Charles Vernon Mobbs, PhD - US grants

These studies will examine regulation of heat-shock-70-like protein (hsp70) isoforms and mRNAs by reproductive hormones. Since the hsp70 family comprises several highly related but distinct beat- shock-induced and constitutive gene products, high-resolution biochemical techniques (2-D gels, Western, slot, and Northern blots) and high-resolution cytochemical techniques (in situ hybridization and immunocytochemistry) will be used in conjunction with 4 antibody and 2 cDNA probes assembled for this project, each with distinct specificities. Heat-shock gene products are induced during gametogenesis in ovaries, testes, and even sporulation, and during early embryonic development; inappropriate expression of heat-shock proteins can lead to developmental defects. In addition, oncogenes induce hsp70 gene products and these products are elevated in transformed cells. Some hsp70 isoforms facilitate transport of secreted proteins into the endoplasmic reticulum and remove clathrin from coated vesicles, suggesting a role in secretion, but the regulation and role of hsp70 gene products in reproduction are not understood. However, estrogen and prolactin induce hsp70 mRNA. Furthermore, the most prominent estrogen- induced protein in ventromedial hypothalamus (EI70) and LHRH- induced protein (LHRH70) in pituitary is apparently the same hsp70- like isoform. Thus hsp70 isoforms may be generally involved in reproductive neuroendocrine processes. (I) The relationship between EI70, LHRH70 specific hsp70 isoforms will be studied using ATP binding, co-migration, immunoblots, peptide maps, and protein sequencing. (II) The induction of hsp70 isoforms and mRNA by estrogen, LHRH, prolactin, and prostaglandin E2 in brain, pituitary, and uterus will be examined using 2-D gels, immunocytochemistry, Northern and slot blots, and in situ hybridization, and correlated with regulation of reproductive functions. (III) The subcellular localization of hsp70 proteins after hormonal stimulation will be assessed with immunocytochemistry and subcellular fractionation, since localization of hsp70 isoforms is regulated by beat-shock and during the cell cycle. (IV) If time permits, hsp24 and hsp108, whose mRNAs are regulated by reproductive steroids, will be studied similarly using available probes. These studies may provide better understanding of the reproductive regulation of heat-shock proteins and their functional relation to reproductive cancers, birth defects, and fertility.

The classic glucostatic hypothesis suggests that energy metabolism is regulated in part by glucose-sensitive cells in or near the ventromedial hypothalamus (VMH), impairments in which can cause obesity. The proposed studies are based on the hypothesis that some hypothalamic cells sense glucose through a mechanism similar to the mechanism in pancreatic beta cells. Glucose induces c-fos and jun-b mRNAs, as well as electrical activity, in the VMH. VMH cells appear to express pancreas-specific glucokinase (pGK), implicated as an important component of the glucose sensing mechanism in pancreatic beta cells. The proposed studies will examine how closely VMH mechanisms parallel pancreatic mechanisms, and if these mechanisms are impaired in genetic obesity. Are VMH responses to glucose mediated by glucose metabolism? VMH in brain slices in vitro will be exposed to glucose or metabolites for 30 minutes, and induction of jun- b and c-fos mRNAs, as well as electrical activity, will be assessed. Are pancreatic glucokinase or other metabolic enzymes involved in glucose sensing by hypothalamic cells? Pancreatic glucokinase activity in hypothalamus will be increased by gene transfer using an adeno-associated virus (AAV) expressing pGK. Conversely, glucokinase expression will be attenuated by anti-sense nucleotides. Rats will be monitored chronically for changes in metabolism and adiposity, after which molecular and electrical responses to glucose will be assessed. Effects of fasting on pyruvate dehydrogenase El alpha and pyruvate carboxylase (and other genes) will be assessed. Is glucose-regulated gene expression in VMH impaired in genetic obesity? Responses of VMH to glucose in fa/fa genetically obese rats will be assessed as in the previous studies. In addition, pGK expression in VMH of fa-fa rats will be increased by gene transfer, and subsequent gene expression and metabolism (including body weight) will be assessed. These studies should clarify mechanisms relevant to the metabolic derangements of obesity.

Broadly speaking a "typical" (though not universal) profile of neuroendocrine change during aging in mammals could be described as often involving decreased plasma luteinizing hormone (LH)and sexual function, decreased plasma growth hormone(GH), decreased plasma thyroxine, and increased plasma glucocorticoid, Interestingly, a similar neuroendocrine profile is exhibited during fasting and in genetically obese mice. Recent studies have indicated that at least some of the neuroendocrine effects of fasting are mediated through the product of the obese gene, leptin, leptin levels fall during fasting, and injection of leptin attenuates the effects of fasting on the hormones described above. It has recently been shown that leptin mRNA and peptide progressively decline with age in C57B1/6J mice. These data suggest the hypothesis that some age-related neuroendocrine impairments are due to relative leptin insufficiency, and therefore that some neuroendocrine impairments may be partially revered by treatment with leptin. There the study will asses if injection of leptin in aging mice, at doses which will restore leptin to youthful levels, will partially restore some neuroendocrine functions toward youthful levels, Six-, 12-, and 24-month-old male and female C57/B1 6j mice will be assessed for sexual behavioral (for males), estrous cycles (for females), metabolic rate, and locomotor activity. Blood will be obtained by reto- orbital puncture to plasma leptin. Mice will then injected for 7 days with either saline or 1 ug/gm b.w. leptin (previously show to produce fed levels of leptin in fasted C57b1/6j mice). During this treatment, behaviors, estous cycles, and metabolic parameters will continue to be assessed. One hour after the last injection of leptin, mice will be sacrificed by decapitation to allow assessment of non-stresses levels of L H, testosterone, GH, T3, glucocoriticoids, insulin, and leptin, and also to allow assessment of leptin-stimulated c-fos mRNA (to assess if aging mice show evidence of leptin resistance), as well as body composition. These studies may provide evidence that some age-related impairments my be treatable by leptin replacement therapy.

Impairments in autonomic function are a major complication of diabetes. The autonomic nervous system involves complex interactions between central neurons and peripheral ganglia. In particular, hypothalamic and brainstem neurons sensitive to glucose and nutritional status play a critical role in regulating the autonomic nervous system. Glucose-sensitive hypothalamic neurons appear to sense glucose through a beta cell-like mechanism. A subset of hypothalamic and brainstem neurons, like beta cells and peripheral neurons but in contrast to other central neurons, are highly sensitive to deleterious effects of and glucose derivatives. Thus some diabetes-induced impairments in autonomic activity may be due to diabetes-induced damage to these glucose-sensitive hypothalamic and brainstem neurons. The proposed studies will characterize effects of diabetes on hypothalamic and brainstem neurons, including effects of diabetes on (i) regulation of key gene products (POMC and CART) thought to be produced by glucose-sensitive neurons; (ii) the ability of these neurons to sense glucose and nutritional status; (iii) structural impairments and possible loss of these neurons. In addition, the proposed studies will assess the correlation between hypothalamic and brainstem impairments and impairments in autonomic ganglia, the vagus nerve, and sympathetic function (regulation of temperature, heart rate, and counterregulatory responses to hypoglycemia). Finally, the proposed studies will assess the role of non-enzymatic glycation in diabetes-induced impairments observed in Studies I and II. Since diabetic neuropathy is thought to entail reversible, presumably metabolic, impairments, and irreversible, presumably structural, impairments, the present study will distinguish between reversible and irreversible impairments by comparing effects of uncorrected diabetes, diabetes corrected with intensive insulin therapy, and diabetes corrected with islet transplants. The present study should clarify the mechanisms and significance of glucose-sensitive hypothalamic and brainstem neurons in autonomic diabetic neuropathy.

The long-term objective of the proposed studies is to assess the role of neuroendocrine responses to caloric restriction in mediating effects of caloric restriction on age- related impairments and life span. The role of neuroendocrine systems in mediating effects of caloric restriction on life span may entail two distinct mechanisms. One possible mechanism, termed "hysteretic", is that nutritional stimulation cumulatively damages essential nutrition-stimulated hypothalamic neurons (especially including neurons that produce POMC). Since nutrition-stimulated hypothalamic neurons produce catabolic effects, erosion of these neurons would lead to the enhanced anabolic tone observed with age, with the consequent deleterious metabolic syndrome, including hyperinsulinemia. An alternate mechanism, which may be viewed as homeostatic, is that caloric restriction produces neuroendocrine responses, such as elevated glucocorticoids and reduced growth hormone, that effectively protect the organism, leading to increased life span. In this case the anabolic tone developed by the aging neuroendocrine system, possibly due to impaired sensitivity to nutritional factors, might actually be protective. The present proposal will address these distinct mechanisms. (1) Why does expression of hypothalamic POMC decrease with age? Degeneration vs. insensitivity. If nutritional stimulation cumulatively damages nutrition-simulated hypothalamic neurons, then expression of nutritionally stimulated hypothalamic genes should preferentially decrease with age. Alternatively, expression of POMC amay decrease due to decreased sensitivity to nutritional sensitivity. To assess these predictions, the number of neurons in the nutrition-stimulated hypothalamic field, especially neurons expressing POMC in 6-, 15-, and 24-month-old mice will be counted using stereological methods. Electrophysiological responsiveness of hypothalamic neurons to glucose, leptin, and insulin at the same ages will also be assessed. Finally, the prediction that nutrition-stimulated hypothalamic mRNAs are specifically susceptible to aging will be assessed using DNA array analysis. (2-4) What are the roles of neuroendocrine responses dependent on POMC, leptin, and glucose in mediating effects of caloric restriction on age-related impairments? If neuroendocrine responses mediate effects of caloric restriction on age-related impairments, then blocking those responses should block those effects. To assess this prediction, transgenic mice have been produced that express POMC, leptin, or glucokinase under control of the neuron-specific enolase promoter; it is anticipated that these transgenes will block these neuroendocrine responses to caloric restriction that depend on POMC, leptin, or glucose, respectively. Effects of these transgenes on age-related impairments and longevity will be assessed in pair-fed and calorically restricted mice. These studies should clarify mechanisms mediating effects of caloric restriction on age- related pathologies and longevity.

DESCRIPTION (provided by applicant): Because the brain is highly dependent on glucose, hypoglycemia is neurotoxic, and would be even more so except that hypoglycemia produces robust neuroprotective and counterregulatory responses. The mechanisms mediating these protective effects to hypoglycemia are unclear. However, DNA microarray data indicate that hypoglycemia induces adenosine, and adenosine receptors are known to mediate many neuroprotective responses to metabolic stresses such as ischemia and hypoxia. On the other hand, drugs that block the both the A1 and A2a adenosine receptors enhance counterregulatory responses in humans, including in diabetic patients. Since A1 adenosine receptors mediate cytoprotective responses to ischemia and hypoxia, A1 receptors may also mediate neuroprotective responses to hypoglycemia. In contrast, A2a receptors can actually potentiate neurotoxicity, possibly by antagonizing A1 receptors. Furthermore, A2a receptors mediate hypoglycemia-induced vasodilation, possibly accounting for the effects of caffeine and theophylline to enhance counterregulation. Therefore specific blockade of A2a receptors and/or activation of A1 receptors might improve neuroprotective and counterregulatory responses to hypoglycemia in patients with diabetes. To assess the feasibility of this approach, the proposed study will assess the influence of A1 and A2a receptors on responses to hypoglycemia, which has not been examined in vivo. Since pharmacological antagonists of the A1 and A2a receptors are of limited specificity and permeability through the blood-brain barrier, the proposed studies will assess responses to hypoglycemia in mice in which the A1 and/or A2a genes are genetically deleted (A1 and A2a knockout mice). These studies could lead to the development of A2a receptor antagonists to improve outcome of hypoglycemia in patients with diabetes.

The long-term objectives of the proposed studies is to discover genes that mediate the development of obesity in humans. Several lines of evidence suggest that neurons sensitive to the neuroendocrine effects of glucose regulate body weight, implying that attenuation of these glucose-sensing mechanisms could cause obesity. For example, attenuation of glucose sensing systems (glucopenia) by the glucose analog 2- deoxyglucose (2-DG) robustly decreases metabolic rate and increases feeding in mammals. However, molecular mechanisms mediating the effects of glucose on body weight regulation have been difficult to study in mammals and are largely not understood. Fortunately, 2-DG, which produces obese phenotypes in mammals, produces rapid and striking obesity in C. elegans. The proposed studies will therefore use RNAi in C. elegans to systematically screen for genes whose ablation blocks 2-DG-induced obesity, focusing specifically on genes which have homologs in both mammals and C. elegans. Specific Aim 1 will assess if ablation of specific genes mediating neuroendocrine regulation (G protein coupled receptors and ligand- regulated ion channels) will block glucopenia-induced obesity. Specific Aim 2 will assess if ablation of specific genes induced by hypoglycemia in mouse hypothalamus will block glucopenia-induced obesity. Specific Aim 3 will assess if ablation of specific genes induced in mouse tissues with diet-induced obesity will block glucopenia-induced obesity. Specific Aim 4 will assess if ablation of genes implicated by the first three Specific Aims in glucopenia-induced obesity will block other forms of obesity (Daf-2, etc.) in C. elegans. Conversely, Specific Aim 4 will also assess if ablation of genes implicated in other forms of obesity in C. elegans will block glucopenia-induced obesity. Of particular interest will be genes whose ablation does not produce an obvious phenotype in standard conditions, but whose ablation blocks glucopenia-induced obesity. These studies will suggest potential targets for anti-obesity drugs.

Hypothalamic neurons can sense and respond to changes in glucose concentration, but the functional significance of this glucose-sensing property remains to be determined. As in pancreatic beta cells, glucokinase (GK) constitutes a key component of the hypothalamic glucose-sensing mechanism. Homozygous whole-body ablation of the GK gene produces lethal neonatal diabetes, and heterozygous whole-body ablation of the GK gene causes impaired glucose homeostasis and obese phenotypes, including reduced POMC and increased AgRP gene expression in hypothalamic neurons. Interestingly, homozygous whole-body ablation of the insulin receptor also produces lethal neonatal diabetes, and neuron-specific expression of the insulin receptor partially rescues this phenotype. We have now demonstrated that heterozygous and homozygous ablation of the GK gene specifically in neurons recapitulates the respective whole-body GK knockout phenotypes. On the other hand, other studies have suggested that GK in hypothalamic tanycytes (a type of glial cell) also plays a role in metabolic homeostasis. We therefore propose that different aspects of metabolic homeostasis are dependent on GK expression in POMC neurons, AgRP neurons, or tanycytes. To address this hypothesis, in Specific Aim 1 we propose to ablate GK specifically from POMC neurons by crossing transgenic mice expressing cre-recombinase under control of the POMC promoter (POMC-cre) with mice in which the GK gene is flanked by lox sites. Complementing these studies, we propose to restore GK specifically in POMC neurons in (homozygous or heterozygous) whole-body GK knockout mice, using the "knock-in" strategy by which Okamoto et al. rescued lethal neonatal diabetes by restoring insulin receptors specifically to neurons in whole-body insulin receptor knockout mice. Mice will be maintained on a low-fat or a high-fat diet. Metabolic phenotypes, including leptin sensitivity, glucose and insulin tolerance tests, food intake, body weight, adiposity, metabolic rate, and temperature will be assessed. Mice will be sacrificed, and gene expression will be determined in hypothalamus and other tissues. We propose similar studies in Specific Aims 2 and 3, using AgRP, or GFAP cre-recombinase (expressed in tanycytes) to ablate or restore GK specifically in in AgRP neurons, or to ablate GK in tanyctes. We anticipate that ablation of GK in specific hypothalamic cell types will partially recapitulate specific metabolic impairments produced by whole-body ablation of GK, and that restoration of GK to POMC or AgRP neurons will reverse specific metabolic impairments caused by whole- body GK deficiency

DESCRIPTION (provided by applicant): We propose the rapid decline of CBP and associated histone acetyltransferase (HAT) activity during maturation serves the function of ending a series of critical periods of organization, especially in the brain, but by reducing transcriptional flexibility leads to a loss of adaptive capacity during aging. We demonstrated significance of the protective effect of the CBP complex during aging in studies showing that this complex is induced by dietary restriction and that blocking this induction blocks several protective effects of dietary restriction. We therefore propose to examine in more detail mechanisms by which CBP functions as a juvenile protective factor whose depletion after maturation drives age-related impairments. I. Causes and consequences of decline in CBP-HAT activity during maturation. Specific lipids after weaning reduce the rapid reduction of CBP during maturation. We therefore propose to assess mRNA of CBP and other transcription factors or histone acetylation in several brain areas liver, and jejunum from male and female mice at birth, and 1, 2, 3, and 4 weeks after birth;mice will be weaned either to a normal high carbohydrate diet or a high-fat diet, Since HDAC inhibitors can compensate for reduced HAT activity, and we have shown that HDAC inhibitors increase lifespan and improve some age-related impairments, an additional group of mice will be treated with an HDAC inhibitor. II. Long-term consequences of diet during maturation Breastfeeding produces long-term health benefits possibly mediated by lipids in milk. We therefore propose to assess if weaning mice onto a diet high in specific lipids until 3 months of age will produce permanent elevation in the CBP transcriptional complex with concomitant improvement in histone acetylation and adaptive capacity (memory, rhythms, and response to nutritional deprivation) during aging. III. Reversal of age-related impairments by HDAC inhibitors 6- and 16-month-old mice will be treated with HDAC inhibitors for 2 months and histone acetylation and memory, rhythms, and response to nutritional deprivation will be assessed. We anticipate that weaning to a diet high in lipids and treating with HDAC inhibitors will cause improve histone acetylation and adaptive capacity during aging. The proposed studies involve assessing if two interventions, post-weaning to a high-fat diet, and a drug that we have shown to be protective in a model organism, will have a protective effect during aging. If successful human trials may ensue.

The purpose of the proposed project is to assess if genetic and pharmacological manipulations that increase lifespan will similarly delay the onset of pathologies in models of Alzheimer's Disease (AD), with the ultimate goal of developing better treatments for this devastating and expensive disease. There are currently no effective treatments for this disease, which is on course to bankrupt the American health system in less than 30 years, despite tremendous and expensive efforts by pharmaceutical companies. The proposed studies take a different approach than taken by pharmaceutical companies, which have been based on specific targets. Instead the proposed studies, in response to NIH PAR-18-596, is based on the concept that since age is the major risk factor for AD, and genes that produce AD in humans produce pathologies in model organisms whose time-course scales with lifespan, manipulations that increase lifespan might also delay the onset of AD in humans. Indeed we and others have already demonstrated that some genetic manipulations and drugs that increase lifespan in the model organ C. elegans also delay symptoms in a standard transgenic model of AD, and some of these discoveries have led to current clinical trials in human AD. However, only a very small fraction of manipulations known to increase lifespan have been assessed for their effects on impairments in models of AD. We therefore propose to address this deficiency by assessing effects of genetic and pharmacological manipulations that reliably increase lifespan on three different C. elegans models of AD: muscle-specific human Abeta 1-42 (standard model), and neuronal-specific human Abeta and Tau, both implicated in human AD. We will also conversely assess if drugs we have already discovered to protect in the muscle-specific Abeta model will also protect in the neuron- specific models of AD and to increase lifespan. Based on the success of the small number of similar studies which we and others have carried out, leading to clinical trials in human AD, we anticipate that the presently proposed studies will vastly increase the available drugs and drug targets promising to treat human AD.