Farida Sohrabji - US grants

This grant addresses estrogen ability to regulate trophic support for forebrain cholinergic circuits known to be at risk in Alzheimer's disease. Cholinergic neurons in the diagonal band of Broca (nDBB) project to, and obtain trophic support from, neurons in the olfactory bulb. Trophic support for cholinergic neurons is provided by a family of growth factor peptides called the neurotrophins and the proposed experiment will test whether estrogen regulates cholinergic function by modulating the availability of neurotrophins. These experiments are based on the hypothesis that reduced trophic support to hormone-sensitive cholinergic neurons resulting from an age- or disease-related decline in estrogen may contribute to the risk of Alzheimer's disease. Experiments in Specific Aim I will test whether estrogen can restore cholinergic function in neurons deprived of olfactory bulb-derived neurotrophins: An in vivo paradigm consisting of intact, ovariectomized and estrogen-replaced animals will be used to assess estrogen's neuroprotective effects on basal forebrain cholinergic neurons following the destruction of bulbar cells by excitotoxic lesion. These studies will also test whether estrogen can, in a lesioned model, stimulate neurotrophin-mediated adaptive responses in other forebrain targets of the septum-diagonal band cholinergic neurons. Experiments in Specific Aim II will examine whether estrogen regulates the ability of cholinergic neurons to bind and transport target-derived growth factors from the olfactory bulb. Using a similar paradigm (intact, ovariectomized, estrogen replaced-ovariectomized animals), these experiments will test whether estrogen modulates the expression of p75, (the pan-neurotrophin receptor) as well as trkA and trkB (the specific tyrosine kinase receptors for NGF and BDNF) in basal forebrain neurons. Furthermore, these experiments will test whether estrogen replacement stimulates receptor mediated transport of neurotrophins from the bulb. Due to its proximity and connections with the nasal mucosa, bulb neurons are especially vulnerable to environmental toxins and viruses, as are its efferent fibers that obtain trophic support from the bulb. Factors that regulate trophic support for cholinergic afferents during injury may significantly impact on the etiology and clinical management of forebrain neurodegenerative diseases. Molecular actions of estrogen on the forebrain are particularly relevant in view of the recent retrospective studies and small-scale clinical trials which suggest that estrogen replacement therapy (ERT) may be beneficial for the management of Alzheimer's disease.

DESCRIPTION (provided by the applicant): The overall goal of this research is to define the mechanisms and consequences of estrogens actions on the forebrain, which we will address in two specific aims. The first aim is to determine the cellular consequences of estrogen-mediated alterations in the ratio of neurotrophin receptors. In the adult forebrain, estrogen increases trk receptors and decreases p75, resulting in a favorable trk/p75 ratio, while in the reproductively senescent forebrain, estrogen replacement, paradoxically, results in an unfavorable trk/p75 ratio. While both neurotrophin receptors stimulate distinct and overlapping signaling pathways, the trks typically promote cell survival, while p75 may stimulate either cell survival or cell death. Here, our in vivo models will be used to assess how estrogen mediated changes in trk/p75 ratios will affect down-stream neurotrophin signaling and, consequently, cell fate, when challenged, in vivo, with neurotrophin stimulation or injury. The second aim is to test the hypothesis that expression/activation of the alpha form of the estrogen receptor (ER-alpha) is detrimental to cell health. Two estrogen receptors have been identified, although the specific contribution of each receptor to neural function is not clearly defined. Recent observations indicate that high ER-alpha expression is associated with decreased estrogen responsiveness on measures such as neurotrophin expression and cell survival. Estrogen receptor specific ligands, in conjunction with ex vivo and in vitro models, will be used to determine the contribution of each estrogen receptor to neurotrophin expression, signal transduction and cell death. These studies will also employ DNA microarray analysis, with the eventual goal of developing strategic gene arrays to facilitate rapid detection of estrogen-mediated cellular changes and to discriminate between the actions of receptor specific ligands. In view of the recent conflicting evidence regarding estrogen use and Alzheimer's disease, understanding the biology of estrogens actions are more critical now than ever. Moreover, identifying hormone-stimulated pathways that initiate cell-degenerative events will be increasingly important to the development of target and receptor specific estrogenic compounds.

The goal of this application is to determine the mechanisms by which reproductive aging and estrogen replacement alter the inflammatory response and consequently the neuronal environment. In a series of studies, we have established that estrogen replacement to young adult animals increases trophic support in the forebrain and attenuates inflammation following neural injury. However estrogen replacement at reproductive senescence, which is physiologically akin to menopause, fails to increase trophic factors and paradoxically, increases inflammatory mediators following neural injury. Collectively these data suggest that the timing of estrogen replacement in relation to reproductive aging may critically determine whether estrogen has a benign or deleterious outcome. Our central hypothesis is that the age-related decline in endogenous hormones triggers compensatory changes in estrogen receptor systems in specific immune cells, thus increasing the central and peripheral inflammatory response. This hypothesis will be tested in three Specific Aims, using animal and human tissue models that span the reproductive spectrum, namely, normally cycling (pre-menopause). irregularly cycling (perimenopause) and reproductive senescent (postmenopause). In Specific Aim 1. we will test the hypothesis that permissive changes in the blood brain barrier will cause a more rapid and robust neural inflammation in reproductive senescent animals as compared to normally cycling or irregularly cycling animals. Animals will be injected systemically with the bacterial pathogen lipopolysaccharide (LPS) and inflammatory mediators will be measured in peripheral organs and the brain. Additionally, we will examine endothelial cells of the blood-brain barrier for reproductive age-related changes in this barrier. In Specific Aim 2. we will determine if the inflammatory response of peripheral blood mononuclear cells (PBMC) is affected by clinically-relevant variables namely, the route of hormone administration (oral versus transdermal) and diet (regular versus high cholesterol). The Response Quotient, derived from an ex vivo LPS challenge assay, will be measured in rat and human blood samples to determine if salient lifestyle variables increase the risks associated with reproductive aging. Finally, in Specific Aim 3 we will test the hypothesis that compensatory alterations of the estrogen receptor system, resulting from ovarian decline, is a principal mechanism underlying estrogen's deleterious effects in reproductive senescence. Changes in the pattern and levels of estrogen receptor (ER)-alpha will be evaluated by immunohistochemistry and Western blots, while functional changes will be evaluated using signaling arrays. Human and rodent PBMC's and rodent cerebral endothelial cells from each reproductive stage will be studied. Collectively, these studies will test the hypothesis that in order for estrogen replacement to be beneficial, therapy must be initiated before compensatory responses to ovarian decline.

DESCRIPTION (provided by applicant): The goal of this application is to determine the impact of ovarian aging on neural inflammation. Several immune cell types are sensitive to gonadal steroids such as estrogen and the loss of these ovarian hormones at menopause significantly affects the immune system and the inflammatory response in target organs such as the brain, bone, skin and cardiovascular system. We are specifically interested in determining the effects of ovarian aging or reproductive senescence on the blood brain barrier and the neural inflammatory response. Inflammation is believed to contribute to the etiology of neurodegenerative diseases such as Alzheimer's and multiple sclerosis, and women as a group appear to be at a higher risk for these diseases. Ordinarily, the brain is protected from circulating immune cells and their cytotoxic products by the blood brain barrier. However, changes in the integrity of the blood brain barrier can exposed fragile, and often irreplaceable, neural networks to proteins that are fatal for them. Using an animal model of reproductive senescence, we have shown that the blood brain barrier of reproductive senescent females is more "permissive" to intravenously injected dye as compared to young adult females. Furthermore, estrogen treatment increases dye transfer into a key brain region of the senescent brain, while suppressing dye transfer in young adult females. A similar dichotomy was seen in estrogen action in our forebrain injury studies, where estrogen suppressed inflammatory cytokine production in young females but exacerbated their levels in the senescent brain. Based on these studies, our central hypothesis is that ovarian aging increases the permeability of the blood brain barrier and increases cytotoxicity in the forebrain. This hypothesis will be tested in two Specific Aims: Specific Aim 1 will test the hypothesis that reproductive aging results in physical and functional changes in the blood brain barrier, causing this structure to become more permissive to circulating immune cells and proteins. Specifically, we will use fluorescent- and radio-labeled tracers to determine permeability of the blood brain barrier, and protein detection assays to determine changes in tight junction proteins, adherence proteins and matrix-cleaving enzymes of the blood brain barrier. Specific Aim 2 will test the hypothesis that systemic insults will result in a rapid and/or prolonged neural inflammation with greater neurodegeneration in reproductive senescent animals as compared to young adult animals. Two systemic insults will be used: an experimental sepsis model using LPS injections and a clinically-relevant inflammatory insult consisting of a high cholesterol diet. We will measure inflammatory mediators in peripheral organs (liver) and specific brain regions, leukocyte trafficking in the brain due to this systemic insult and neuronal cell death.

DESCRIPTION (provided by applicant): Stroke is the leading cause of disability in the US and with heart disease, the leading cause of death. The risk for stroke with consequent functional disability is increased with age, and in women this risk is elevated after the menopause. Paradoxically, hormone therapy at menopause increases the risk for stroke. Animal models of stroke confirm that stroke severity is worse in aged animals as compared to younger animals. In middle age, our recent data shows that female rats sustain a greater degree of tissue damage in the cortex and striatum as compared to younger females. Middle aged males, on the other hand, do not differ significantly from younger males in the extent of cortical infarction. This age difference in cortical cell loss is also paralleled by functional changes in astrocytes, a specific brain support cell. Astrocytes play a key role in normal and pathological conditions. Following stroke, astrocytes are rapidly mobilized to the peri-infarct area, detoxify the injured brain via glutamate uptake and fluid efflux and secrete growth factors known to promote angiogenesis and neuronal survival and neurogenesis. Astrocytes culled from the ischemic cortex of middle aged female rats show profound loss of protective functions including a reduced ability to sequester glutamate, decreased growth factor release, increased release of chemokines and increased ability to recruit leukocytes. These changes are consistent with increased infarct volume observed in older females. Hence in this proposal we will determine age and sex-specific epigenomic changes in astrocytes obtained from the ischemic cortex, to determine critical translational and transcriptional modulators. In Specific Aim 1 we will determine age-related changes in the expression of small non-coding RNA. MicroRNA, a key translation regulatory element, regulates large gene networks, and have been shown to play a central role in cell senescence and injury (stroke). In Specific Aim 2 we will determine age-related changes in DNA and histone methlyation patterns. Methylation patterns of specific leucines associated with activation (H3K4me3 and H3K9ac) or repression (H3K9me3 and H3K27me3) of gene transcription will be targeted. These complementary approaches will allow us to develop a molecular fingerprint of the aging astrocyte. Finally, in Specific Aim 3, select molecular targets will be manipulated using (1) miRNA mimetics or antagomirs and (2) demethylases to reverse age-specific patterns in astrocytes. Data gathered from these studies is expected to aid in the eventual identification of epigenomic changes that predict disease severity and facilitate discovery of therapeutic targets. PUBLIC HEALTH RELEVANCE: The risk and disability associated with stroke increases with age. In order to develop more effective therapies for this disease, this application will focus on age-related changes in a specific brain cell called the astrocyte. Our studies using an animal model show that middle-aged females sustain more brain damage after stroke than younger females and this is associated with functional changes in the neuroprotective ability of astrocytes. We will seek to understand global age-related changes in this cell type so as to develop markers for disease severity as well as new therapeutic targets.

DESCRIPTION (provided by applicant): In women, the disruption of the endocrine environment during menopause amplifies the risk for stroke and neuro-inflammatory disease. Using acyclic female rats to model the postmenopausal state, our studies indicate that these animals sustain greater tissue damage following experimental ischemic stroke as compared to mature (cyclic) adult females. Furthermore, while estrogen treatment is protective to mature adult females, it paradoxically increases tissue damage in older acyclic females. These data are congruent with the WHI study, where the risk for stroke was elevated in older women receiving hormone therapy, and underscores the need for novel therapeutic approaches for this group. Based on our recent studies, we hypothesize that estrogens neuroprotective actions require the cooperative action of insulin-like growth factor (IGF)-1, a neuroprotective peptide hormone, and that age-related decline in IGF-1, which occurs in virtually all species, destabilizes the neuroprotective actions of estrogen. This hypothesis is supported by our studies in that (1) IGF-1 levels are reduced in the middle aged (reproductive senescent) female rat, and (2) in a stroke model, IGF-1 infusion to older females overcomes the neurotoxic effects of estrogen in this group. Here we will examine the cooperative interaction of estrogen and IGF-1 on stroke recovery and infarct volume and the extent to which this interaction is altered in reproductive senescence. Secondly, we will examine the extent that estrogen/IGF-1 can improve astrocyte function post stroke in reproductive senescent females. Astrocytes are responsive to both IGF-1 and estrogen and play a crucial role in detoxification of the ischemic environment. Finally we will determine if stroke outcomes can be improved in older females by manipulating epigenetic regulators of IGF-1 namely, microRNA. MiRNA, a type of small non-coding RNA, regulate large gene networks, and play a central role in cell senescence, proliferation (cancer) and injury (stroke). Specifically, we will identify and manipulate miRNA that regulate IGF-1 to improve stroke recovery on older females. Collectively these studies will provide an understanding of estrogen's age-delimited neuroprotective effects and form the foundation for pre-clinical studies of stroke therapy tailored to older females.

Project(Summary( Fetal Alcohol Spectrum Disorders (FASD) result in life-long systemic disabilities that contribute to disease and premature mortality in FASD adults. We recently found that prenatal alcohol-exposure (PAE) led to long-term deficits in cranially-directed vascular function in aging mice. PAE also diminished neurological recovery in young adult mice following cerebrovascular ischemic stroke. Preliminary data indicate that middle-aged PAE animals experience larger stroke infarcts compared to age-matched controls or young PAE adults. Moreover, reduced levels of the peptide hormone, IGF1, and epigenetic re-programming of IGF pathways contribute to ischemia-induced brain damage and disability, while intracranial IGF1 delivery after stroke improves tissue survival and behavior. Therefore, we hypothesize that ?PAE accelerates the age-dependent increase in brain vulnerability to ischemic stroke by epigenetically programming IGF1 signaling pathways??. We plan to assess effects of PAE on brain adaptation to ischemia in aging male and female adults in rat models, and consistent with stroke research guidelines, use two models for ischemic stroke by intraluminal suture-occlusion and by endtothelin-1-mediated vasoconstriction of the middle cerebral artery. Aim 1 will determine the extent to which PAE influences brain damage, sensorimotor impairment, and blood brain barrier (BBB) permeability, in aging adults following ischemia. Our working hypothesis is that the middle- aged PAE brain will exhibit a larger infarct volume following ischemia, compared to age-matched controls, and comparable to the aged non-PAE adult brain. Middle-aged and aged PAE animals will also exhibit increased sensorimotor impairment, accompanied by prolonged BBB permeability following an ischemic episode compared to age-matched, non-PAE controls. Aim 2 will assess the contribution of PAE to aging-related epigenetic reprogramming of IGF1 pathways. Our working hypothesis is that PAE epigenetically reprograms liver and brain resulting in aging-related loss of IGF1 in adulthood. We expect that PAE will result in chromatin silencing or miRNA-mediated translation-repression of IGF1 signaling. Aim 3 will determine the impact of exogenous IGF1, or epigenetic stimulators of hepatic or brain IGF1, on ischemia outcomes in PAE adults. Our working hypothesis is that IGF1 supplementation after ischemia will ameliorate effects of PAE on the BBB, infarct volume, and sensorimotor function in aging animals. We will test the extent to which effects of PAE on stroke-induced impairment are ameliorated by post-stroke treatment with IGF1, or with agents that promote IGF function, like sodium butyrate, a histone deacetylase inhibitor, and an antagomir to the microRNA Let7. This proposal tests an innovative hypothesis that PAE increases risk for adverse outcomes due to adult-onset disease, in an experimentally rigorous way. It is significant because it addresses a critical knowledge gap about brain vulnerability in aging adults with FASD. The investigators have a history of collaboration, and bring complementary expertise to studies that will inform clinical care of adults with FASD.

Circadian clocks throughout the body provide for the local coordination of tissue- or cell-specific processes including the homeostatic regulation of key endocrine signals, growth factors and cytokines that mediate cardiovascular health. Circadian rhythm desynchronization, in the form of jet lag, shift work and irregular sleep patterns, has been shown to increase the risk for stroke as well as exacerbate stroke severity/recovery. At present, little is known about how circadian dysregulation interacts with other non-modifiable risk factors such as biological sex and advancing age to modulate the pathophysiology of stroke. The proposed experiments will use an established rat model to study how interactions between circadian rhythm dysregulation and two non- modifiable risk factors, biological sex and aging, modulate pathogenic responses to ischemic stroke. The primary objectives of these studies are to determine whether the effects of circadian rhythm dysregulation during early adulthood persist after intervening stable light-dark entrainment (for 3mo) so as to promote a chronic basal pro-inflammatory state and thus exacerbate the pathophysiological severity of strokes that occur in middle age (10mo of age), when stroke susceptibility increases in both sexes. Comparisons of adult (5mo) male and female rats will be used to identify sex differences in the ?after? or diathetic effects of circadian rhythm desynchronization on the differential activation of pro-inflammatory cytokines, and on stroke-induced brain damage and functional deficits. Overall, the objectives of this project are to establish a translational model that closely replicates epidemiologic data, and to promote the development of therapeutic interventions or other strategies (e.g., adaptive changes in industry standards for managing shift work duration and schedules) for reducing stroke risk and severity in individuals with a history of shift work or irregular schedules.