpha, and activity-dependent neurotrophic factor) and cytokines (e.g., tumor necrosis factor-alpha) in synaptic terminals can exert synaptoprotective actions that either can be transduced locally or may require signals to the nucleus and back. In addition to their roles in synaptic degeneration and neuron death, apoptotic cascades may play roles in synaptic plasticity. For example, we found that caspase activation can lead to proteolysis of certain glutamate receptor subunits and that this action of capases is correlated with reduced calcium responses to glutamate. We propose that apoptotic cascades function in a continuum in which low levels of activation play roles in adaptive responses to "stressors," whereas higher levels of activation mediate synaptic degeneration and cell death., (Copyright 1999 Wiley-Liss, Inc.)

The localization of signal transduction machinery at synapses is a fundamental organizational feature of the nervous system that allows for highly complex integration of information coding processes. Synaptic communication evolved as multicellular organisms became more complex, and as selection pressures were placed on such organisms such that those capable of responding rapidly and specifically to environmental demands survived. Two obvious advantages of synaptic transmission (as opposed to endocrine or paracrine signaling) are that it provides for rapid intercellular communication over great distances and that it provides a high level of spatial specificity. There are several structural and functional aspects of synapses that set them apart from other cellular compartments, with many of the specializations subserving roles in synaptic signal transduction (e.g., neurotransmitter release from the presynaptic terminal and postsynaptic receptor activation and second messenger production). However, studies of developing nervous systems have shown that many synaptic signaling mechanisms are operative prior to synaptogenesis and play important roles in regulating growth cone behaviors, synaptogenesis, and even programmed cell death. Indeed, the concept that "ontogeny recapitulates phylogeny" can be effectively applied to the evolution of the synapse. As the embryo rapidly grows, neurons must elaborate axons and dendrites, establish functional synaptic connections, and maintain and adjust those connections as the organism matures. The purpose of this introductory article is to set the stage for the following articles by briefly reviewing fundamental aspects of the molecular and cellular biology of synapses in an evolutionary context., (Copyright 1999 Wiley-Liss, Inc.)

Dysfunction and death of midbrain dopaminergic neurons underlies the clinical features of Parkinson's disease (PD). Increasing evidence suggests roles for oxidative stress and a form of cell death called apoptosis in the pathogenesis of PD. We recently identified a 38-kd protein called prostate apoptosis response-4 (Par-4), which is rapidly induced in cultured neurons after exposure to apoptotic insults, and appears to play a necessary role in the cell death process. We now report that Par-4 levels increase dramatically in midbrain dopaminergic neurons of monkeys and mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The increase in Par-4 levels occurs in both neuronal cell bodies in the substantia nigra and their axon terminals in the striatum, and precedes loss of tyrosine hydroxylase immunoreactivity and cell death. In the monkey model, Par-4 levels were also increased in several brain regions (red nucleus, lateral geniculate nucleus, and cerebral cortex) in which functional alterations have previously been documented in PD patients and MPTP-treated monkeys. Exposure of cultured human dopaminergic neural cells to the complex I inhibitor rotenone, or to Fe2+, resulted in Par-4 induction, mitochondrial dysfunction, and subsequent apoptosis. Blockade of Par-4 induction by antisense treatment prevented rotenone- and Fe2+-induced mitochondrial dysfunction and apoptosis demonstrating a critical role for Par-4 in the cell death process. The data suggest that Par-4 may be involved in the neurodegenerative process in PD.

Neurons are an unusual type of cell in that they send processes (axons and dendrites) over great distances. This elaborate morphology, together with their excitability, places neurons at risk for multiple insults. Recent studies have demonstrated that apoptotic and excitotoxic mechanisms not only contribute to neuronal death, but also to synaptic dysfunction and a breakdown in neural circuitry (see Mattson and Duan [1999] J. Neurosci. Res. 58:152-166, this issue). Proteases of the caspase and calpain families have been implicated in neurodegenerative processes, as their activation can be triggered by calcium influx and oxidative stress. Caspases and calpains are cysteine proteases that require proteolytic cleavage for activation. The substrates cleaved by caspases include cytoskeletal and associated proteins, kinases, members of the Bcl-2 family of apoptosis-related proteins, presenilins and amyloid precursor protein, and DNA-modulating enzymes. Calpain substrates include cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. Many of the substrates of caspases and calpains are localized in pre- and/or postsynaptic compartments of neurons. Emerging data suggest that, in addition to their roles in neurodegenerative processes, caspases and calpains play important roles in modulating synaptic plasticity. The present article provides a review of the properties of the different caspases and calpains, their roles in cell death pathways, and the substrates upon which they act. Emerging data are considered that suggest key roles for these proteases in the regulation of synaptic plasticity., (Copyright 1999 Wiley-Liss, Inc.)

Alzheimer's disease (AD) is an age-related disorder that involves degeneration of synapses and neurons in brain regions involved in learning and memory processes. Some cases of AD are caused by mutations in presenilin-1 (PS1), an integral membrane protein located in the endoplasmic reticulum. Previous studies have shown that PS1 mutations increase neuronal vulnerability to excitotoxicity and apoptosis. Although dietary restriction (DR) can increase lifespan and reduce the incidence of several age-related diseases in rodents, the possibility that DR can modify the pathogenic actions of mutations that cause AD has not been examined. The vulnerability of hippocampal neurons to excitotoxic injury was increased in PS1 mutant knockin mice. PS1 mutant knockin mice and wild-type mice maintained on a DR regimen for 3 months exhibited reduced excitotoxic damage to hippocampal CA1 and CA3 neurons compared to mice fed ad libitum; the DR regimen completely counteracted the endangering effect of the PS1 mutation. The magnitude of increase in levels of the lipid peroxidation product 4-hydroxynonenal following the excitotoxic insult was lower in DR mice compared to mice fed ad libitum, suggesting that suppression of oxidative stress may be one mechanism underlying the neuroprotective effect of DR. These findings indicate that the neurodegeneration-promoting effect of an AD-linked mutation is subject to modification by diet.

Stroke, an age-related disorder involving degeneration of neurons resulting from cerebral ischemia, is a major cause of disability and mortality. Although dietary restriction (DR) extends lifespan and reduces levels of cellular oxidative stress in several different organ systems including the brain, the impact of DR on ischemic brain injury is unknown. We report that maintenance of adult rats on a DR regimen resulted in reduced brain damage and improved behavioral outcome in a middle cerebral artery occlusion-reperfusion (MCAO-R) stroke model. Administration of 2-deoxyglucose (2-DG), a nonmetabolizable analogue of glucose, to rats fed ad libitum resulted in reduced ischemic brain damage and improved behavioral outcome following MCAO-R. 2-DG protected cultured hippocampal neurons against chemical hypoxia, demonstrating a direct protective action on neurons. DR and 2-DG administration resulted in an increase in the level of the stress protein heat-shock protein 70 (HSP-70) in striatal cells in vivo, and 2-DG treatment induced HSP-70 in cultured neurons suggesting involvement of a preconditioning stress response in the neuroprotective actions of DR and 2-DG. The neuroprotective effect of DR and 2-DG in this focal cerebral ischemia model suggests that outcome following stroke may be improved in individuals who follow a regimen of reduced food intake., (Copyright 1999 Wiley-Liss, Inc.)

This study examined the accumulation of the cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE) after lateral fluid percussion (FP) brain injury in rats. A diffuse distribution of HNE-immunoreactivity (HNE-ir) was observed in the cortex and hippocampus of the ipsilateral, but not of the contralateral, hemisphere at 30 min, 6 h, 24 h, and 48 h after brain injury. The HNE-ir was well-localized in cell bodies of the ipsilateral cortex and the CA3 pyramidal layer in the ipsilateral hippocampus. Because HNE's interaction with certain proteins causes protein dysfunction and HNE, in vitro, causes neuronal cell damage, the present results suggest that HNE's interaction with neuronal proteins may contribute to neuronal damage in the ipsilateral cortex and hippocampus after brain injury.

The rapid increase in basic fibroblast growth factor (bFGF) production following spinal cord injury (SCI) in rats is thought to serve a role in the cellular processes responsible for the functional recovery often observed. In this study, bFGF was intrathecally administered continuously for 1 week beginning 30 min after a moderate (12.5 mm) spinal cord contusion in adult rats using the New York University impactor device. Osmotic minipumps were implanted into the lateral ventricle and lumbar thecal sac to deliver bFGF at a rate of 3 microg or 6 microg per day versus control vehicle. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and histologically assessed for both tissue sparing and glial reactivity rostral and caudal to the lesion. Rats treated with bFGF regained coordinated hindlimb movements earlier than controls and demonstrated consistent coordination from 4 to 6 weeks. Vehicle-treated rats showed only modest improvements in hindlimb function. The amount of spared tissue was significantly higher in bFGF-treated rats than in controls. Astrocyte and microglial reactivity was more pronounced in bFGF-treated animals versus controls. In summary, intrathecal infusion of exogenous bFGF following SCI significantly reduces tissue damage and enhances functional recovery. Early pharmacological intervention with bFGF following SCI may serve a neuroprotective role and/or create a proregenerative environment, possibly by modulating the neuroglial response.

A fundamental feature of neurons is that they possess a polarized morphology, typified by a single long axon and several short dendrites. This cellular polarity forms the basis for directionalized rapid signaling, and for bi-directional trophic signaling, in neuronal circuits. While a catalog of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in the establishment of neuronal polarity (also referred to as axogenesis) are not well understood. Cytoskeletal components, including microtubules and actin filaments, and cytoskeleton-regulating proteins such as microtubule-associated proteins (MAPs), actin-binding proteins, and "motor" proteins, likely play important roles in the establishment and maintenance of neuronal polarity. Polarized sorting of membranes and proteins occurs in neurons, and a better understanding of such sorting mechanisms will likely provide insight into the process of axogenesis. Calcium plays a pivotal role in the regulation of neurite elongation and growth cone motility. Studies in which intracellular calcium levels are measured and manipulated in embryonic neurons during the process of axogenesis suggest a necessary role for calcium gradients in the establishment of neuronal polarity. Experimental models in which developing neurons are induced to switch their polarity support central roles for calcium-regulated cytoskeletal reorganization and protein sorting in the process of axogenesis. Finally, recent findings suggest that mitochondria play an important role in organizing neuronal polarity, possibly by controlling local calcium and/or energy gradients. While considerable progress is being made in elucidating mechanisms that regulate neuronal polarity, the seminal event(s) underlying this process remain a mystery. J. Neurosci. Res. 57:577-589., (Copyright 1999 Wiley-Liss, Inc.)

Apolipoprotein E (apoE)-related synthetic peptides, the 22 kDa N-terminal thrombin-cleavage fragment of apoE (truncated apoE), and full-length apoE have all been shown to exhibit neurotoxic activity under certain culture conditions. In the present study, protease inhibitors reduced the neurotoxicity and proteolysis of full-length apoE but did not block the toxicity of truncated apoE or a synthetic apoE peptide, suggesting that fragments of apoE may account for its toxicity. Additional experiments demonstrated that both truncated apoE and the apoE peptide elicit an increase in intracellular calcium levels and subsequent death of embryonic rat hippocampal neurons in culture. Similar effects on calcium were found when the apoE peptide was applied to chick sympathetic neurons. The rise in intracellular calcium and the hippocampal cell death caused by the apoE peptide were significantly reduced by receptor-associated protein, removal of extracellular calcium, or administration of the specific NMDA glutamate receptor antagonist MK-801. These results suggest that apoE may be a source of both neurotoxicity and calcium influx that involves cell surface receptors. Such findings strengthen the hypothesis that apoE plays a direct role in the pathology of Alzheimer's disease.

Synaptic dysfunction and degeneration are believed to underlie the cognitive deficits that characterize Alzheimer's disease, and overactivation of glutamate receptors under conditions of increased oxidative stress and metabolic compromise may contribute to the neurodegenerative process in many different disorders. The secreted form of amyloid precursor protein (sAPPalpha), which is released from neurons in an activity-dependent manner, can modulate neurite outgrowth, synaptic plasticity, and neuron survival. We now report that sAPPalpha can enhance glucose and glutamate transport in synaptic compartments. Treatment of cortical synaptosomes with nanomolar concentrations of sAPPalpha resulted in an attenuation of impairment of glutamate and glucose transport induced by exposure to amyloid beta-peptide and Fe2+. The protective effect of sAPPalpha was mimicked by treatment with 8-bromo-cyclic GMP and blocked by a cyclic GMP-dependent protein kinase inhibitor, suggesting that protective action of sAPPalpha is mediated by cyclic GMP. Our data suggest that glucose and glutamate transport can be regulated locally at the level of the synapse and further suggest important roles for sAPPalpha and cyclic GMP in modulating synaptic physiology under normal and pathophysiological conditions.

Patients with Alzheimer's disease (AD) exhibit alterations in glucose metabolism and dysregulation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) neuroendocrine system. The mechanisms responsible for these alterations and their possible contributions to the neurodegenerative process in AD are unknown. We now report that transgenic mice expressing a mutant form of human amyloid precursor protein (APP) that causes inherited early-onset AD exhibit increased sensitivity to physiological stressors, which is associated with aberrancies in HPA function and regulation of blood glucose levels. Specifically, APP mutant mice exhibit severe hypoglycemia and death following food restriction, and sustained elevations of plasma glucocorticoid levels and hypoglycemia following restraint stress. The alterations in HPA function and glucose regulation were evident in relatively young mice prior to overt deposition of amyloid beta-peptide (A beta). However, diffuse accumulations of A beta were present in the hypothalamus of older mice, suggesting a role for soluble forms of A beta in dysregulation of HPA function. Our data demonstrate disturbances in neuroendocrine function in APP mutant mice similar to those seen in AD patients. These impairments in stress response, glucocorticoid signaling, and regulation of blood glucose should be considered in interpretations of data from past and future studies of APP mutant mice.

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein initially identified as the product of a gene upregulated in prostate tumor cells undergoing apoptosis. Par-4 contains both a death domain and a leucine zipper domain, and has been shown to interact with several proteins known to modulate apoptosis, including protein kinase Czeta, Bcl-2, and caspase-8. A rapid increase in Par-4 levels occurs in neurons undergoing apoptosis in a variety of paradigms, including trophic factor withdrawal, and exposure to oxidative and metabolic insults. Par-4, which can be induced at the translational level, acts at an early stage of the apoptotic cascade prior to caspase activation and mitochondrial dysfunction. The mechanism whereby Par-4 promotes apoptosis may involve inhibition of the antiapoptotic transcription factor NF-kappaB and suppression of Bcl-2 expression and/or function. Studies of postmortem tissues from patients and animal models of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, amyotrophic lateral sclerosis (ALS), and HIV encephalitis, have documented increased levels of Par-4 in vulnerable neurons. Manipulations that block Par-4 expression or function prevent neuronal cell death in models of each disorder, suggesting a critical role for Par-4 in the neurodegenerative process. Interestingly, Par-4 levels rapidly increase in synaptic terminals following various insults, and such local increases in Par-4 levels appear to play important roles in synaptic dysfunction and degeneration. A better understanding of the molecular and cellular biology of Par-4 will help clarify mechanisms of neuronal apoptosis, and may lead to the development of novel preventative and therapeutic strategies for neurodegenerative disorders.

In Alzheimer's disease (AD) synapses degenerate and neurons die in brain regions involved in learning and memory processes. Although the cellular and molecular mechanisms underlying the neurodegenerative process in AD are unclear, increasing evidence suggests roles for amyloid beta-peptide (Abeta) and biochemical cascades associated with a form of programmed cell death called apoptosis. Cysteine proteases of the caspase family are activated in neurons undergoing apoptosis and apparently play a major role in the cell death process by cleaving yet-to-be-identified substrates. We now report that caspase activity is increased in brain tissue and neurons from AD patients, and in cultured hippocampal neurons undergoing apoptosis after exposure to amyloid beta-peptide (Abeta). Western blot analyses using antibodies against different subunits of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types of ionotropic glutamate receptors indicate that AMPA receptor subunits (GluR1, GluR2/3, and GluR4), but not NMDA receptor subunits (NR1 and NR2A), are proteolytically cleaved after exposure of hippocampal neurons to apoptotic insults, including Abeta, and that the caspase inhibitor zVAD-fmk suppresses such cleavage. Western blot analysis of brain tissue from AD patients and age-matched controls revealed evidence for increased proteolysis of AMPA receptor subunits in AD. Our data suggest roles for caspase-mediated cleavage of AMPA receptor subunits in modifying neuronal responsivity to glutamate and in the neurodegenerative process in AD., (Copyright 1999 Wiley-Liss, Inc.)

Spinal cord trauma can cause a marked release of free fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free fatty acids, and AA by itself, may lead to secondary damage to spinal cord neurons. To study this hypothesis, cultured spinal cord neurons were exposed to increasing concentrations of AA (0.01-10 microM). AA-induced injury to spinal cord neurons was assessed by measurements of cellular oxidative stress, intracellular calcium levels, activation of nuclear factor-KB (NF-kappaB), and cell viability. AA treatment increased intracellular calcium concentrations and decreased cell viability. Oxidative stress increased significantly in neurons exposed to 1 and 10 microM AA. In addition, AA treatment activated NF-kappaB and decreased levels of the inhibitory subunit, IKB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.

Tumor necrosis factor alpha (TNFalpha) is widely expressed in both neurons and glia and has been shown to be upregulated after traumatic brain injury (TBI). TNFalpha receptor activation results in activation of the transcription factor nuclear factor kappaB (NF-kappaB), which may serve an antiapoptotic role via the induction of target genes manganese superoxide dismutase (MnSOD) and/or calbindin. In the present study, we used a controlled cortical impact model of TBI with pertinent lines of transgenic mice to combine both morphological characterization and molecular analysis to elucidate the role of TNFalpha after TBI. Measurements of both the lesion volume and the blood-brain barrier breach indicated exacerbations in mice rendered genetically deficient in both the p55 and p75 TNFalpha receptors (TNFR-KO) compared with wild-type animals. Additionally, animals genetically altered to overexpress MnSOD showed a significant decrease in lesion volume compared with that of control littermates, whereas no alterations were observed in mice lacking the calcium-binding protein calbindin D28k. Analysis of NF-kappaB activation and relative levels of MnSOD revealed delayed responses in the injured cortex of TNFR-KO animals compared with wild-type animals, implying that endogenous TNFalpha may be neuroprotective after TBI.

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.