Increased tau phosphorylation and impaired brain insulin/IGF signaling in mice fed a high fat/high cholesterol diet.

Previous studies demonstrated that a high fat/high cholesterol (HFC) diet results in a loss of working memory in mice correlated with neuroinflammatory changes and increased AßPP processing (Thirumangalakudi et al. (2008) J Neurochem 106, 475-485). To further explore the nature of the molecular correlates of cognitive impairment, in this study, we examined changes in tau phosphorylation, insulin/IGF-1 signaling (IIS) including GSK3, and levels of specific synaptic proteins. Immunoblot analysis of hippocampal tissue from C57BL/6 mice fed HFC for 2 months with anti-phospho-tau (i.e., PHF1 and phospho-Thr-231 tau) antibodies demonstrated the presence of hyperphosphorylated tau. The tau phosphorylation correlated with activated GSK3, a prominent tau kinase normally kept inactive under the control of IIS. That IIS itself was impaired due to the hyperlipidemic diet was confirmed by a down-regulation of insulin receptor substrate-1 and phospho-Akt levels. Although no significant changes in the levels of the pre-synaptic protein (i.e., synaptophysin) in response to HFC were apparent in immunoblot analysis, there was a clear down-regulation of the post-synaptic protein, PSD95, and drebrin, a dendritic spine-specific protein, indicative of altered synaptic plasticity. The results, in concert with previous findings with the same model, suggest that high dietary fat/cholesterol elicits brain insulin resistance and altered IIS leading to Alzheimer's disease-like cognitive impairment in 'normal' mice.

Role of hippocampal neurogenesis in mnemonic segregation: implications for human mood disorders.

OBJECTIVES: Although hippocampal neurogenesis has been implicated in mood disorders, the precise role new neurons play in mood regulation is not fully elucidated. Here we examine whether neurogenesis improves mood by facilitating segregation of novel experiences that conflict with older maladaptive memories. METHODS: Study 1: Four groups (N = 9 each) of adult male rats (exposed to stress or control conditions plus antidepressant or placebo) underwent active training on the place-avoidance task (PAT) on week 0; tested on recalling the "Initial PAT" on weeks 4 and 8; learning a subtly "Altered PAT" on week 8; and euthanazed on week 9. Study-2: Two groups (N = 12 each) rats tested either on the Initial-PAT or Altered-PAT 3 days post-training and immediately euthanized. RESULTS: Stressed subjects treated with placebo were slower in learning the week 8 Altered Task and had lower neurogenesis rates than non-stressed animals and Stressed subjects given drug (Study 1). Synaptic activation of mature hippocampal neurons inversely correlated with Altered-PAT performance and with neurogenesis rates (Study 2). CONCLUSIONS: Increasing neurogenesis enhances acquisition of novel experiences possibly by suppressing activation of mature hippocampal neurons that mediate established, conflicting memories. Therefore, antidepressants may improve mood by stimulating new hippocampal neurogenesis that facilitate detection of positive experiences while suppressing interference from recurring depressogenic thought patterns.

Correlations between hippocampal neurogenesis and metabolic indices in adult nonhuman primates.

Increased neurogenesis in feeding centers of the murine hypothalamus is associated with weight loss in diet-induced obese rodents (Kokoeva et al., 2005 and Matrisciano et al., 2010), but this relationship has not been examined in other species. Postmortem hippocampal neurogenesis rates and premortem metabolic parameters were statistically analyzed in 8 chow-fed colony-reared adult bonnet macaques. Dentate gyrus neurogenesis, reflected by the immature neuronal marker, doublecortin (DCX), and expression of the antiapoptotic gene factor, B-cell lymphoma 2 (BCL-2), but not the precursor proliferation mitotic marker, Ki67, was inversely correlated with body weight and crown-rump length. DCX and BCL-2 each correlated positively with blood glucose level and lipid ratio (total cholesterol/high-density lipoprotein). This study demonstrates that markers of dentate gyrus neuroplasticity correlate with metabolic parameters in primates.

Necessity of hippocampal neurogenesis for the therapeutic action of antidepressants in adult nonhuman primates.

BACKGROUND: Rodent studies show that neurogenesis is necessary for mediating the salutary effects of antidepressants. Nonhuman primate (NHP) studies may bridge important rodent findings to the clinical realm since NHP-depression shares significant homology with human depression and kinetics of primate neurogenesis differ from those in rodents. After demonstrating that antidepressants can stimulate neurogenesis in NHPs, our present study examines whether neurogenesis is required for antidepressant efficacy in NHPs. MATERIALS/METHODOLOGY: Adult female bonnets were randomized to three social pens (N = 6 each). Pen-1 subjects were exposed to control-conditions for 15 weeks with half receiving the antidepressant fluoxetine and the rest receiving saline-placebo. Pen-2 subjects were exposed to 15 weeks of separation-stress with half receiving fluoxetine and half receiving placebo. Pen-3 subjects 2 weeks of irradiation (N = 4) or sham-irradiation (N = 2) and then exposed to 15 weeks of stress and fluoxetine. Dependent measures were weekly behavioral observations and postmortem neurogenesis levels. RESULTS: Exposing NHPs to repeated separation stress resulted in depression-like behaviors (anhedonia and subordinance) accompanied by reduced hippocampal neurogenesis. Treatment with fluoxetine stimulated neurogenesis and prevented the emergence of depression-like behaviors. Ablation of neurogenesis with irradiation abolished the therapeutic effects of fluoxetine. Non-stressed controls had normative behaviors although the fluoxetine-treated controls had higher neurogenesis rates. Across all groups, depression-like behaviors were associated with decreased rates of neurogenesis but this inverse correlation was only significant for new neurons in the anterior dentate gyrus that were at the threshold of completing maturation. CONCLUSION: We provide evidence that induction of neurogenesis is integral to the therapeutic effects of fluoxetine in NHPs. Given the similarity between monkeys and humans, hippocampal neurogenesis likely plays a similar role in the treatment of clinical depression. Future studies will examine several outstanding questions such as whether neuro-suppression is sufficient for producing depression and whether therapeutic neuroplastic effects of fluoxetine are specific to antidepressants.

RANTES upregulation in the Alzheimer's disease brain: a possible neuroprotective role.

Numerous studies demonstrate inflammatory proteins in the brain and microcirculation in Alzheimer's disease (AD) and implicate inflammation in disease pathogenesis. However, emerging literature suggests that neuroinflammation can also be neuroprotective. The chemokine RANTES has been implicated in neurodegenerative diseases including AD. The objectives of this study are to determine the expression of RANTES in AD microvessels, its regulation in endothelial cells and its effects on neuronal survival. Our data show elevated expression of RANTES in the cerebral microcirculation of AD patients. Treatment of neurons in vitro with RANTES results in an increase in cell survival and a neuroprotective effect against the toxicity of thrombin and sodium nitroprusside. Oxidative stress upregulates RANTES expression in rat brain endothelial cells. Developing strategies to augment neuroprotection and diminish inflammatory activation of multifunctional mediators such as RANTES holds promise for the development of novel neuroprotective therapeutics in AD.

Involvement of PGE2 and PGDH but not COX-2 in thrombin-induced cortical neuron apoptosis.

The pathways that contribute to thrombin-induced neuron death have been incompletely defined. Induction of cyclooxygenase 2 (COX-2), the enzyme that catalyzes the first step in prostaglandin synthesis, promotes neuronal injury. PGE2, a downstream product of COX-2 metabolism, is neurotoxic in vitro and in vivo, and is thought to be the bioactive mediator responsible for COX-2 neurotoxicity. The objective of this study is to determine the ability of thrombin to affect PGE2 metabolism in cultured neurons. The data show that in thrombin-induced apoptosis of cultured neurons, PGE2 release increases when COX-2 is absent, and is regulated by prostaglandin dehydrogenase (PGDH), a key enzyme that degrades PGE2. NS398, a COX-2 specific inhibitor, protects neurons against thrombin toxicity, by inducing active PGDH. These data implicate PGDH in thrombin-mediated neuronal cell death.

Cyclin C and cyclin dependent kinases 1, 2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis.

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200nM) for 30min to 4.5h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice.

Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer's disease-like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer's disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)-deficient mice were fed a high fat/cholesterol diet for an 8-week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR-/- mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat-fed C57BL/6 mice and LDLR-/- mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor-alpha, interleukin-1beta and -6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. beta-site APP cleaving enzyme 1 in both high fat/cholesterol-fed C57BL/6 and LDLR-/- mice accompanied by an increased generation of C-terminal fragments of APP. Although ELISA for beta-amyloid failed to record significant changes in the non-transgenic mice, a threefold increase in beta-amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild-type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non-transgenic mouse model.

IL-8 induces expression of matrix metalloproteinases, cell cycle and pro-apoptotic proteins, and cell death in cultured neurons.

Neuronal cell loss is a critical feature of age-related neurodegenerative diseases such as Alzheimer's disease (AD). In the AD brain, a marked increase in pro-inflammatory cytokines and chemokines, including IL-8, has been documented. The objective of this study was to determine the effect of IL-8 on cell viability and expression of neurotoxic, apoptotic, and cell cycle proteins in cultured neurons. Incubation of cultured neurons with IL-8 for 24 h resulted in neuronal cell death. RT-PCR analysis of primary rat neuronal cultures treated with IL-8 for 24 h showed induction of genes for matrix metalloproteinases (MMP-2 and MMP-9), proinflammatory proteases with neurotoxic properties. Gelatin zymography demonstrated IL-8 induced MMP-2 and MMP-9 activity. Western blot analysis showed that IL-8 also increased levels of the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death). In addition, message levels of the cell cycle protein cyclin D1, an early marker for G1/S transition and a protein implicated as a regulator of neuronal apoptosis, were elevated after IL-8 exposure. These results suggest that IL-8 could be an important mediator of neuronal death in AD both via its effects on release of neurotoxins such as MMPs as well as by induction of cell cycle and pro-apoptotic proteins.

Expression of macrophage inflammatory protein 1-alpha is elevated in Alzheimer's vessels and is regulated by oxidative stress.

Inflammatory mediators are highly expressed in the Alzheimer's disease (AD) brain. We have shown that in AD the cerebral microcirculation is a rich source of cytokines and chemokines including interleukins (IL) 1beta, IL-6, IL-8, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1. However, the factors that regulate expression of these inflammatory proteins have not been defined. The objective of this study is to compare expression of macrophage inflammatory protein 1-alpha (MIP-1alpha) in brain microvessels isolated from AD patients to vessels from age-matched controls and further to determine whether expression of MIP-1alpha in brain endothelial cells is altered by oxidative stress. The data show that brain AD-derived microvessels express high levels of MIP-1alpha mRNA and release high levels of MIP-1alpha protein compared to brain microvessels isolated from controls. Treatment of brain endothelial cell cultures with menadione, a superoxide releasing compound, hydrogen peroxide, lipopolysacharride, or oxidatively modified low density lipoproteins (LDL) (Ox-LDL, HNE-LDL) results in a dose- dependent increase in MIP-1alpha mRNA levels and MIP-1alpha release into the media. These results suggest that oxidative and lipid insults to the brain microvasculature are likely to contribute to the inflammatory milieu of the AD brain.

Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons.

Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.

Angiogenic proteins are expressed by brain blood vessels in Alzheimer's disease.

Data are emerging to support the idea that mediators of angiogenesis are found in the Alzheimer's disease (AD) brain. The objective of this study is to compare the expression of the angiogenic mediators vascular endothelial growth factor (VEGF), angiopoietin, and matrix metalloproteinases (MMPs) in the microcirculation of AD patients and age-matched controls. Our results indicate that angiopoietin-2 and VEGF are expressed by AD- but not control-derived microvessels. AD-derived microvessels also release higher levels of MMP-2 and MMP-9 compared to controls. The data show that despite high levels of MMP-9, assessed by western blot, MMP-9 activity is not detectable in AD microvessels. In this regard we find high levels of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in AD, but not control vessels. Furthermore, we explore the ability of thrombin, previously shown to be present in AD microvessels, to affect TIMP expression in cultured brain endothelial cells and find that thrombin causes up regulation of TIMP-1. These data show that angiogenic changes occur in the microcirculation of the AD brain and suggest that if these changes are contributory to disease pathogenesis, targeting the abnormal brain endothelial cell would provide a novel therapeutic approach for the treatment of this disease.

Thrombin and inflammatory proteins are elevated in Alzheimer's disease microvessels: implications for disease pathogenesis.

The notion that microvascular abnormalities contribute to deleterious changes in the Alzheimer's disease (AD) brain is supported by work from our laboratory and others demonstrating biochemical and functional alterations of the microcirculation in AD. The objective of this study is to determine whether levels of neurotoxic (thrombin) and inflammatory (interleukin 8 (IL-8), integrins alphaVbeta3 and alphaVbeta5) proteins are altered in microvessels isolated from AD patients compared to levels in vessels obtained from non-demented age-matched controls. We also evaluate in AD and control microvessels expression of the transcription factor hypoxia-inducible factor 1-alpha(HIF1-alpha), which regulates pro-inflammatory gene expression, and the regulation of HIF1-alpha expression by thrombin in cultured brain endothelial cells. Our results indicate that in AD there are high levels of expression of the neurotoxic protease thrombin and the inflammation-associated proteins IL-8 and alphaVbeta3 and alphaVbeta5 integrins. HIF1-alpha is higher in AD microvessels compared to control and thrombin treatment of cultured brain endothelial cells results in increased expression of HIF1-alpha. These data suggest that in AD the cerebral microcirculation is a source of neurotoxic and inflammatory mediators and as such contributory to pathologic processes ongoing in the AD brain.