Louise D. McCullough, MD, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Despite advances in our understanding of neuronal responses to cellular stress, interventions for acute neuronal injury remain elusive. One novel avenue of investigation is to identify changes in neuronal metabolism incurred as a consequence of cerebral ischemia. AMP-activated protein kinase (AMPK) is a protein kinase that plays a key regulatory role in energy metabolism in both the brain and peripheral tissues. AMPK is activated via phosphorylation in times of energy demand, as monitored by increasing AMP and declining ATP levels. AMPK reduces fatty acid, cholesterol, and protein biosynthesis and increases catabolic, ATP generating pathways. Recently, numerous stimuli including hypoxia, ischemia/reoxygenation injury, NAD, peroxynitrite (ONOO) and nitric oxide (NO) have been shown to activate AMPK suggesting that AMPK plays a critical role in the response to oxidative stress. We have shown that robust activation of AMPK occurs after middle cerebral artery occlusion (MCAO). Interestingly, this stroke-induced elevation in AMPK is detrimental, as pharmacological inhibition of AMPK is dramatically neuroprotective. In aim 1 we will test the hypothesis that reduction in AMPK activation leads to sustained neuroprotection and functional improvement after stroke. The effects of pharmacological agents that decrease AMPK activation will be examined. Aim 2 will evaluate the response to pharmacological activation of AMPK after MCAO. In Aim 3 we will examine animals with targeted deletions of the catalytic subunit of AMPK to confirm our pharmacological data and determine the isoform responsible for the neurotoxicity seen after MCAO. In Aim 4 we will examine the hypothesis that AMPK activation leads to neuronal damage specifically via activation of neuronal NOS (nNOS). Elucidation of the role of AMPK in ischemia may lead to the development of novel treatments for stroke. Relevance: Stroke is the leading cause of disability in the U.S. Interfering with metabolic pathways could prevent or delay cell death by reducing energy demand in the damaged brain. Delaying cell death could prolong the "therapeutic window" in stroke. A multi-faceted approach is clearly needed to develop efficacious neuroprotective agents that will benefit stroke patients. Manipulation of AMPK levels represents a novel approach to neuroprotection. [unreadable] [unreadable] [unreadable]

Clinically, ischemic stroke is recognized as a sexually dimorphic disease. Reproductive hormones are a major contributor to differences in male and female pathobiology. However, emerging data clearly show that outcome from stroke is shaped by biologic sex in addition to hormone exposure. The major hypothesis of this proposal is that distinct gender-based cell death programs are activated after an ischemic insult. We will investigate and identify molecular cell death pathways that are differentially regulated by gender. The overall goal of this proposal is to identify and evaluate the major pathways of cell death activated in male and female brain after in vivo middle cerebral artery occlusion (MCAO). We hypothesize that male cell death is mediated primarily by the caspase-independent activation of nitric oxide (NO), peroxynitrite formation, oxidant-induced DMA damage, and activation of PARP-1, leading to the release of apoptosis inducing factor (AIF) from mitochondria. In contrast, cell death in females is predominately caspase- dependent and secondary to ischemia-induced release of cytochrome C, activation of caspase-9 and formation of the apoptosome. Selective interference with each of these pathways will lead to different outcomes in males and females. In Aim 1we will examine hormonally controlled wild type (WT) and PARP-/- mice to determine the extent of AIF translocation. We will reduce AIF levels in animals of both genders and assess outcomes after stroke. Aim 2 will evaluate cytochrome C release and caspase formation and determine the effect of caspase inhibition in both genders. Gender differences in X-linked inhibitor of apoptosis (XIAP) will be examined in Aim 3. Finally, in Aim 4 we will examine the production of reactive oxygen species (ROS) and bioenergetic markers in males and females. Relevance: Emerging pre-clinical data suggests that the fundamental pathways leading to cell death differ in males and females. Recently, gender differences in the response to pharmaceutical agents used to both prevent (aspirin) and treat (thrombolytics) stroke have been described. Understanding these differences will allow us to develop novel and more efficacious therapies that will enhance the health of stroke patients of both sexes.

DESCRIPTION (provided by applicant): Clinically, ischemic stroke is recognized as a sexually dimorphic disease. Most international databases consistently demonstrate that women enjoy a lower stroke incidence relative to men until advanced age. Reproductive hormones contribute to such differences in male and female pathobiology, as a wealth of data show that estrogens and progestins alter neuronal survival after injury both in vivo and in vitro. However, it is becoming increasingly evident that the hormonal environment does not fully account for ischemic sexual dimorphism. Tissue damage and functional outcome after ischemic damage are shaped by biologic sex in addition to the hormonal milieu. Sex differences in stroke have been well documented in pediatric populations;both stroke incidence and stroke-related disability are higher in boys despite equivalent levels of circulating hormones. Similar findings are seen in experimental models of neonatal hypoxic- ischemic encephalopathy). The most convincing evidence for intrinsic biological differences in stroke sensitivity between the sexes is that sex-specificity can also be modeled in cell culture when sex steroids are removed from the media. One of the new concepts that must be considered is that stroke operates in a different genetic background in females (XX) and males (XY). We hypothesize that basic mechanisms of ischemic cell death differ in males and females based on the chromosomal complement. We will test this hypothesis by examining outcome in two recently developed mouse strains that have a single X chromosome (X0) secondary to meiotic non-disjunction. The 39, XO mice will be compared to 40, XX and 40 XY littermates to provide data on X-linked gene dosage effects. Key X-linked cell death proteins (X linked inhibitor of apoptosis (XIAP) and Apoptosis Inducing Factor (AIF)) will be evaluated in XX, XO and XY mice to determine the contribution of the second X chromosome to ischemic sensitivity. PUBLIC HEALTH RELEVANCE: Over the past five years, data are emerging that basic cell death mechanisms activated after ischemic insults are strongly influenced by biological sex. The contribution of the sex chromosomes (XX vs. XY) to stroke sensitivity is not known, but could account in part for the dramatic sex differences seen in stroke incidence and outcome. We hypothesize that basic mechanisms of ischemic cell death differ in males and females based on the chromosomal complement. We will evaluate stroke outcome in mice with a single X chromosome (XO) secondary to meiotic non-disjunction. The 39, XO mice will be compared to 40, XX and 40, XY littermates to provide data on X-linked gene dosage effects.

DESCRIPTION (provided by applicant): Inflammatory processes have a fundamental role in the pathophysiology of stroke. A key initial event is the rapid activation of resident immune cells, primarily the microglia. This cell population is an important target for new therapeutic approaches to limit stroke damage. Activation of microglia is normally held in check by strictly controlled mechanisms involving neuronal-glial communication. CD200 is an important, but understudied regulator of microglia activation. CD200 is expressed on a variety of cell types, including endothelium and neurons. Neuronal CD200 induces an inhibitory signal by interacting with CD200 receptors (CD200Rs) on microglia reducing injury-induced inflammation. Disruption of CD200-CD200R signaling aggravates injury in models of neuroinflammation but this signaling pathway has not been well investigated in stroke. Aging is associated with an increase in the number and activation state of microglia. We hypothesize that stroke decreases the normal inhibitory constraints of neuronal CD200 on microglia and that this is further exacerbated in the aged brain. We will utilize a well-established experimental model of stroke, middle cerebral artery occlusion (MCAO), to determine if CD200 signaling is activated after an ischemic insult (Aim 1) and manipulate this signaling pathway to determine the effects on infarct size (Aim 2).The main goal of this proposal is to determine if CD200 signaling is altered in experimental models of stroke, the time- course of this activation, and if it differs in the aged brain.

DESCRIPTION (provided by applicant): Considerable evidence from clinical studies has shown that outcomes after stroke are strongly influenced by psychosocial factors. Patients with high levels of social support or large social networks exhibit more rapid and extensive functional recovery after stroke than socially isolated individuals. In contrast, perceived social isolation predicts morbidity and mortality from cerebrovascular disease. These same effects can be reproducibly demonstrated in animals; social interaction improves behavioral deficits and reduces histological damage after experimental stroke, whereas social isolation enhances ischemic damage. The mechanisms that mediate the interactions between the social environment, behavioral responses, and disease outcomes remain unclear. Advancing age is associated with an increased risk for the development of many chronic diseases including cerebrovascular disease. With our aging population, the incidence and prevalence of stroke will continue to rise, leading to increasing numbers of stroke survivors in our communities. Many older individuals are exposed to significant psychosocial stress and isolation (loss of spouse, depression, frailty etc.), however no studies have investigated the mechanisms by which psychosocial factors influence behavioral and outcomes in aged animals. We will utilize a transient focal ischemia model in socially isolated and pair housed males. We will examine the contribution of aging to the detrimental effects of isolation on the amount of stroke damage, the immune response, and behavioral recovery. Components of the peripheral and central immune system will be examined and manipulated.

DESCRIPTION (provided by applicant): Clinically, ischemic stroke is recognized as a sexually dimorphic disease. Most international databases consistently demonstrate that women have lower stroke incidence relative to men until advanced age. However, elderly women have higher morbidity and mortality compared to age-matched men once a stroke occurs. Aging enhances the inflammatory response to stroke, and recent data demonstrate that this effect is significantly more pronounced in females. Reproductive hormones clearly contribute to such differences in male and female pathobiology, however, the hormonal environment does not fully account for ischemic sexual dimorphism as tissue damage and functional outcome after an induced stroke are influenced by biologic sex in addition to the hormonal milieu. Emerging data has shown that the mechanisms that trigger cell death differ in males and females. We will utilize genetically manipulated (Four Core Genotype) mice to dissociate the effects of chromosomal sex from that of gonadal hormones on stroke outcome in young animals (Aim 1); determine the effect of manipulating neonatal hormone levels on adult infarct damage (Aim 2); and investigate sex and hormone contributions to post-stroke inflammation in the 4CG mice (Aim 3) using a well established middle cerebral artery occlusion (MCAO) model of stroke. The overall goal of this proposal is to determine the genetic and hormonal (organizational and activational effects) contributions to stroke sensitivity across the lifespan. Identification of sex selective cell death mechanisms has significant translational relevance, as neuroprotective agents that are efficacious in one sex may exacerbate injury in the other. As recent clinical trials have shown variable efficacy of drugs in male and female patients, developing sex- specific therapeutic targets may improve our ability to treat stroke patients of both sexes.

DESCRIPTION (provided by applicant): Ischemic stroke is now the most frequent cause of persistent neurologic disability in the US. Despite considerable effort there are no therapies that can reduce injury or restore function once a stroke occurs. Over the past several years, our view of stroke as a neuronal disease has been transformed into the concept of stroke as a neurovascular disease, and more recently into the novel theory that stroke is truly a systemic disease in which peripheral inflammatory processes play a fundamental role. Secondary injury from the infiltration of peripheral immune cells is increasingly recognized as a major contributor to brain injury, edema and hemorrhagic transformation. These cells require proteases to transmigrate into the brain, a process mediated primarily by activation of matrix metalloproteinases (MMPs). Extracellular Matrix Metalloproteinase Inducer (EMMPRIN; CD 147) is a 58-kDa cell surface glycoprotein that regulates leukocyte trafficking into the brain. The proposed work will examine regulation of CD147 after stroke and determine if a function blocking antibody can reduce injury or blood brain barrier (BBB) breakdown. These effects will be confirmed in aged animals (Aim 1) and with chronic functional assessments (Aim 2). This preliminary work will set the stage for an investigation of the potential for other biologically based therapies to treat stroke. These exploratory studies will hopefully identify new biological targets for therapeutic intervention for patients with stroke.

? DESCRIPTION (provided by applicant): Current stroke therapies approved for human use are limited. The one drug clinically available, the thrombolytic agent tissue plasminogen activator (t-PA), is indicated only if administered within 4.5 hours of symptom onset and carries with it a significant risk of intracranial hemorrhage. Consequently, only a small percentage of patients receive this therapy. Emerging data suggest that post-ischemic oxidative stress and inflammatory mediators contribute to brain injury and expansion of the ischemic lesion. As the inflammatory response is delayed (hours to days), it is an attractive target for therapeutic intervention. Inter-alpha inhibitor proteins (IAIP) are complex proteins (250 and 125 kDa) circulating in blood, which consist of multiple subunits. In Japan and China, one subunit (the light chain, also known as bikunin) has been isolated from urine and is used clinically to treat acute pancreatitis and other inflammatory diseases. However, the half-life of this subunit is very short (3-10 min), requiring large amounts of protein and constant intravenous infusion. We propose to evaluate the blood-derived complexes form of IAIP (half-life of 8-12 hrs.) as a potential neuroprotective agent, as this form is more feasible for clinical use. This formulation may also have additive protective effects due to the presence of other subunits (the heavy chain) in the complex. Preliminary work in our lab using exogenously administered blood-derived human IAIP at a dose of 30mg/kg (a dose that has shown protection in sepsis) found that IAIP is strikingly neuroprotective after experimental stroke in young adult mice. Infarct volumes are reduced and functional outcomes improved even when IAIP was given six hours after ischemic onset. We have now shown that the protection is sustained at 30 days, and that this agent is also protective in aged animals. Based on these very promising initial results we propose to systematically determine the optimal dose, examine the pharmacokinetics, and directly evaluate brain and serum levels of IAIP (Aim1). We will determine if neuroprotection and behavioral improvements are sustained at chronic (1 and 3 month) endpoints in aged mice of both sexes (Aim 2) and determine if protection is also seen in an embolic model of stroke (Aim 3). These studies were designed in response to the NINDS Exploratory/Developmental Projects in Translational Research (PAR13-023) program.

DESCRIPTION (provided by applicant): Ischemic stroke is now the most frequent cause of persistent neurologic disability in the US. Despite considerable effort there are no therapies that can reduce injury or restore function once a stroke occurs. Over the past several years, our view of stroke as a neuronal disease has been transformed into the concept of stroke as a neurovascular disease, and more recently into the novel theory that stroke is truly a systemic disease in which peripheral inflammatory processes play a fundamental role. This peripheral immune response is a target for stroke therapy, as reducing peripheral infiltration of circulating leukocytes, specifically monocytes and neutrophils, decreases ischemic injury. Transforming growth factor ¿ activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family has been recently recognized as an indispensible signaling molecule in the innate immune response to brain injury. The proposed work will examine the effects of loss of TAK signaling on post-stroke inflammation using selective deletion of TAK in myeloid cells. Mechanistic studies will be performed in TAK1 knockout animals (Aim 1). We will then determine the neuroprotective efficacy of pharmacologically inhibiting TAK in aged mice, a clinically relevant animal model for stroke (Aim 2). These exploratory studies will hopefully identify new biological targets for therapeutic intervention for patients with stroke.

PROJECT SUMMARY In recent years, it has become apparent that a ?gut-brain? axis exists where communication occurs between the gut, its microbiota, and the brain. Although not fully understood, this axis has a major role in the onset and severity of many neurological diseases. In direct response to PAR-17-029, we propose to study the gut-brain axis in aging and in a model of cerebral amyloid angiopathy (CAA), a neurodegenerative condition that is characterized by the deposition of toxic amyloid-beta aggregates into the basement membrane of brain arterioles and capillaries, leading to recurrent stroke, cerebral hemorrhage and cognitive decline. Results will be compared to an animal model that demonstrates both parenchymal and vascular amyloid deposition (Tg2576) to determine if the microbiome has a preferential effect on vascular amyloid. We will develop the idea that the gut microbiota constitutes a fundamental source for the initiation and maintenance of inflammation associated with aging. It is this ?age-inflamed brain? that sustains an environment conducive for the onset and progression of CAA and other dementias. We propose to test the following hypotheses. (1) Age-related dysbiosis initiates and sustains brain inflammation in WT mice through the gut-brain axis, which can be reversed by manipulation of the biome. (2) Preventing dysbiosis delays the onset of amyloid deposition in animal models and restoration of a balanced microbiome after the onset of disease can decrease severity, behavioral deficits, and slow disease progression. In Aim 1, we will determine if neuroinflammation can be decreased in aged WT mice by altering the microbiome. If age-related dysbiosis is responsible for enhanced neuroinflamation, then creating a ?young? microbiome in aged mice should reduce brain and systemic inflammation, this will be assessed with several methods including flow cytometry and immunohistochemistry. In Aim 2, we will determine if bacteria and bacterial components translocate the gut epithelial barrier, gain access to the systemic circulation, and ultimately traffic to the brain to induce neuroinflammation in aged WT mice. We will determine if aged-related dysbiosis increases translocation of labeled bacteria and bacterial toxins/metabolites from the gut to brain. In Aim 3, we will determine if preventing dysbiosis delays the onset of amyloid deposition and behavioral deficits in animal models. We will use fecal microbiota transfer to alter the biome in two lines of mice, one that deposits amyloid primarily in the parenchyma (neurons), and the other that preferentially deposits amyloid in the cerebral blood vessels and models CAA. This latter strain may be more sensitive to biome manipulation as the neurovascular niche may be the ?first line? of cells exposed to bacterial antigens or metabolites, and the first to generate an immune response. As sex differences are present in longevity/aging, inflammation and immunity, vascular disease, dementia, and the biome, all studies will be performed in both sexes. These studies represent a ?translatable? foundation for the potential treatment of age-related neurodegenerative diseases in humans.

PROJECT SUMMARY In recent years, it has become apparent that a ?microbiota-gut-brain? axis exists where bidirectional communications occur between the gut, its microbiota contents, and the brain. In this proposal, we will develop the hypothesis that this ?microbiota-gut-brain? axis is operational after stroke. That is, stroke produces gut dysbiosis, a pathological change in the gut microbiota, and gut dysbiosis, as occurs with aging, negatively affects outcomes following stroke. Our overall hypothesis is that age-related dysbiosis contributes to the high mortality and poor functional recovery seen after stroke in aged animals. Reversing dysbiosis in aged mice by manipulating the resident bacterial population (?the microbiota?) will lead to enhanced recovery after experimental stroke. We support our hypothesis with strong preliminary data. a) Gut dysbiosis occurred with aging in mice. (b) Changes in the innate and adaptive immunity, occurring with age, were reversed with transfer of young microbiota into aged mice. (c) Gavaging the gut microbiota from young mice (3 months) into aged mice (18-20 months) after experimental stroke improved recovery. (d) A deficit of short chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, in the gut of aged mice is associated with poor outcome after stroke. (e) Post-stroke gavage of short chain fatty acid producing bacteria (probiotics) enhanced recovery in aged mice. We will develop the idea that age-related changes in T-cell subsets in both the gut and the brain mediate these detrimental effects and that deficiency of SCFAs is responsible for these detrimental T-cell changes. The main goal of this proposal is to understand how the components of the ?microbiota-gut-brain? axis change with age and stroke with a focus on brain inflammation (microglia and brain resident T-cells; Aim 1), gut inflammation (regulatory and gamma delta T-cells; Aim 2) and bacterial products (the short chain fatty acids, butyrate, acetate, and propionate) to enhance the recovery from stroke in aging mice (Aim 3). We will manipulate the biome in entirety (heterochronic fecal transfers), with bacterial metabolites (short chain fatty acids), and with targeted next-generation probiotics. Furthermore, we will determine the role of each short chain fatty acid on stroke recovery by directly infusing into the cecum and colon through a chronically indwelling cannula. Stroke is now the most common cause of long-term disability in the US and the incidence continues to rise with our aging population. The ?microbiota-gut-brain axis? after stroke is a critical and novel area of investigation to fully understand the pathophysiology of stroke and offers a promising approach to therapeutic interventions to improve recovery from stroke, especially in the elderly.

PROJECT SUMMARY The overall goal of this proposal is to determine the sex chromosome genes that regulate ischemic stroke sensitivity in the aged brain, and to explore the mechanisms underlying their regulatory role. It has been increasingly recognized that stroke is a sexually dimorphic disease, however, the mechanisms underlying these sex differences are not known. The elderly constitute the majority of stroke victims, and aged women have a higher incidence, higher morbidity and higher mortality compared to age-matched men, and these differences cannot be explained solely by exposure to gonadal hormones. Previous work has shown the sex chromosome complement contributes to stroke sensitivity selectively in aged animals, when gonadal hormones are equivalent between the sexes. We have found that there is an effect of the X chromosome dosage (one X or XX) on microglial activation and immune responses. A prominent feature of the aged X chromosome is that genetic silencing of genes on the second X chromosome becomes incomplete, allowing for genes to escape from X-chromosome inactivation (XCI). This results in higher expression of these X escapee genes in XX vs. XY cells in many tissues. Kdm6a and Kdm5c are two X escapees that can regulate expression of interferon regulatory factors (IRFs) that are responsible for microglial activation through epigenetic modification. Recent work has found Kdm6a and Kdm5c are more highly expressed in microglia derived from aged female vs. male ischemic brain. Our CENTRAL HYPOTHESIS is that X chromosome complement contributes to stroke sensitivity in the aged brain, AND that the X escapee genes Kdm6a and Kdm5c epigenetically modify IRF1/3/4/5/8 in aged microglia leading to sex-specific inflammatory responses. In Aim 1 we will use the XY* mouse model to determine if the X chromosome contributes to stroke sensitivity in aged animals. Aim 2 will use an inducible conditional knock out (ICKO) animal model to test the hypothesis that Kdm6a and Kdm5c sex specifically impact on stroke outcomes through a mechanism of epigenetic modification, i.e. demethylation of H3K27me3 and H3K4me3 marks respectively. Aim 3 will test the hypothesis that X chromosome and Kdm6a/Kdm5c regulate microglial activation and immune responses through mediation of IRF1/3/4/5/8 expression. These proposed studies will investigate the Kdm6a/5c- H3k27me3/H3K4me3-IRFs signaling axes, a very innovative and novel area. We hypothesize that this pathway plays a critical role in inducing sex differences in stroke in the aged.