Adam D. Bachstetter - US grants

DESCRIPTION (provided by applicant): A primary pathological component of Alzheimer's disease (AD) is brain inflammation. Activation of microglia, the "macrophages of the brain", is known to occur in AD and believed to contribute to the neuronal damage. Studies of peripheral macrophages have determined that multiple distinct activation states of macrophages exist, and recent studies have confirmed these findings in microglia. Macrophages/microglia can be broadly defined as being classically activated or alternatively activated. Classically activated macrophages are typified by the release of proinflammatory cytokines (e.g. IL-12, TNF-1) and reactive oxygen and nitrogen species, which are components of the inflammatory response that is known to cause neuronal damage. However, not all forms of microglia activation are detrimental. Some forms of microglia activation are beneficial, such as alternative activation responses that lead to removal or clearance of amyloid beta (A2). This proposal will test the hypothesis that the p381 MAP kinase signaling cascade leads to the detrimental forms of microglia activation, and that blocking the p381 pathway will decrease the detrimental responses of activated microglia without affecting the beneficial responses. Aim 1 will test the hypothesis that activation of the p381 MAP kinase signaling cascade occurs in microglia during the early phase of pathogenesis in an AD-relevant transgenic mouse model (the Tg6799 mouse). Aim 2 will use a novel, brain-penetrant, small molecule inhibitor of p381 to test the hypothesis that suppression of p381 activity will slow the pathology in the Tg6799 mouse. This aim will provide training in the use of pharmacological tools, transgenic models of neurodegenerative disease, and therapeutic target validation. Aim 3 will evaluate the relative contribution of p381 in microglia vs. other neural cell types in the damage produced by A2. To test this aim, we will use an A2 infusion AD-relevant model in a mouse with a genetic deletion of p381 only in the microglia. This aim will provide training in the use of conditional knockout mouse models using the cre/lox system, and stereotaxic surgery in mice. Successful completion of this project will provide mechanistic insight into how the key regulatory protein p381 is involved in CNS pathophysiology mechanisms and intervention responses, and will form the foundation for follow-on CNS therapeutic development campaigns targeting this important protein kinase. A Career Development Plan has been formalized, and includes a combination of formal classroom and specialty workshop participation, regular presentations of proposed research plans and results at the laboratory and research group level, presentation of independent research results in seminar format, participation in national and international scientific meetings, preparation of research proposals and publications, and development of additional career- enhancing skills. An experienced mentor, a rich scientific environment, and an organized educational and training plan will assure that the applicant has optimal opportunities for scientific growth, career enhancement and development into an independent academic investigator. PUBLIC HEALTH RELEVANCE: Neuroinflammation is increasingly being recognized as a contributor to pathology in many neurodegenerative diseases, such as Alzheimer's disease, a devastating disease of aging with no effective treatment or cure. Successful completion of this project will provide mechanistic insight into how the key regulatory protein, p381 MAPK, is involved in brain pro-inflammatory responses and CNS dysfunction caused by disease-relevant stressors. In addition, the results will delineate the relative importance of microglial p381 MAPK to the disease-relevant pathological responses. Longer term, the insights and knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease- modifying therapeutics and fuller interpretation of disease progression investigations.

DESCRIPTION (provided by applicant): This K99/R00 application provides career development training and a research plan to further the understanding of glial responses in white matter in the context of Alzheimer's disease (AD). The hypothesis to be tested is that a progressive loss of myelin integrity occurs in AD, activating microglia towards a proinflammatory feed-forward loop, which mediates hyperphosphorylation of the microtubule-associated protein tau and traditional AD pathology: neuritic plaques (NP) and neurofibrillary tangles (NFTs). Conventionally considered a disease of the CNS gray matter, AD also has pronounced and progressive deterioration of cerebral white matter. Recent evidence suggests that changes in myelin integrity could be an early factor driving AD pathology, through stimulation of inflammatory microglia activation and subsequent axonal damage. Extensive work has been done to understand the glial response in AD gray matter yet very little is known about microglia activation in AD white matter, despite the fact that we and others have found a more robust activation of microglia and inflammatory response in AD white matter compared to gray matter. No studies have systematically and quantitatively examined myelin changes and inflammatory profiles as a function of disease progression. Our project will fill this gap by using human autopsy tissue and a mouse model that exhibits loss of myelin integrity to test our hypothesis. Our specific aims are: 1) Quantify the relationship between myelin integrity, microglia activation, proinflammatory cytokine levels, and traditional measures of AD burden (NPs and NFTs) in the white matter of autopsy samples; 2) Determine if loss of myelin integrity, induced by mutation in PLP, in hTau mice will accelerate hyper-phosphorylated tau pathology, and if this pathology can be rescued by suppressing the chronic neuroinflammation using a glia cytokine inhibitor. This project takes advantage of a strong scientific environment and extensive resources at the University of Kentucky, including the Alzheimer's Disease Center, clinically well-characterized autopsy cases that span the disease pathology continuum, and renowned scientific expertise of an enthusiastic and committed mentoring team. A comprehensive training and career development plan has been developed for the K99 phase that includes further scientific training in oligodendrocyte/myelin biology and human neuropathology; formal coursework and participation in local, national and international scientific meetings; evaluative meetings with th mentoring team; and activities designed to improve communication, writing, teaching, and management skills. Overall, there is an outstanding intellectual environment and access to relevant expertise in the applicant's project area, multiple opportunities for career growth, and substantial institutional commitment. This rich and supportive environment will enable a highly promising young scientist to further develop his research expertise, pursue his structured training and career development plan, and launch his career as an independent academic investigator.

ABSTRACT Management of neuroinflammation is a promising target for improving patient outcomes following a traumatic brain injury (TBI), and substantial evidence suggests therapies targeting the interleukin-1 receptor (IL-1R1) pathway may control neuroinflammation. Despite the promise, there have been limited attempts to move anti-interleukin-1 (IL-1) drugs forward for TBI neuroprotection. We hold that a critical reason for the lack of progress on this promising target is the incomplete understanding of the mechanistic underpinnings of IL-1 signaling after a TBI. It is well-recognized that the clinical picture of TBI is a spectrum of different primary injury mechanisms and injury severities, and that it is necessary to understand the secondary injury mechanism as they relate to the primary injury. Over 75% of TBIs are classified as mild. While not all TBIs lead to neurodegeneration, a mild TBI can result in progressive brain atrophy and persistent cognitive dysfunction, and is a known risk factor for the development of Alzheimer?s disease and related dementias. The current knowledge of IL-1 / IL-1R1 signaling after a TBI is almost exclusively following a moderate-to-severe injury. Using our novel genetic mouse models that allow for cell-type regulation of IL-1R1 signaling, and our model of mild TBI caused by a closed head injury (CHI) we will address this fundamental gap in our knowledge by testing the role of IL-1R1 following a mild TBI, and for the first time, define a cellular mechanism for the pathological effects of IL-1R1 following a mild TBI. Importantly our exciting preliminary data has uncovered a critical role for the brain endothelium in regulating neuroinflammation, which is dependent on IL-1R1. Our preliminary results have led us to propose the overall hypothesis: Secondary neuronal injury following a mild TBI is driven by neuroinflammation and vascular dysfunction, which can be reduced through suppression of IL-1R1. The actions of IL-1R1 following a mild TBI will require the involvement of endothelial cells. We will test our hypothesis in the following aims: Aim 1: Assess the role of endothelial IL-1R1 signaling in the neuroinflammatory feedforward loop. Aim 2: Define the role of endothelial IL-1R1 signaling in the vascular response to a CHI. Aim 3: Delineate the role of endothelial IL-1R1 signaling on synaptic plasticity and spatial learning and memory following a CHI. Successful completion of these studies will increase our understanding of the role of IL-1R1 after a mild TBI, and define the role of the brain endothelium in the neuroinflammatory response to a mild TBI. Our results will fill a critical knowledge gap concerning how best to target neuroinflammation to achieve neuroprotection after a mild TBI, and potential for other disease associated with neuroinflammation (i.e., Alzheimer?s disease).

Age-related neurologic disease is a significant and growing burden on our society. Although the largest share of research effort has typically been devoted to the common neurodegenerative illnesses (such as Alzheimer's disease, or AD), the reality is that nearly all cases of neurodegenerative disease possess elements of mixed pathology. Individuals diagnosed with AD frequently harbor neuropathologic hallmarks common in other diseases. For example, tau pathology is also found in some forms of frontotemporal dementia, ALS, and other forms of neurodegenerative disease, and is also believed to be a key form of neuropathology that develops following traumatic brain injury. Cerebrovascular disease (CVD) is abundant in individuals with a history of obesity (and type 2 diabetes, or T2D), which have a well known elevated risk of dementia. In general, it is actually quite rare to identify AD cases lacking elements of co-morbid cerebrovascular pathology. It is unclear as to whether these elements of pathology contribute to dementia in an additive or synergistic manner. In recent studies in our lab, we have observed an intriguing relationship between various aspects of neuropathology that could potentially connect to AD and CVD. We have identified a membrane ion exchanger, NHE1 (SLC9A1), as potentially involved in both pathologic processes. NHE1 has been shown to be involved with neuronal injury, and tau pathology as our preliminary data indicates. This project seeks to determine whether the function of this exchanger is important for either, or both, of these pathologies. This project combines both genetic and pharmacologic approaches to explore this exciting new target that has not previously been examined as a major player in age-related neurodegenerative disease. In specific aim 1 (SA1), we will investigate the mechanism of the membrane ion exchanger NHE1 in a unique mouse model combining AD- and CVD-related pathology, using a highly specific pharmacologic agent. In SA2, we will investigate how this membrane ion exchanger drives the formation of tau pathology, by over expressing tau on a background of NHE1 genetic reduction. Efficacy will be determined using a range of immunohistochemical, molecular, and biochemical markers of pathology, as well as gauging changes in cognitive function. We hypothesize that NHE1 will be responsible for multiple aspects of neurodegenerative disease pathology, and that interfering with its activity will ameliorate these problems, and will alleviate cognitive dysfunction. This is a highly innovative hypothesis that, to our knowledge, has not been previously explored. Another strength of this proposal is the use of a novel mouse model with unique features. This project has the capacity to significantly improve our understanding of co-morbid neuropathologies, and could have significant implications for the treatment and prevention of age-related neurodegenerative disease.

Evidence from genetic, biochemical, and neuropathological studies has shown that loss of homeostatic microglia function contributes to Alzheimer?s disease (AD) pathogenesis. From our work and the work of others, dystrophic microglia are now known to be a common feature of AD and other neurodegenerative diseases, but little is known about the causes and consequences of dystrophic microglia in AD. Our pilot data from an unbiased proteomic screening of AD brains has identified ferritin light chain (FTL) as a top candidate protein. FTL is used to store excess iron in microglia. Prior studies have shown that dystrophic microglia in the AD brain express high amounts of ferritin. Regulation of iron homeostasis occurs by the influx, storage, and efflux of iron from cells. Our project is a departure from prior work on microglia in AD, as we will define the mechanisms leading to a primary glial pathology ? dystrophic microglia. Our hypothesis that dysregulation of iron homeostasis is a driving force in microglial degeneration is novel. By using complementary histological, biochemical, and flow cytometry methods in human brain tissue, we will define this human-specific glia degeneration. We hypothesize that a dysregulation of iron homeostasis is a driver of dystrophic microglia in neurodegenerative disease. Using biosamples from the University of Kentucky Alzheimer?s Disease Center biobank of well-characterized tissue encompassing the full spectrum of disease severity, we will address the following specific aims: Aim 1: Define iron influx pathway in dystrophic microglia Aim 2: Define iron storage pathway in dystrophic microglia Aim 3: Define iron efflux pathway in dystrophic microglia Overall impact: Successful completion of this project will lead to the discovery of a novel target, in microglia iron homeostasis, to restore the healthy function of microglia, and prevent the degeneration of these cells.

Chronic sleep disruption, resulting from work schedules, noise exposure, family obligations, sleep disorders, or lifestyle choices, is a pervasive feature of contemporary life. Sleep problems affect up to 40% of AD patients, may precede cognitive impairments by more than a decade, and worsen as the disease progresses. As well as affecting mood and well-being, sleep disruption may drive the development of AD neuropathology for instance, by reducing clearance of amyloid-? (A?) and by promoting a neurotoxic proinflammatory state involving astrocytes and microglia. Sleep disruption can include reduced total sleep (sleep restriction [SR]), loss of deep sleep (also known as slow-wave sleep [SWS], marked by large amplitude, low frequency electrical activity), and fragmentation of sleep (SF) into shorter bouts. Fragmentation of the daily sleep-wake rhythm is associated with greater risk of incident AD and earlier cognitive decline in older humans. In spite of these correlative studies, whether or how chronic SF impacts the progression of AD has not been experimentally investigated. SF may be a better model of the sleep disruption associated with AD than the traditional approach of SR. Our studies of AD mouse models show that spontaneously occurring SF is associated with more severe A? accumulation and that experimentally-induced SF leads to A? accumulation and neuroinflammation. Besides SF, loss of SWS may exacerbate AD, and improving SWS may be beneficial in mild cognitive impairment (MCI) or even in AD. Since sleep disruption adversely affects the development of AD-related neuropathology, it is surprising that sleep enhancement (SE) strategies to consolidate sleep and increase SWS have not been adequately explored to slow or reverse these effects. Our overall working hypothesis is that a change in the quality of sleep, especially sleep fragmentation and loss of SWS, is more important than the quantity of sleep. Further, we hypothesize that the mechanism underlying these effects is primarily neuroinflammation, at least in part mediated by A? peptide deposition. We will use a unique, well-characterized mouse model, that exhibits AD-related A? pathology, neuroinflammation, and cognitive deficits. This project has three specific aims: (1) that SF will accelerate (and SE decelerate) AD progression; (2) that increases in A? accumulation mediates SF-induced neuroinflammation, neuropathology, and cognitive decline; and (3) that increases in neuroinflammation mediate SF-induced neuropathology and cognitive decline. We will use multiple novel approaches, including thermoneutral temperature manipulation, and a unique anti-inflammatory compound that has recently entered early stage clinical trials. Thus, these studies will elucidate the underlying mechanisms by which sleep disruption is linked to AD and will lay the groundwork for new therapeutic strategies.