Showing papers by "Mark P. Mattson published in 2003"

Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake

Abstract: Dietary restriction has been shown to have several health benefits including increased insulin sensitivity, stress resistance, reduced morbidity, and increased life span. The mechanism remains unknown, but the need for a long-term reduction in caloric intake to achieve these benefits has been assumed. We report that when C57BL/6 mice are maintained on an intermittent fasting (alternate-day fasting) dietary-restriction regimen their overall food intake is not decreased and their body weight is maintained. Nevertheless, intermittent fasting resulted in beneficial effects that met or exceeded those of caloric restriction including reduced serum glucose and insulin levels and increased resistance of neurons in the brain to excitotoxic stress. Intermittent fasting therefore has beneficial effects on glucose regulation and neuronal resistance to injury in these mice that are independent of caloric intake.

Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders.

Abstract: Activation of glutamate receptors can trigger the death of neurons and some types of glial cells, particularly when the cells are coincidentally subjected to adverse conditions such as reduced levels of oxygen or glucose, increased levels of oxidative stress, exposure to toxins or other pathogenic agents, or a disease-causing genetic mutation. Such excitotoxic cell death involves excessive calcium influx and release from internal organelles, oxyradical production, and engagement of programmed cell death (apoptosis) cascades. Apoptotic proteins such as p53, Bax, and Par-4 induce mitochondrial membrane permeability changes resulting in the release of cytochrome c and the activation of proteases, such as caspase-3. Events occurring at several subcellular sites, including the plasma membrane, endoplasmic reticulum, mitochondria and nucleus play important roles in excitotoxicity. Excitotoxic cascades are initiated in postsynaptic dendrites and may either cause local degeneration or plasticity of those synapses, or may propagate the signals to the cell body resulting in cell death. Cells possess an array of anti-excitotoxic mechanisms including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins, and inhibitor of apoptosis proteins. Considerable evidence supports roles for excitotoxicity in acute disorders such as epileptic seizures, stroke and traumatic brain and spinal cord injury, as well as in chronic age-related disorders such as Alzheimer’s, Parkinson’s, and Huntington’s disease and amyotrophic lateral sclerosis. A better understanding of the excitotoxic process is not only leading to the development of novel therapeutic approaches for neurodegenerative disorders, but also to unexpected insight into mechanisms of synaptic plasticity.

Calcium orchestrates apoptosis.

Abstract: Apoptosis is a feature of many diseases and is critical for the sculpting and maintenance of tissues. New work demonstrates that calcium released from the endoplasmic reticulum synchronizes the mass exodus of cytochrome c from the mitochondria, a phenomenon that coordinates apoptosis.

Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice

Abstract: In addition to neurological deficits, Huntington's disease (HD) patients and transgenic mice expressing mutant human huntingtin exhibit reduced levels of brain-derived neurotrophic factor, hyperglycemia, and tissue wasting. We show that the progression of neuropathological (formation of huntingtin inclusions and apoptotic protease activation), behavioral (motor dysfunction), and metabolic (glucose intolerance and tissue wasting) abnormalities in huntingtin mutant mice, an animal model of HD, are retarded when the mice are maintained on a dietary restriction (DR) feeding regimen resulting in an extension of their life span. DR increases levels of brain-derived neurotrophic factor and the protein chaperone heat-shock protein-70 in the striatum and cortex, which are depleted in HD mice fed a normal diet. The suppression of the pathogenic processes by DR in HD mice suggests that mutant huntingtin promotes neuronal degeneration by impairing cellular stress resistance, and that the body wasting in HD is driven by the neurodegenerative process. Our findings suggest a dietary intervention that may suppress the disease process and increase the life span of humans that carry the mutant huntingtin gene.

Glucagon-like peptide-1 decreases endogenous amyloid-β peptide (Aβ) levels and protects hippocampal neurons from death induced by Aβ and iron

Abstract: Glucagon-like peptide-1(7-36)-amide (GLP-1) is an endogenous insulinotropic peptide that is secreted from the gastrointestinal tract in response to food. It enhances pancreatic islet beta-cell proliferation and glucose-dependent insulin secretion and lowers blood glucose and food intake in patients with type 2 diabetes mellitus. GLP-1 receptors, which are coupled to the cyclic AMP second messenger pathway, are expressed throughout the brains of rodents and humans. It was recently reported that GLP-1 and exendin-4, a naturally occurring, more stable analogue of GLP-1 that binds at the GLP-1 receptor, possess neurotrophic properties and can protect neurons against glutamate-induced apoptosis. We report here that GLP-1 can reduce the levels of amyloid-beta peptide (Abeta) in the brain in vivo and can reduce levels of amyloid precursor protein (APP) in cultured neuronal cells. Moreover, GLP-1 and exendin-4 protect cultured hippocampal neurons against death induced by Abeta and iron, an oxidative insult. Collectively, these data suggest that GLP-1 can modify APP processing and protect against oxidative injury, two actions that suggest a novel therapeutic target for intervention in Alzheimer's disease.

Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport

Abstract: Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimer's disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3beta (GSK3beta). In vivo, GSK3beta phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/- and PS1 knock-inM146V (KIM146V) mice and cultured cells. We show that relative levels of GSK3beta activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3beta activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146V mice. Thus PS1 appears to modulate GSK3beta activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.

Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms

Abstract: Although all cells in the body require energy to survive and function properly, excessive calorie intake over long time periods can compromise cell function and promote disorders such as cardiovascular disease, type-2 diabetes and cancers. Accordingly, dietary restriction (DR; either caloric restriction or intermittent fasting, with maintained vitamin and mineral intake) can extend lifespan and can increase disease resistance. Recent studies have shown that DR can have profound effects on brain function and vulnerability to injury and disease. DR can protect neurons against degeneration in animal models of Alzheimer's, Parkinson's and Huntington's diseases and stroke. Moreover, DR can stimulate the production of new neurons from stem cells (neurogenesis) and can enhance synaptic plasticity, which may increase the ability of the brain to resist aging and restore function following injury. Interestingly, increasing the time interval between meals can have beneficial effects on the brain and overall health of mice that are independent of cumulative calorie intake. The beneficial effects of DR, particularly those of intermittent fasting, appear to be the result of a cellular stress response that stimulates the production of proteins that enhance neuronal plasticity and resistance to oxidative and metabolic insults; they include neurotrophic factors such as brain-derived neurotrophic factor (BDNF), protein chaperones such as heat-shock proteins, and mitochondrial uncoupling proteins. Some beneficial effects of DR can be achieved by administering hormones that suppress appetite (leptin and ciliary neurotrophic factor) or by supplementing the diet with 2-deoxy-d-glucose, which may act as a calorie restriction mimetic. The profound influences of the quantity and timing of food intake on neuronal function and vulnerability to disease have revealed novel molecular and cellular mechanisms whereby diet affects the nervous system, and are leading to novel preventative and therapeutic approaches for neurodegenerative disorders.

Membrane properties of rat embryonic multipotent neural stem cells.

Abstract: We have characterized several potential stem cell markers and defined the membrane properties of rat fetal (E10.5) neural stem cells (NSC) by immunocytochemistry, electrophysiology and microarray analysis. Immunocytochemical analysis demonstrates specificity of expression of Sox1, ABCG2/Bcrp1, and shows that nucleostemin labels both progenitor and stem cell populations. NSCs, like hematopoietic stem cells, express high levels of aldehyde dehydrogenase (ALDH) as assessed by Aldefluor labeling. Microarray analysis of 96 transporters and channels showed that Glucose transporter 1 (Glut1/Slc2a1) expression is unique to fetal NSCs or other differentiated cells. Electrophysiological examination showed that fetal NSCs respond to acetylcholine and its agonists, such as nicotine and muscarine. NSCs express low levels of tetrodotoxin (TTX) sensitive and insensitive sodium channels and calcium channels while expressing at least three kinds of potassium channels. We find that gap junction communication is mediated by connexin (Cx)43 and Cx45, and is essential for NSC survival and proliferation. Overall, our results show that fetal NSCs exhibit a unique signature that can be used to determine their location and assess their ability to respond to their environment.

Reversal of behavioral and metabolic abnormalities, and insulin resistance syndrome, by dietary restriction in mice deficient in brain-derived neurotrophic factor.

Abstract: Dietary restriction (DR) extends life span and improves glucose metabolism in mammals. Recent studies have shown that DR stimulates the production of brain-derived neurotrophic factor (BDNF) in brain cells, which may mediate neuroprotective and neurogenic actions of DR. Other studies have suggested a role for central BDNF signaling in the regulation of glucose metabolism and body weight. BDNF heterozygous knockout (BDNF+/-) mice are obese and exhibit features of insulin resistance. We now report that an intermittent fasting DR regimen reverses several abnormal phenotypes of BDNF(+/-) mice including obesity, hyperphagia, and increased locomotor activity. DR increases BDNF levels in the brains of BDNF(+/-) mice to the level of wild-type mice fed ad libitum. BDNF(+/-) mice exhibit an insulin-resistance syndrome phenotype characterized by elevated levels of circulating glucose, insulin, and leptin; DR reduces levels of each of these three factors. DR normalizes blood glucose responses in glucose tolerance and insulin tolerance tests in the BDNF(+/-) mice. These findings suggest that BDNF is a major regulator of energy metabolism and that beneficial effects of DR on glucose metabolism are mediated, in part, by BDNF signaling. Dietary and pharmacological manipulations of BDNF signaling may prove useful in the prevention and treatment of obesity and insulin resistance syndrome-related diseases.

Intermittent Food Deprivation Improves Cardiovascular and Neuroendocrine Responses to Stress in Rats

Abstract: Stressful events may trigger disease processes in many different organ systems, with the cardiovascular system being particularly vulnerable. Five-mo-old male rats had ad libitum (AL) access to food or were deprived of food every other day [intermittent food deprivation (IF)] for 6 mo, during which time their heart rate (HR), blood pressure (BP), physical activity and body temperature were measured by radiotelemetry under nonstress and stress (immobilization or cold-water swim) conditions. IF rats had significantly lower basal HR and BP, and significantly lower increases in HR and BP after exposures to the immobilization and swim stressors. Basal levels of adrenocorticotropic hormone (ACTH) and corticosterone were greater in the IF rats. However, in contrast to large stress-induced increases in ACTH, corticosterone and epinephrine levels in AL rats, increases in these hormones in response to repeated immobilization stress sessions were reduced or absent in IF rats. Nevertheless, the IF rats exhibited robust hypothalamic/pituitary and sympathetic neuroendocrine responses to a different stress (swim). The IF treatment improved glucose metabolism, as indicated by lower basal levels of circulating glucose and insulin, but with maintenance of glucose and insulin responses to stress. We concluded that improvements in cardiovascular risk factors and cardiovascular and neuroendocrine stress adaptation occur in response to IF.

RNA Interference in Biology and Medicine

Abstract: First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complex that degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins--RNAi technology may eventually make that dream a reality.

Intermittent fasting and dietary supplementation with 2-deoxy-d-glucose improve functional and metabolic cardiovascular risk factors in rats

Abstract: Hypertension and insulin resistance syndrome are risk factors for cardiovascular disease, and it is therefore important to identify interventions that can reduce blood pressure and improve glucose metabolism. We performed experiments aimed at determining whether intermittent fasting (IF) can improve cardiovascular health and also tested the hypothesis that beneficial effects of IF can be mimicked by dietary supplementation with 2-deoxy-D-glucose (2DG) a non-metabolizable glucose analog. Four-month-old male rats were implanted with telemetry probes to allow continuous monitoring of heart rate, blood pressure, physical activity, and body temperature. Rats were then maintained for 6 months on one of three different dietary regimens: ad libitum feeding, IF, or 2DG supplementation. Rats on the IF regimen consumed 30% less food over time and had reduced body weights compared with rats fed ad libitum, whereas rats on the 2DG regimen did not reduce their food intake and maintained their body weight. Heart rate and blood pressure were significantly decreased within 1 month in rats on IF and 2DG diets and were maintained at reduced levels thereafter. Body temperature was significantly decreased in group IF, but not in group 2DG. Levels of serum glucose and insulin were significantly decreased in rats maintained on IF and 2DG-supplemented diets, suggesting that IF and 2DG diets affect insulin sensitivity in a similar manner. Finally, rats in groups IF and 2DG exhibited increased levels of plasma adrenocorticotropin and corticosterone, indicating that these diets induced a stress response. We conclude that reductions in blood pressure, heart rate, and insulin levels, similar to or greater than those obtained with regular physical exercise programs, can be achieved by IF and by dietary supplementation with 2DG by a mechanism involving stress responses.

Gene-diet interactions in brain aging and neurodegenerative disorders.

Abstract: While there are many examples of people who live for 100 years or more with little evidence of a decline in brain function, many others are not so fortunate and experience a neurodegenerative disorder, such as Alzheimer disease or Parkinson disease. Although an increasing number of genetic factors that may affect the risk for neurodegenerative disorders are being identified, emerging findings suggest that dietary factors play major roles in determining whether the brain ages successfully or experiences a neurodegenerative disorder. Dietary factors may interact with disease-causing or predisposing genes in molecular cascades that either promote or prevent the degeneration of neurons. Epidemiologic findings suggest that high-calorie diets and folic acid deficiency increase the risk for Alzheimer disease and Parkinson disease; studies of animal models of these disorders have shown that dietary restriction (reduced calorie intake or intermittent fasting) and dietary supplementation with folic acid can reduce neuronal damage and improve behavioral outcome. Animal studies have shown that the beneficial effects of dietary restriction on the brain result in part from increased production of neurotrophic factors and cytoprotective protein chaperones in neurons. By keeping homocysteine levels low, folic acid can protect cerebral vessels and prevent the accumulation of DNA damage in neurons caused by oxidative stress and facilitated by homocysteine. Although additional studies are required in humans, the emerging data suggest that high-calorie diets and elevated homocysteine levels may render the brain vulnerable to age-related neurodegenerative disorders, particularly in persons with a genetic predisposition to such disorders.

Will caloric restriction and folate protect against AD and PD

Abstract: Recent epidemiologic studies of different sample populations have suggested that the risk of AD and PD may be increased in individuals with high-calorie diets and in those with increased homocysteine levels. Dietary restriction and supplementation with folic acid can reduce neuronal damage and improve behavioral outcome in mouse models of AD and PD. Animal studies have shown that the beneficial effects of dietary restriction result, in part, from increased production of neurotrophic factors and cytoprotective protein chaperones in neurons. By keeping homocysteine levels low, folic acid can protect cerebral vessels and can prevent the accumulation of DNA damage in neurons caused by oxidative stress and facilitated by homocysteine. Although further studies are required in humans, the emerging data suggest that high-calorie diets and elevated homocysteine levels may render the brain vulnerable to neurodegenerative disorders.

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons.

Abstract: Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.

Autoantibodies to amyloid β-peptide (Aβ) are increased in Alzheimer’s disease patients and Aβ antibodies can enhance Aβ neurotoxicity

Abstract: Studies of amyloid precursor protein transgenic mice suggest that immune responses to amyloid β peptide (Aβ) may be instrumental in the removal of plaques from the brain, but the initial clinical trial of an Aβ vaccine in patients with Alzheimer’s disease (AD) was halted as the result of serious neurological complications in some patients. We now provide evidence that AD patients exhibit an enhanced immune response to Aβ and that, contrary to expectations, Aβ antibodies enhance the neurotoxic activity of the peptide. Serum titers to Aβ were significantly elevated in AD patients and Aβ antibodies were found in association with amyloid plaques in their brains, but there was no evidence of cell-mediated immune responses to Aβ in the patients. Aβ antibodies were detected in the serum of old APP mutant transgenic mice with plaque-like Aβ deposits, but not in the serum of younger transgenic or nontransgenic mice. Serum from APP mutant mice potentiated the neurotoxicity of Aβ. Our data suggest that a humoral immune response to Aβ in AD patients may promote neuronal degeneration, a process with important implications for the future of vaccine-based therapies for AD.

TERT suppresses apoptotis at a premitochondrial step by a mechanism requiring reverse transcriptase activity and 14-3-3 protein-binding ability.

Abstract: The catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that adds a six-base DNA repeat onto chromosome ends and prevents their shortening during successive cell divisions. Telomerase is associated with cell immortality and cancer, which may by related to the ability of TERT to prevent apoptosis by stabilizing telomeres. However, fundamental information concerning the antiapoptotic function of TERT is lacking, including whether RT activity and/or nuclear localization are required and where telomerase acts to suppress the cell death process. Here, we show that overexpression of wild-type human TERT in HeLa cells, and in a cells lacking TERT but containing the telomerase RNA template, increases their resistance to apoptosis induced by the DNA damaging agent etoposide or the bacterial alkaloid staurosporine. In contrast, TERT mutants with disruptions of either the RT domain or a 14-3-3 binding domain fail to protect cells against apoptosis, and overexpression of TERT in cells lacking the telomerase RNA template is also ineffective in preventing apoptosis. Additional findings show that TERT suppresses apoptosis at an early step before release of cytochrome c and apoptosis-inducing factor from mitochondria. We conclude that both RT activity and 14-3-3 protein binding ability are required for the antiapoptotic function of TERT in tumor cells and that TERT can suppress a nuclear signal(s) that is an essential component of apoptotic cascades triggered by diverse stimuli.

Alzheimer's amyloid beta-peptide enhances ATP/gap junction-mediated calcium-wave propagation in astrocytes.

Abstract: Alzheimer's disease (AD) involves the progressive extracellular deposition of amyloid beta-peptide (Abeta), a self-aggregating 40-42 amino acid protein that can damage neurons resulting in their dysfunction and death Studies of neurons have shown that Abeta perturbs cellularcalcium homeostasis so that calcium responses to agonists that induce calcium influx or release from internal stores are increased The recent discovery of intercellular calcium waves in astrocytes suggests intriguing roles for astrocytes in the long-range transfer of information in the nervous system We now report that Abeta alters calcium-wave signaling in cultured rat cortical astrocytes Exposure of astrocytes to Abeta1-42 resulted in an increase in the amplitude and velocity of evoked calcium waves, and increased the distance the waves traveled Suramin decreased wave propagation in untreated astrocytes and abrogated the enhancing effect of Abeta on calciumwave amplitude and velocity, indicating a requirement for extracellular ATP in wave propagation Treatment of astrocytes with an uncoupler of gap junctions did not significantly reduce the amplitude, velocity, or distance of calcium waves in control cultures, but completely abolished the effects of Abeta on each of the three wave parameters These findings reveal a novel action of Abeta on the propagation of intercellular calcium signals in astrocytes, and also suggests a role for altered astrocyte calcium-signaling in the pathogenesis of AD

Involvement of mitochondrial K+ release and cellular efflux in ischemic and apoptotic neuronal death

Abstract: We measured and manipulated intracellular potassium (K+) fluxes in cultured hippocampal neurons in an effort to understand the involvement of K+ in neuronal death under conditions of ischemia and exposure to apoptotic stimuli. Measurements of the intracellular K+ concentration using the fluorescent probe 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI) revealed that exposure of neurons to cyanide (chemical hypoxia), glutamate (excitotoxic insult) or staurosporine (apoptotic stimulus) results in efflux of K+ and cell death. Treatment of neurons with 5-hydroxydecanoate (5HD), an inhibitor of mitochondrial K+ channels, reduced K+ fluxes in neurons exposed to each insult and increased the resistance of the cells to death. K+ efflux was attenuated, levels of oxyradicals were decreased, mitochondrial membrane potential was stabilized and release of cytochrome c from mitochondria was attenuated in neurons treated with 5HD. K+ was rapidly released into the cytosol from mitochondria when neurons were exposed to the K+ channel opener, diazoxide, or to the mitochondrial uncoupler, carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), demonstrating that the intramitochondrial K+ concentration is greater than the cytosolic K+ concentration. The release of K+ from mitochondria was followed by efflux through plasma membrane K+ channels. In vivo studies showed that 5HD reduces ischemic brain damage without affecting cerebral blood flow in a mouse model of focal ischemic stroke. These findings suggest that intracellular K+ fluxes play a key role in modulating neuronal oxyradical production and cell survival under ischemic conditions, and that agents that modify K+ fluxes may have therapeutic benefit in stroke and related neurodegenerative conditions.

p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults

Abstract: A form of programmed cell-death called apoptosis occurs in neurons during development of the nervous system, and may also occur in a variety of neuropathological conditions. Here we present evidence obtained in studies of adult mice and neuronal cell cultures showing that p53 protein is present in synapses where its level and amount of phosphorylation are increased following exposure of the cells to the DNA-damaging agent etoposide. We also show that levels of active p53 increase in isolated cortical synaptosomes exposed to oxidative and excitotoxic insults. Increased levels of p53 also precede loss of synapsin I immunoreactive terminals in cultured hippocampal neurons exposed to etoposide. Synaptosomes from p53-deficient mice exhibit increased resistance to oxidative and excitotoxic insults as indicated by stabilization of mitochondrial membrane potential and decreased production of reactive oxygen species. Finally, we show that a synthetic inhibitor of p53 (PFT-alpha) protects synaptosomes from wild-type mice against oxidative and excitotoxic injuries, and preserves presynaptic terminals in cultured hippocampal neurons exposed to etoposide. Collectively, these findings provide the first evidence for a local transcription-independent action of p53 in synapses, and suggest that such a local action of p53 may contribute to the dysfunction and degeneration of synapses that occurs in various neurodegenerative disorders.

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons.

Abstract: 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.

Presenilin-1 mutation sensitizes oligodendrocytes to glutamate and amyloid toxicities, and exacerbates white matter damage and memory impairment in mice.

Abstract: Damage to white matter occurs in the brains of patients with Alzheimer’s disease (AD), but it is not known if and how oligodendrocytes are affected in AD, nor whether white matter alterations contribute to the cognitive dysfunction in this disease. Mutations in the gene encoding presenilin-1 (PS1) cause some cases of early-onset inherited AD. These mutations may promote neuronal degeneration by increasing the production of neurotoxic forms of amyloid β-peptide and by perturbing cellular calcium homeostasis. Damage to oligodendrocytes induced by a demyelinating agent is enhanced, and spatial learning is impaired in PS1 mutant knockin mice. Oligodendrocytes from PS1 mutant knockin mice are more vulnerable to being killed by glutamate and amyloid β-peptide, and exhibit an abnormality in calcium regulation which is responsible for their death. These findings demonstrate an adverse effect of a disease-causing PS1 mutation in oligodendrocytes, and suggest a mechanism responsible for white matter damage in AD and a contribution of such damage to cognitive impairment.

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation

Abstract: Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimer's disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show here that an FTDP-17 tau mutation (V337M) in SH-SY5Y cells reduces microtubule polymerization, increases voltage-dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L-type Ca channel blocker), whereas ω-conotoxin GVIA (N-type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L-type calcium channel activity. The depolarization-induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH-SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule-binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.