Mark P. Mattson, PhD, University of Iowa - US grants

Although the mechanisms responsible for the death of neurons in age-related disorders such as Alzheimer?s and Parkinson?s diseases and stroke are not well understood, increasing evidence suggests that neurons may die by a form of programmed cell death called apoptosis. A major effort in the LNS is aimed at identifying the factors that may trigger neuronal apoptosis in neurodegenerative disorders, as well as the specific molecular cascades responsible for executing the cell death program. The work described below has identified novel neuroprotective strategies for suppressing the neurodegenerative process in Alzheimer?s disease, Parkinson?s disease and stroke. A Novel Protein Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis: 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. We have found 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. The Transcription Factor NF-kappaB and Neuronal Apoptosis: Peroxidation of membrane lipids occurs in many different neurodegenerative conditions including stroke, and Alzheimer's and Parkinson's diseases. Recent findings suggest that lipid peroxidation can promote neuronal death by a mechanism involving production of the toxic aldehyde 4-hydroxy-2,3-nonenal (HNE), which may act by covalently modifying proteins and impairing their function. The transcription factor NF-kappa B can prevent neuronal death in experimental models of neurodegenerative disorders by inducing the expression of anti-apoptotic proteins including Bcl-2 and manganese superoxide dismutase. We found that HNE selectively suppresses basal and inducible NF-kappa B DNA binding activity in cultured rat cortical neurons. Immunoprecipitation-immunoblot analyses using antibodies against HNE-conjugated proteins and p50 and p65 NF-kappa B subunits indicate that HNE does not directly modify NF-kappa B proteins. Moreover, HNE did not affect NF-kappa B DNA-binding activity when added directly to cytosolic extracts, suggesting that HNE inhibits an upstream component of the NF-kappa B signaling pathway. Inhibition of the survival-promoting NF-kappa B signaling pathway by HNE may contribute to neuronal death under conditions in which membrane lipid peroxidation occurs. The activation of NF-kappaB has been closely associated with changes in intracellular calcium levels, but the relationship between the two remains unclear. We have discovered that inhibition of endoplasmic reticulum (ER) d-myo-inositol 1,4,5-trisphosphate (IP(3))-gated calcium release caused decreased basal NF-kappaB DNA-binding activity in cultured rat cortical neurons. Activation of NF-kappaB in response to tumor necrosis factor-alpha and glutamate was completely abolished when IP(3) receptors were blocked, and NF-kappaB activation in response to depletion of ER calcium by thapsigargin treatment was also decreased by IP(3) receptor blockade. We further investigated the relationship between IP(3) receptor activation and NF-kappaB activity using a cell-free system. Microsomes enriched in the ER were isolated from adult rat cerebral cortex, resuspended, and treated with agents that induce or inhibit ER calcium release. They were then recentrifuged, and the supernatant was added to cytoplasmic extract isolated from the same source tissue. We found that microsomes released an NF-kappaB-stimulating signal in response to activation of IP(3) receptors or inhibition of the ER Ca(2+)-ATPase, but not in response to ryanodine. Studies of intact cells and cell-free preparations indicated that the signal released from the ER was not calcium and was heat- and trypsin-sensitive. Our data suggest that activation of IP(3) receptors is required for a major component of both constitutive and inducible NF-kappaB binding activity in neurons and that decreasing ER intraluminal calcium levels triggers release of a diffusible NF-kappaB-activating signal from the ER. Mechanisms of Neuronal Apoptosis in Stroke: Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. The signaling cascades activated by cerebral ischemia that may either promote or protect against neuronal death are not well understood. One injury-responsive signaling pathway that has recently been characterized in studies of non-neural cells involves cleavage of membrane sphingomyelin by acidic and/or neutral sphingomyelinase (ASMase) resulting in generation of the second messenger ceramide. We found that transient focal cerebral ischemia induces large increases in ASMase activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase. Neurons lacking ASMase exhibit decreased vulnerability to excitotoxicity and hypoxia, which is associated with decreased levels of intracellular calcium and oxyradicals. Treatment of mice with a drug that inhibits ASMase activity and ceramide production reduces ischemic neuronal injury and improves behavioral outcome, suggesting that drugs that inhibit this signaling pathway may prove beneficial in stroke patients. After a stroke many neurons in the ischemic brain tissue die by a process called apoptosis, a form of cell death that may be preventable. The specific molecular cascades that mediate ischemic neuronal death are not well understood. The authors recently identified prostate apoptosis response-4 (Par-4) as a protein that participates in the death of cultured hippocampal neurons induced by trophic factor withdrawal and exposure to glutamate. We have found that Par-4 levels increase in vulnerable populations of hippocampal and striatal neurons in rats after transient forebrain ischemia; Par-4 levels increased within 6 hours of reperfusion and remained elevated in neurons undergoing apoptosis 3 days later. After transient focal ischemia in mice, Par-4 levels were increased 6 to 12 hours after reperfusion in the infarcted cortex and the striatum, and activation of caspase-8 occurred with a similar time course. Par-4 immunoreactivity was localized predominantly in cortical neurons at the border of the infarct area. A Par-4 antisense oligonucleotide protected cultured hippocampal neurons against apoptosis induced by chemical hypoxia and significantly reduced focal ischemic damage in mice. The current data suggest that early up-regulation of Par-4 plays a pivotal role in ischemic neuronal death in animal models of stroke and cardiac arrest. DNA Damage Respons

Approximately 5 million Americans currently suffer from Alzheimers disease (AD) a neurodegenerative disorder characterized by progressive impairment of cognitive function and emotional and sleep disturbances. This laboratory has developed cell culture and mouse models of AD, and have used these models to elucidate the biochemical and molecular events responsible for neuronal dysfunction and death in AD. We have found that there are abnormalities in lipid metabolism in the brains of patients with AD. Specifically, levels of cholesterol and long-chain ceramides are increased. Studies of experimental animal and cell culture models of AD suggest that increased oxidative stress, associated with amyloid deposition is responsible for the lipid abnormalities. Antioxidants and drugs that prevent the production of ceramides protect neurons from being damaged and killed by amyloid suggesting an important role for the lipid abnormalities in the disease process. Membrane lipid peroxidation appears to play an important role in amyloidogenic processing of the amyloid precursor protein as the lipid peroxidation product 4-hydroxynonenal covalently modifies the protein nicastrin and thereby increases gamma-secretase activity. We have also found that redox enzymes in the plasma membrane play important roles in protecting neurons against membrane lipid peroxidation and Abeta toxicity. In other studies we have provided evidence that activation of certain toll-like receptors (TLRs) in neurons and glial cells renders neurons vulnerable to Abeta toxicity and energy deprivation. Moreover,TLRs 2, 3 and 4 have interesting and disparate roles in the regulation of behaviors, including learning and memory and anxiety. Additional findings suggest that there is a defect in DNA base excision repair in brain cells of AD patients and subjects with amnestic mild cognitive impairment. Moreover, we have found that dietary restriction can reduce amyloid deposition and protect neurons from being damaged and killed in animal models of AD, and that this beneficial effect of dietary restriction involves stimulation of the production of brain-derived neurotrophic factor (BDNF). Antidepressant serotonin reuptake inhibitors can reduce amyloid deposition and improve cognitive function in a mouse model of AD, suggesting a potential prophylactic/therapeutic use of such drugs. We have shown that diabetes causes a deficit in cognitive function which is associated with impaired hippocampal synaptic plasticity and neurogenesis;exercise and dietary energy restriction can counteract these adverse effects of diabetes. We recently demonstrated a therapeutic benefit of drugs used to improve glycemic control in animal models of diabetes and Alzheimer's disease, and we are moving forward with a clinical trial of one of these drugs, Exenatide, in human subjects with mild cognitive impairment or early stage Alzheimer's disease.

We have identified several growth factors and cytokines that can protect neurons against dysfunction and death in experimental models of Alzheimers disease, Parkinsons disease and stroke. These trophic factors activate signaling pathways that stimulate the expression of genes whose encoded proteins increase resistance of neurons to oxidative and metabolic stress. Neuroprotective Actions of BDNF. We have found that brain-derived neurotrophic factor (BDNF) is a key mediator of the neuroprotective effects of dietary restriction in animal models of Parkinsons and Huntingtons diseases. In other studies we have found that caloric restriction reduces damage to dopaminergic neurons and improves functional outcome in a non-human primate model of Parkinsons disease. The beneficial effect of CR is associated with increased amounts of BDNF and glial cell line-derived neurotrophic factor (GDNF), a growth factor which is now in early clinical trials in patients with Parkinsons disease. In related studies we have found that the antidepressant paroxetine can suppress neuronal degeneration and improve motor function and survival in a mouse model of Hungtingtons disease by a mechanism involving increased production of BDNF. In addition, we have identified GLP-1 (glucagon-like peptide 1) as a neuroprotective neuropeptide with the potential to ameliorate neuronal dysfunction and degeneration in some neurodegenerative conditions. More recently, we have demonstrated a neuroprotective role for the mitochondrial uncoupling protein UCP4, which acts by reducing levels of oxidative stress. UCP4 expression increases in response to dietary restriction and BDNF treatment, suggesting a role for UCP4 in the neuroprotective effects of dietary restriction and neurotrophic factors. In preclinical studies we have developed novel analogs of uric acid and histidine as neuroprotective agents in a mouse model of stroke. We have also shown that intravenous immunoglobulin and gamma-secretase inhibitors improve outcome following a stroke in mice, by a mechanism involving inhibition of the complement cascade. In addition, we have developed high throughput screens to identify chemicals that activate adaptive cellular stress response pathways, with several novel neuroprotective agents emerging from these screens.

In previous studies we had shown that intermittent fasting (IF) is neuroprotective in rodent models of Alzheimers and Parkinsons diseases and stroke. The neuroprotective mechanism involves induction of a mild beneficial cellular response as indicated by increased expression of heat-shock proteins and brain-derived neurotrophic factor (BDNF). We have found that IF increases BDNF levels in the brain, ameliorates diabetes, suppresses neuronal degeneration in the striatum and cortex, and extends survival in a mouse model of Huntingtons disease. In a more recent study we have shown that dietary restriction is beneficial in a monkey model of Parkinsons disease. We have recently provided evidence that dietary lipids may modulate risk of AD and ALS. Levels of cholesterol and long-chain ceramides are increased in membranes of cells in the brains of AD patients and spinal cords of ALS patients. Additional data in studies of cell culture and animal models of AD and ALS suggest that ceramides may play an important role in the cell death process in these disorders. Because levels of cholesterol, sphingolipids and ceramides can be modulated by changes in diet, our data suggest that dietary lipids may modify the vulnerability of neurons to age-related diseases. In other studies we have shown that IF can improve glucose metabolism (increased insulin sensitivity) and cardiovascular risk factors (decreased blood pressure and superior cardiovascular stress adaptation) in rats. The latter effects of IF were mimicked by intermittent feeding of rats a diet supplemented with 2-deoxyglucoe, a non-metabolizable analog of glucose. Interestingly, IF and caloric restriction also increase heart rate variability in a manner suggesting that these diets increase parasympathetic tone, while decreasing sympathetic tone. Thus, IF and caloric restriction exert physiological actions that would be expected to reduce the risk of diabetes and cardiovascular disease. In our efforts to establish the mechanism by which dietary restriction protects neurons we have found that dietary restriction increases the expression of mitochondrial uncoupling proteins and enzymes of the plasma membrane redox system, resulting in a decrease in oxidative stress and stabilization of cellular energy homeostasis in neurons. In human studies we have found that an alternate day caloric restriction diet improves symptoms and decreases markers of oxidative stress and inflammation in asthma patients. In a meal frequency study, we found that consuming one large meal versus three smaller meals each day results in complex changes in physiology, some of which may be beneficial and others detrimental for health.

We have made considerable progress towards understanding the cellular and molecular mechanisms that regulate the proliferation, differentiation and survival of neural progenitor cells in the developing and adult central nervous system. We found that SDFalpha, activates CXCR4 in glial progenitor cells resulting in increased migration and differentation of those cells. Our recent research has revealed a new molecular signaling system that regulates the fate of neural stem cells in the cerebral cortex. We used antibody-blocking and genetic experiments to reveal an requirement for laminin/integrin interactions in apical process adhesion and neural stem cell regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal cerebral cortex development. Using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling resulted in substantial layering defects in the postnatal neocortex. Apart from protecting telomeres, nuclear TRF2 interacts with the master neuronal gene-silencer repressor element 1-silencing transcription factor (REST), and disruption of this interaction induces neuronal differentiation. We discovered the existence of a developmental switch from the expression of TRF2 in proliferating neural progenitor cells to expression of a unique short nontelomeric isoform of TRF2 (TRF2-S) as neurons establish a fully differentiated state. Unlike nuclear TRF2, which enhances REST-mediated gene repression, TRF2-S is located in the cytoplasm where it sequesters REST, thereby maintaining the expression of neuronal genes, including those encoding glutamate receptors, cell adhesion, and neurofilament proteins. In neurons, TRF2-S-mediated antagonism of REST nuclear activity is greatly attenuated by either overexpression of TRF2 or administration of the excitatory amino acid kainic acid. Overexpression of TRF2-S rescues kainic acid-induced REST nuclear accumulation and its gene-silencing effects. Thus, TRF2-S acts as part of a unique developmentally regulated molecular switch that plays critical roles in the maintenance and plasticity of neurons. Recently,we found that TLR3 protein is present in brain cells in early embryonic stages of development, and in cultured neural stem/progenitor cells (NPC). NPC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells, as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling, were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NPCs. Our findings reveal a novel role for TLR3 in the negative regulation of NPC proliferation in the developing brain. Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF1), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and III-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD. Although high amounts of reactive oxygen species (ROS) can damage cells, ROS can also play roles as second messengers, regulating diverse cellular processes. Here, we report that embryonic mouse cerebral cortical neural progenitor cells (NPCs) exhibit intermittent spontaneous bursts of mitochondrial superoxide (SO) generation (mitochondrial SO flashes) that require transient opening of membrane permeability transition pores (mPTP). This quantal SO production negatively regulates NPC self-renewal. Mitochondrial SO scavengers and mPTP inhibitors reduce SO flash frequency and enhance NPC proliferation, whereas prolonged mPTP opening and SO generation increase SO flash incidence and decrease NPC proliferation. The inhibition of NPC proliferation by mitochondrial SO involves suppression of extracellular signal-regulated kinases. Moreover, mice lacking SOD2 (SOD2-/- mice) exhibit significantly fewer proliferative NPCs and differentiated neurons in the embryonic cerebral cortex at midgestation compared with wild-type littermates. Cultured SOD2-/- NPCs exhibit a significant increase in SO flash frequency and reduced NPC proliferation. Taken together, our findings suggest that mitochondrial SO flashes negatively regulate NPC self-renewal in the developing cerebral cortex. We also found that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). Mitochondrial SO flash activity in NPCs increased during the first 6 24 hours of exposure to aggregating amyloid beta-peptide (Abeta1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Abeta1-42, during which time global cellular ROS increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Abeta1-42 on NPC proliferation and survival.

The ability of synapses to change their properties in response to environmental demands (synaptic plasticity) is essential for learning and memory. Abnormalities in synaptic plasticity are involved in Alzheimers disease and related disorders. In our continuing efforts to understand the molecular mechanisms involved in synaptic plasticity, in the contexts of aging and neurodegenerative disorders, we have made two major advances. 1) During development of the nervous system, the fate of stem cells is regulated by a cell surface receptor called Notch. Notch is also present in the adult mammalian brain;however, because Notch null mice die during embryonic development, it has proven difficult to determine the functions of Notch. Here, we used Notch antisense transgenic mice that develop and reproduce normally, but exhibit reduced levels of Notch, to demonstrate a role for Notch signaling in synaptic plasticity. Mice with reduced Notch levels exhibit impaired long-term potentiation (LTP) at hippocampal CA1 synapses. A Notch ligand enhances LTP in normal mice and corrects the defect in LTP in Notch antisense transgenic mice. Levels of basal and stimulation-induced NF-kappa B activity were significantly decreased in mice with reduced Notch levels. These findings suggest an important role for Notch signaling in a form of synaptic plasticity known to be associated with learning and memory processes. 2) Although ATP is reported to modulate synaptic plasticity, the mechanism of action of ATP on synaptic transmission is not fully understood. Here we show that ATP enhances long-term potentiation (LTP), and P2X receptor antagonists inhibit this ATP effect, but do not affect paired pulse facilitation (PPF) in rat hippocampal slices. ATP rapidly increases intracellular calcium, and P2X receptor antagonists inhibit this increase in cultured dissociated neurons. These results indicate that ATP enhances LTP via activation of postsynaptic P2X receptors. In additional studies, we have found that intermittent fasting and caloric restriction ameliorate age-related learning and memory deficits in a novel transgenic mouse model of Alzheimers disease. We also found that the antidepressant drug paroxetine was effective in suppressing amyloid pathology and preserving learning and memory ability in the same mouse model of Alzheimers disease. Other experiments have shown that diabetes impairs hippocampal neurogenesis and synaptic plasticity as the result of a chronic elevation in the level of adrenal glucocorticoids. We have found that perturbed membrane sphingolipid metabolism occurs in the brain in aging, Alzheimer's disease and HIV dementia. Studies of experimental models suggest that excessive activation of sphingomyelinases result in aberrant production of ceramides and perturbed membrane excitability and synaptic plasticity.

Chronic stresses such as loss of a spouse or sleep deprivation, may cause memory impairments and increase susceptibility to AD. Experimental models of stress demonstrate impairments in spatial memory, contextual memory and object recognition in response to psychosocial or environmental stress. Yet, it remains to be determined if and how environmental stress modifies the cellular and molecular alterations that result in cognitive deficits in normal aging and in AD. We are employing mouse models to test the hypothesis that chronic psychosocial stress and sleep deprivation will accelerate the development of cognitive impairment in normal aging and in AD. Using the triple-transgenic AD mouse model (3xTgAD mice) we are determining the effects of chronic stress on amyloidogenes, tau pathology, synaptic dysfunction and learning and memory impairment. We are testing the hypothesis that aging and AD compromise adaptive cellular stress response pathways resulting in increased oxidative stress associated with reduced expression of neuroprotective proteins such as brain-derived neurotrophic factor (BDNF) and antioxidant enzymes. In related studies we have found that, in a model of type 2 diabetes, overeating results in hyperactivation of the neuroendocrine stress system, and that elevated levels of adrenal glucocorticoids impair hippocampal synaptic plasticity and neurogenesis, and that these stress-related alterations are associated with a deficit in cognitive function. Interestingly, regular exercise and dietary energy restriction can counteract the adverse effects of diabetes on hippocampal plasticity by a mechanism involving up-regulation of the expression of the neurotrophic factor BDNF. Chronic stress may be a risk factor for developing Alzheimer's disease (AD), but most studies of the effects of stress in models of AD utilize acute adverse stressors of questionable clinical relevance. We therefore undertook a study to determine how chronic psychosocial stress affects behavioral and pathological outcomes in an animal model of AD, and to elucidate underlying mechanisms. A triple-transgenic mouse model of AD (3xTgAD mice) and nontransgenic control mice were used to test for an affect of chronic mild social stress on blood glucose, plasma glucocorticoids, plasma insulin, anxiety, and hippocampal amyloid, phosphorylated tau (ptau), and brain-derived neurotrophic factor (BDNF) levels. Despite the fact that both control and 3xTgAD mice experienced rises in corticosterone during episodes of mild social stress, at the end of the 6-week stress period 3xTgAD mice displayed increased anxiety, elevated levels of amyloid; oligomers and intraneuronal amyloid;, and decreased brain-derived neurotrophic factor levels, whereas control mice did not. Our findings suggest 3xTgAD mice are more vulnerable than control mice to chronic psychosocial stress, and that such chronic stress exacerbates amyloid; accumulation and impairs neurotrophic signaling. Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of -synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of -synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of -synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of -synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals. Age-associated dysregulation of sleep can be worsened by Alzheimer's disease (AD). AD and sleep restriction both impair cognition, yet it is unknown if mild chronic sleep restriction modifies the proteopathic processes involved in AD. The goal of this work was to test the hypothesis that sleep restriction worsens memory impairments, and amyloid β-peptide (Aβ) and pTau accumulations in the brain in a mouse model of AD, with a focus on a role for circulating glucocorticoids (GC). Male 3xTgAD mice were subjected to sleep restriction (SR) for 6h/day for 6 weeks using the modified multiple platform technique, and behavioral (Morris water maze, fear conditioning, open field) and biochemical (immunoblot) outcomes were compared to mice undergoing daily cage transfers (large cage control; LCC) as well as control mice that remained in their home cage (control; CTL). At one week, both LCC and SR mice displayed significant elevations in plasma corticosterone compared to CTL (p<0.002). By four weeks, SR mice displayed a two-fold increase in circulating corticosterone levels compared to CTL. Behavioral data indicated deficits in contextual and cued memory in SR mice that were not present for LCC or CTL (p<0.04). Both Aβ and pTau levels increased in the cortex of SR mice compared to CTL and LCC; however these changes were not noted in the hippocampus. Significant positive correlations between cortical Aβ and pTau levels and circulating corticosterone indicate a potential role for GCs in mediating behavioral and biochemical changes observed after sleep restriction in a mouse model of AD. The ability to control impulses varies greatly, and difficulty with impulse control can have severe consequences; in the extreme, it is the defining feature of many psychiatric disorders. Evidence from disparate lines of research suggests that uric acid is elevated in psychiatric disorders characterized by high impulsivity, such as attention-deficit/hyperactivity disorder and bipolar disorder. The present research tests the hypothesis that impulsivity is associated with higher uric acid in humans and mice. Using two longitudinal, nonclinical community samples (total n = 6883), we tested whether there is an association between uric acid and normal variation in trait impulsivity measured with the Revised NEO Personality Inventory. We also examined the effect of uric acid on behavior by comparing wild-type mice, which naturally have low levels of uric acid, with mice genetically modified to accumulate high levels of uric acid. In both human samples, the emotional aspects of trait impulsivity, specifically impulsiveness and excitement seeking, were associated with higher levels of uric acid concurrently and when uric acid was measured 3 to 5 years later. Consistent with the human data, the genetically modified mice displayed significantly more exploratory and novelty-seeking behavior than the wild-type mice. Higher uric acid was associated with impulsivity in both humans and mice. The identification of biological markers of impulsivity may lead to a better understanding of the physiological mechanisms involved in impulsivity and may suggest potential targets for therapeutic intervention.

We developed a bioassay to screen a panel of botanical insecticides to identify those that activate adaptive stress responses in neurons at subtoxic doses. Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neural cell models, and identified plumbagin as a potent activator of the nuclear factor E2-related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway. Subtoxic concentrations of plumbagin increase nuclear localization and transcriptional activity of Nrf2 and induce the expression of the Nrf2/ARE-dependent gene heme oxygenase 1 (HO-1) in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary cortical neurons from ARE-human placental alkaline phosphatase (hPAP) reporter mice. The activation of the ARE and the induction of HO-1 are abolished by RNA interference-mediated knockdown of Nrf2 expression. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The induction of HO-1 and the neuroprotective effects of plumbagin involve the PI3K/Akt signaling pathway upstream of Nrf2 activation. Intravenous administration of plumbagin significantly reduces the amount of brain damage and ameliorates associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways. Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca(2+) influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca(2+) chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors. In pathological conditions such as ischemic stroke, excessive DNA damage can trigger the death of neurons. Oxidative DNA damage is mainly repaired by base excision repair (BER), a process initiated by DNA glycosylases that recognize and remove damaged DNA bases. Endonuclease VIII-like 1 (NEIL1) is a DNA glycosylase that recognizes a broad range of oxidative lesions. Here, we show that mice lacking NEIL1 exhibit impaired memory retention in a water maze test, but no abnormalities in tests of motor performance, anxiety, or fear conditioning. NEIL1 deficiency results in increased brain damage and a defective functional outcome in a focal ischemia/reperfusion model of stroke. The incision capacity on a 5-hydroxyuracil-containing bubble substrate was lower in the ipsilateral side of ischemic brains and in the mitochondrial lysates of unstressed old NEIL1-deficient mice. These results indicate that NEIL1 plays an important role in learning and memory and in protection of neurons against ischemic injury. Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Because electroconvulsive shock (ECS) can stimulate the production of brain-derived neurotrophic factor (BDNF) and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration. In related studies we found that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT-mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT-induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT-induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntington's disease models and open new avenues for the development of neuroprotective strategies in Huntington's disease.

One major component of this project involves efforts to understand the functions of toll-like receptors (TLRs)in neuronal plasticity and neurodegenerative disorders. We discovered that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-gamma stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death in stroke. Toll-like receptors (TLRs) are innate immune receptors that have recently emerged as regulators of neuronal survival and developmental neuroplasticity. Adult TLR3-deficient mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition, and contextual fear-conditioning tasks. In contrast, TLR3-deficient mice demonstrated impaired amygdala-related behavior and anxiety in the cued fear-conditioning, open field, and elevated plus maze tasks. Further, TLR3-deficient mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis, and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Direct activation of TLR3 by intracerebroventricular infusion of a TLR3 ligand impaired working memory, but not reference memory. Our findings reveal previously undescribed roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention. In studies of relevance to Alzheimer's disease (AD) we found that TLR4 expression increases in neurons when exposed to amyloid beta-peptide (Abeta1-42) or the lipid peroxidation product 4-hydroxynonenal (HNE). Neuronal apoptosis triggered by Abeta and HNE was mediated by jun N-terminal kinase (JNK);neurons from TLR4 mutant mice exhibited reduced JNK and caspase-3 activation and were protected against apoptosis induced by Abeta and HNE. Levels of TLR4 were decreased in inferior parietal cortex tissue specimens from end-stage AD patients compared to aged-matched control subjects, possibly as the result of loss of neurons expressing TLR4. Our findings suggest that TLR4 signaling increases the vulnerability of neurons to Abeta and oxidative stress in AD, and identify TLR4 as a potential therapeutic target for AD. Other novel findings suggest important roles for TLRs in development of the nervous system. TLR3 protein is present in brain cells in early embryonic stages of development, and in cultured neural stem/progenitor cells (NPC). NPC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells, as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling, were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NPCs. Our findings reveal a novel role for TLR3 in the negative regulation of NPC proliferation in the developing brain. More recently we have obtained evidence that TLR3 plays important roles in adult neurogenesis and synaptic plasticity. Brain ischemia induces an inflammatory response involving activated complement fragments. In a preclinical study we showed that i.v. Ig (IVIG) treatment, which scavenges complement fragments, protects brain cells against the deleterious effects of experimental ischemia and reperfusion (I/R) and prevents I/R-induced mortality in mice. Animals administered IVIG either 30 min before ischemia or after 3 h of reperfusion exhibited a 50-60% reduction of brain infarct size and a 2- to 3-fold improvement of the functional outcome. Even a single low dose of IVIG given after stroke was effective. IVIG was protective in the nonreperfusion model of murine stroke as well and did not exert any peripheral effects. Human IgG as well as intrinsic murine C3 levels were significantly higher in the infarcted brain region compared with the noninjured side, and their physical association was demonstrated by immuno-coprecipitation. C5-deficient mice were significantly protected from I/R injury compared with their wild-type littermates. Exposure of cultured neurons to oxygen/glucose deprivationresulted in increased levels of C3 associated with activation of caspase 3, a marker of apoptosis;both signals were attenuated with IVIG treatment. Our data suggest a major role for complement-mediated cell death in ischemic brain injury and the prospect of using IVIG in relatively low doses as an interventional therapy for stroke. Another example of our efforts on this project involves studies of the effects of age and dietary energy intake on stroke outcome. Age and excessive energy intake/obesity are risk factors for cerebrovascular disease, but it is not known if and how these factors affect the extent of brain damage and outcome in ischemic stroke. We utilized a novel microchip-based immunoaffinity capillary electrophoresis technology to measure a panel of neurotrophic factors, cytokines and cellular stress resistance proteins in brain tissue samples from young, middle age and old mice that had been maintained on control or energy restricted diets prior to middle cerebral artery occlusion and reperfusion (I/R). Mortality from focal ischemic stroke was increased with advancing age and reduced by an intermittent fasting (IF) diet. Brain damage and functional impairment were reduced by IF in young and middle age mice, but not in old mice. The basal and post-stroke levels of neurotrophic factors (BDNF and bFGF), protein chaperones (HSP70 and GRP78) and the antioxidant enzyme HO-1 were decreased, while levels of inflammatory cytokines were increased in the cerebral cortex and striatum of old mice compared to younger mice. IF coordinately increased levels of protective proteins and decreases inflammatory cytokines in young, but not in old mice. We conclude that a reduction of dietary energy intake differentially modulates neurotrophic and inflammatory pathways to protect neurons against ischemic injury, and these beneficial effects of IF are compromised during aging resulting in increased brain damage and poorer functional outcome.

We have identified several growth factors and cytokines that can protect neurons against dysfunction and death in experimental models of Alzheimers disease, Parkinsons disease and stroke. These trophic factors activate signaling pathways that stimulate the expression of genes whose encoded proteins increase resistance of neurons to oxidative and metabolic stress. Neuroprotective Actions of BDNF. We have found that brain-derived neurotrophic factor (BDNF) is a key mediator of the neuroprotective effects of dietary restriction in animal models of Parkinsons and Huntingtons diseases. Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of -synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of -synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of -synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of -synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals. The Sonic hedgehog (Shh) signaling pathway is well known in patterning of the neural tube during embryonic development, but its emerging role in differentiated neurons is less understood. Here we report that Shh enhances autophagy in cultured hippocampal neurons. Microarray analysis reveals the upregulation of multiple autophagy-related genes in neurons in response to Shh application. Through analysis of the autophagy-marker LC3 by immunoblot analysis and immunocytochemistry, we confirm activation of the autophagy pathway in Shh-exposed neurons. Using electron microscopy, we find autophagosomes and associated structures with a wide range of morphologies in synaptic terminals of Shh-exposed neurons. Moreover, we show that Shh-triggered autophagy depends on class III Phosphatidylinositol 3-kinase complexes (PtdIns3K). These results identify a link between Shh and autophagy pathways and, importantly, provide a lead for further understanding the physiology of Shh signaling activity in neurons. The formation, maintenance and reorganization of synapses are critical for brain development and the responses of neuronal circuits to environmental challenges. Here we describe a novel role for peroxisome proliferator-activated receptor; alpha co-activator PGC-1alpha;, a master regulator of mitochondrial biogenesis, in the formation and maintenance of dendritic spines in hippocampal neurons. In cultured hippocampal neurons, PGC-1alpha; overexpression increases dendritic spines and enhances the molecular differentiation of synapses, whereas knockdown of PGC-1alpha; inhibits spinogenesis and synaptogenesis. PGC-1alpha; knockdown also reduces the density of dendritic spines in hippocampal dentate granule neurons in vivo. We further show that brain-derived neurotrophic factor stimulates PGC-1alpha;-dependent mitochondrial biogenesis by activating extracellular signal-regulated kinases and cyclic AMP response element-binding protein. PGC-1alpha; knockdown inhibits brain-derived neurotrophic factor-induced dendritic spine formation without affecting expression and activation ofthe brain-derived neurotrophic factor receptor tyrosine receptor kinase B. Our findings suggest that PGC-1alpha; and mitochondrial biogenesis have important roles in the formation and maintenance of hippocampal dendritic spines and synapses. Based on our own research and evolutionary considerations, we developed a new hypothesis to explain the health benefits of plant consumption, namely, that some phytochemicals exert disease-preventive and therapeutic actions by engaging one or more adaptive cellular response pathways in cells. The evolutionary basis for this hypothesis is based on the fact that plants produce natural antifeedant/noxious chemicals that discourage insects and other organisms from eating them. However, in the amounts typically consumed by humans, the phytochemicals activate one or more conserved adaptive cellular stress response pathways and thereby enhance the ability of cells to resist injury and disease. Examples of such pathways include those involving stress-responsive transcription factors , as well as the production and action of trophic factors and hormones. Translational research to develop interventions that target these pathways may lead to new classes of therapeutic agents that act by stimulating adaptive stress response pathways to bolster endogenous defenses against brain injury and disease. The impact of mitochondrial protein acetylation status on neuronal function and vulnerability to neurological disorders is unknown. Here we show that the mitochondrial protein deacetylase SIRT3 mediates adaptive responses of neurons to bioenergetic, oxidative, and excitatory stress. Cortical neurons lacking SIRT3 exhibit heightened sensitivity to glutamate-induced calcium overload and excitotoxicity and oxidative and mitochondrial stress; AAV-mediated Sirt3 gene delivery restores neuronal stress resistance. In models relevant to Huntington's disease and epilepsy, Sirt3(-/-) mice exhibit increased vulnerability of striatal and hippocampal neurons, respectively. SIRT3 deficiency results in hyperacetylation of several mitochondrial proteins, including superoxide dismutase 2 and cyclophilin D. Running wheel exercise increases the expression of Sirt3 in hippocampal neurons, which is mediated by excitatory glutamatergic neurotransmission and is essential for mitochondrial protein acetylation homeostasis and the neuroprotective effects of running. Our findings suggest that SIRT3 plays pivotal roles in adaptive responses of neurons to physiological challenges and resistance to degeneration. The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons.

Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of -synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals. In another study we found that mortality from focal ischemic stroke was increased with advancing age and reduced by an intermittent fasting (IF) diet. Brain damage and functional impairment were reduced by IF in young and middle-aged mice, but not in old mice. The basal and poststroke levels of neurotrophic factors (brain-derived neurotrophic factor and basic fibroblast growth factor), protein chaperones (heat shock protein 70 and glucose regulated protein 78), and the antioxidant enzyme heme oxygenase-1 were decreased, whereas levels of inflammatory cytokines were increased in the cerebral cortex and striatum of old mice compared with younger mice. IF coordinately increased levels of protective proteins and decreased inflammatory cytokines in young, but not in old mice. We further found that intermittent fasting suppresses activation of the so-called 'inflammasome' in brain cells, which was associated with improved functional outcome in the mouse stroke model. We conclude that dietary energy intake differentially modulates neurotrophic and inflammatory pathways to protect neurons against ischemic injury, and these beneficial effects of IF are compromised during aging, resulting in increased brain damage and poorer functional outcome. The 3xTgAD mouse model was used to test the hypothesis that a ketone ester-based diet can ameliorate AD pathogenesis. Beginning at a presymptomatic age, 2 groups of male 3xTgAD mice were fed a diet containing a physiological enantiomeric precursor of ketone bodies (KET) or an isocaloric carbohydrate diet. The results of behavioral tests performed at 4 and 7 months after diet initiation revealed that KET-fed mice exhibited significantly less anxiety in 2 different tests. 3xTgAD mice on the KET diet also exhibited significant, albeit relatively subtle, improvements in performance on learning and memory tests. Immunohistochemical analyses revealed that KET-fed mice exhibited decreased Abeta; deposition in the subiculum, CA1 and CA3 regions of the hippocampus, and the amygdala. KET-fed mice exhibited reduced levels of hyperphosphorylated tau deposition in the hippocampus and amygdala. These findings demonstrate a therapeutic benefit of a diet containing a ketone ester in a mouse model of Alzheimer's disease. The impact of dietary factors on brain health and vulnerability to disease is increasingly appreciated. The results of epidemiological studies, and intervention trials in animal models suggest that diets rich in phytochemicals can enhance neuroplasticity and resistance to neurodegeneration. Here we describe how interactions of plants and animals during their co-evolution, and resulting reciprocal adaptations, have shaped the remarkable characteristics of phytochemicals and their effects on the physiology of animal cells in general, and neurons in particular. Based on our own research and evolutionary considerations, we developed a novel hypothesis to explain the beneficial effects of diets rich in fruits and vegetables on health, including brain health. Plants do not have the option of fleeing predators. As a consequence, they have developed an elaborate set of chemical defenses to ward off insects and other creatures that want to make them into a meal. Toxins that plants use against predators are consumed by us at low levels in fruits and vegetables. Exposure to these chemicals causes a mild stress reaction that lends resilience to cells in our bodies. Adaptation to these stresses, a process called hormesis, accounts for a number of health benefits, including protection against brain disorders, that we receive from eating vegetables and fruits. Survival advantages were conferred upon plants capable of producing noxious bitter-tasting chemicals, and on animals able to tolerate the phytochemicals and consume the plants as an energy source. The remarkably diverse array of phytochemicals present in modern fruits, vegetables spices, tea and coffee may have arisen, in part, from the acquisition of adaptive cellular stress responses and detoxification enzymes in animals that enabled them to consume plants containing potentially toxic chemicals. Interestingly, some of the same adaptive stress response mechanisms that protect neurons against noxious phytochemicals are also activated by dietary energy restriction and vigorous physical exertion, two environmental challenges that shaped brain evolution. We have elucidated some of the signaling pathways relevant to cellular energy metabolism that are modulated by 'neurohormetic phytochemicals' (potentially toxic chemicals produced by plants that have beneficial effects on animals when consumed in moderate amounts). We highlight the cellular bioenergetics-related sirtuin, adenosine monophosphate activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and insulin-like growth factor 1 (IGF-1) pathways. The inclusion of dietary neurohormetic phytochemicals in an overall program for brain health that also includes exercise and energy restriction may find applications in the prevention and treatment of a range of neurological disorders. We fpimd that IF ameliorates cognitive deficits in a rat model of sepsis by a mechanism involving NF-B activation, suppression of the expression of pro-inflammatory cytokines, and enhancement of neurotrophic support. Treatment of rats with LPS resulted in deficits in cognitive performance in the Barnes maze and inhibitory avoidance tests, without changing locomotor activity, that were ameliorated in rats that had been maintained on the IF diet. IF also resulted in reduced levels of mRNAs encoding the LPS receptor TLR4 and inducible nitric oxide synthase (iNOS) in the hippocampus. Moreover, IF prevented LPS-induced elevation of IL-1, IL-1 and TNF- levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1, IFN-, RANTES, TNF- and IL-6 levels. Taken together, our results suggest that IF induces adaptive responses in the brain and periphery that can suppress inflammation and preserve cognitive function in an animal model of systemic bacterial infection.

Approximately 5 million Americans currently suffer from Alzheimers disease (AD) a neurodegenerative disorder characterized by progressive impairment of cognitive function and emotional and sleep disturbances. This laboratory has developed cell culture and mouse models of AD, and have used these models to elucidate the biochemical and molecular events responsible for neuronal dysfunction and death in AD. Our findings suggest that during aging neurons become increasingly prone to dysfunction as a result of impaired cellular energy metabolism and destabilization of calcium-regulating systems. Amyloid beta-peptide can exacerbate these age-related changes in neurons resulting in their degeneration and consequent cognitive deficits. Membrane lipid peroxidation appears to play an important role in amyloidogenic processing of the amyloid precursor protein as the lipid peroxidation product 4-hydroxynonenal covalently modifies the protein nicastrin and thereby increases gamma-secretase activity. We have also found that redox enzymes in the plasma membrane play important roles in protecting neurons against membrane lipid peroxidation and Abeta toxicity. The latter findings reveal previously unknown molecular targets for the development of novel therapeutic interventions in AD. We have found that dietary restriction can reduce amyloid deposition and protect neurons from being damaged and killed in animal models of AD, and that this beneficial effect of dietary restriction involves stimulation of the production of brain-derived neurotrophic factor (BDNF). Antidepressant serotonin reuptake inhibitors can reduce amyloid deposition and improve cognitive function in a mouse model of AD, suggesting a potential prophylactic/therapeutic use of such drugs. In addition, we found that a drug called diazoxide, previously used to treat hypertension,reduces amyloid and tau pathologies and improves cognitive function in our 3xTgAD mouse model of AD. In addition, dietary supplementation with nicotinamide retards the disease process in a mouse model of AD by a mechanism involving sustenance of neuronal energy levels and enhanced clearance of abnormal forms of amyloid and tau. We have shown that diabetes causes a deficit in cognitive function which is associated with impaired hippocampal synaptic plasticity and neurogenesis; exercise and dietary energy restriction can counteract these adverse effects of diabetes. Our recent findings suggest that an excitatory imbalance, resulting from reduced GABAergic inhibition, is an early and pivotal event in AD pathogenesis. We recently demonstrated a therapeutic benefit of drugs used to improve glycemic control in animal models of diabetes and Alzheimer's disease, and we have initiated a clinical trial of one of these drugs, Exenatide, in human subjects with mild cognitive impairment or early stage Alzheimer's disease. Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline and the pathologic accumulations of amyloid -peptide (A) and hyperphosphorylated tau in vulnerable brain regions. The 3xTgAD mouse model was used to test the hypothesis that a ketone ester-based diet can ameliorate AD pathogenesis. Beginning at a presymptomatic age, 2 groups of male 3xTgAD mice were fed a diet containing a physiological enantiomeric precursor of ketone bodies (KET) or an isocaloric carbohydrate diet. The results of behavioral tests performed at 4 and 7 months after diet initiation revealed that KET-fed mice exhibited significantly less anxiety in 2 different tests. 3xTgAD mice on the KET diet also exhibited significant, albeit relatively subtle, improvements in performance on learning and memory tests. Immunohistochemical analyses revealed that KET-fed mice exhibited decreased Abeta; deposition in the subiculum, CA1 and CA3 regions of the hippocampus, and the amygdala. KET-fed mice exhibited reduced levels of hyperphosphorylatedtau deposition in the same regions of the hippocampus, amygdala, and cortex. Thus, a novel ketone ester can ameliorate proteopathic and behavioral deficits in a mouse AD model. We explored the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase . A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Pol(+/-) mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations. AD patients typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase- (Pol) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild type and 3xTgAD mice with normal or reduced Pol levels. Compared to wild type control mice, Pol heterozygous (Pol+/-) and 3xTgAD mice, 3xTgAD/Pol+/- mice exhibited impaired performance in a buried food test of olfaction. Pol deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Pol+/- and 3xTgAD mice, and by over 60% in the 3xTgAD/Pol+/- mice compared to wild type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Pol+/- mice compared to 3xTgAD mice. Levels of amyloid -peptide (A) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Pol+/- mice, and cultured Pol-deficient neurons exhibited increased vulnerability to A-induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD. Recently, we found that intermittent fasting has an anxiolytic effect in mice, and that this effect is mediated by upregulation of GABAergic tone and requires the mitochondrial deacetylase SIRT3. In another study we found that brain regional synchronous activity is associated with Tau pathology, but not Abeta pathology in the 3xTgAD mouse model of AD

The impact of mitochondrial protein acetylation status on neuronal function and vulnerability to neurological disorders is unknown. We found that the mitochondrial protein deacetylase SIRT3 mediates adaptive responses of neurons to bioenergetic, oxidative, and excitatory stress. Cortical neurons lacking SIRT3 exhibit heightened sensitivity to glutamate-induced calcium overload and excitotoxicity and oxidative and mitochondrial stress; AAV-mediated Sirt3 gene delivery restores neuronal stress resistance. In models relevant to Huntington's disease and epilepsy, Sirt3(-/-) mice exhibit increased vulnerability of striatal and hippocampal neurons, respectively. SIRT3 deficiency results in hyperacetylation of several mitochondrial proteins, including superoxide dismutase 2 and cyclophilin D. Running wheel exercise increases the expression of Sirt3 in hippocampal neurons, which is mediated by excitatory glutamatergic neurotransmission and is essential for mitochondrial protein acetylation homeostasis and the neuroprotective effects of running. Our findings suggest that SIRT3 plays pivotal roles in adaptive responses of neurons to physiological challenges and resistance to degeneration. Intermittent food deprivation/fasting (IF) improves mood and cognition and protects neurons against excitotoxic degeneration in animal models of epilepsy and Alzheimers disease (AD). The mechanisms by which neuronal networks adapt to IF and how such adaptations impact neuropathological processes are unknown. We found that hippocampal neuronal networks adapt to IF by enhancing GABAergic tone, which is associated with reduced anxiety-like behaviors. These adaptations involve increased expression of the mitochondrial protein deacetylase SIRT3 and are abolished in SIRT3-deficient mice, demonstrating a pivotal role for mitochondrial protein deacetylation in behavioral and neuronal network adaptations to IF. In the AppNL-G-F mouse model of AD, IF reduces neuronal network hyperexcitability and ameliorates deficits in cognition and hippocampal synaptic plasticity. These findings demonstrate a pivotal role for mitochondrial protein deacetylation in behavioral and neuronal network adaptations to IF, and suggest a potential application of intermittent fasting in neurological disorders that involve hyperactivity of neuronal networks.

The ability of synapses to change their properties in response to environmental demands (synaptic plasticity) is essential for learning and memory. Abnormalities in synaptic plasticity are involved in Alzheimers disease and related disorders. In our continuing efforts to understand the molecular mechanisms involved in synaptic plasticity, in the contexts of aging and neurodegenerative disorders, we have made several major advances. We used Notch antisense transgenic mice that develop and reproduce normally, but exhibit reduced levels of Notch, to demonstrate a role for Notch signaling in synaptic plasticity. Mice with reduced Notch levels exhibit impaired long-term potentiation (LTP) at hippocampal CA1 synapses. A Notch ligand enhances LTP in normal mice and corrects the defect in LTP in Notch antisense transgenic mice. Levels of basal and stimulation-induced NF-kappa B activity were significantly decreased in mice with reduced Notch levels. These findings suggest an important role for Notch signaling in a form of synaptic plasticity known to be associated with learning and memory processes. We found that Notch1 and its ligand Jagged1 are present at the synapse, and that Notch signaling in neurons occurs in response to synaptic activity. In addition, neuronal Notch signaling is positively regulated by Arc/Arg3.1, an activity-induced gene required for synaptic plasticity. In Arc/Arg3.1 mutant neurons, the proteolytic activation of Notch1 is disrupted both in vivo and in vitro. Conditional deletion of Notch1 in the postnatal hippocampus disrupted both long-term potentiation (LTP) and long-term depression (LTD), and led to deficits in learning and short-term memory. Our findings show that Notch signaling is dynamically regulated in response to neuronal activity, Arc/Arg3.1 is a context-dependent Notch regulator, and Notch1 is required for the synaptic plasticity that contributes to memory formation. The formation, maintenance and reorganization of synapses are critical for brain development and the responses of neuronal circuits to environmental challenges. Here we describe a novel role for peroxisome proliferator-activated receptor alpha; co-activator gamma (PGC-1alpha) ;, a master regulator of mitochondrial biogenesis, in the formation and maintenance of dendritic spines in hippocampal neurons. In cultured hippocampal neurons, PGC-1alpha overexpression increases dendritic spines and enhances the molecular differentiation of synapses, whereas knockdown of PGC-1alpha inhibits spinogenesis and synaptogenesis. PGC-1alpha; knockdown also reduces the density of dendritic spines in hippocampal dentate granule neurons in vivo. We further show that brain-derived neurotrophic factor stimulates PGC-1alpha-dependent mitochondrial biogenesis by activating extracellular signal-regulated kinases and cyclic AMP response element-binding protein. PGC-1alpha knockdown inhibits brain-derived neurotrophic factor-induced dendritic spine formation without affecting expression and activation of the brain-derived neurotrophic factor receptor tyrosine receptor kinase B. Our findings suggest that PGC-1alpha and mitochondrial biogenesis have important roles in the formation and maintenance of hippocampal dendritic spines and synapses. Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior. We found that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release. Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth.