Julia Kofler, MD, University of Vienna, PhD, University of Vienna - US grants

Alzheimer's disease (AD) is associated with activation of microglia in the vicinity of amyloid plaques with both detrimental results due to release of neurotoxic and pro-inflammatory mediators and beneficial effects due to amyloid phagocytosis. It is increasingly recognized that compared to normal aging, AD is associated with impairment of both innate and adaptive arms of the immune system. As key innate immune cells, microglial senescence may contribute to the development or progression of neurodegenerative diseases. Peripheral macrophages and microglia can adopt different stimulus-dependent activation states, termed classical and alternative activation, with different associated functions. Little is known about the contribution of these different microglial activation states to AD pathogenesis and disease course. This proposal will test the hypothesis that microglial activation patterns and phenotypes are differentially affected in healthy and pathological aging and contribute to impaired amyloid phagocytosis In AD. We will first establish a microarray gene signature for classical and alternative activation states by exposing cultured postmortem human microglial cells to appropriate cytokine stimuli. Using flow cytometry, real-time PCR and immunohistochemical techniques, we will then compare the activation pattern and potential of microglia derived from aged patients with no AD pathology, mild AD pathology but no dementia and from demented AD patients. We expect to see no differences in classical activation potential but an age- and disease-dependent decline in alternative activation. Lastly, we will evaluate activation-state dependent functional differences between these patient groups by performing amyloid phagocytosis assays, and measuring the release of chemokines, cytokines and neurotrophic factors. Together these studies will contribute to our understanding of distinct microglial functions and activation patterns, will begin to elucidate the degree to which microglia from aged and diseased brain are amenable to cytokine stimulation in vitro and will point to potential therapeutic means of modulating microglial phenotype in vivo.

Core D: Neuropathology Care Abstract The Neuropathology (NP) Core fulfills several important roles within the University of Pittsburgh ADRC (PITT-ADRC). Postmortem neuropathological confirmation of the clinical diagnosis of Alzheimer's disease (AD) and related disorders and the assessment of co-existing neurodegenerative and vascular disease processes is essential to promote progress in diagnosis, treatment and prevention of AD. Since its inception, the NP Core has collected and characterized >600 brains from PITT-ADRC subjects at various disease stages. This resource has been extensively utilized for teaching purposes and for research studies by local and national investigators to develop amyloid imaging agents and correlate in vivo amyloid imaging results with postmortem findings, improve our understanding of genetic factors contributing to AD risk and advance our knowledge regarding the neurobiology of psychiatric comorbidities and neuroinflammatory processes. Our proposed approach to fulfill the diagnostic, tissue banking, research and educational roles of the NP Core is outlined in five specific aims: 1) The NP Core will continue to maintain and expand a well-catalogued bank of frozen and formalin-fixed tissue samples of ADRC subjects. A special emphasis will be placed on harvesting brains from cognitively normal and mildly impaired subjects, which represent a main recruitment focus of our Clinical Core. 2) All banked cases will undergo detailed diagnostic evaluation using the latest consensus criteria. Results will be shared with ADRC clinical staff, families and uploaded to centralized databases (NACC). 3) The NP core will increasingly take advantage of new digital microscopy technologies by utilizing whole slide scanning systems and virtual slides for didactic purposes, and, in combination with digital image analysis tools, for quantitative assessments of tau and amyloid burdens. 4) The NP Core will continue to participate in local and national research efforts by providing brain bank tissue materials, data from diagnostic evaluations and technical expertise. The NP core will closely collaborate with other PITT-ADRC cores and projects in joint research studies and will engage in NP Core-driven research projects. Most of these studies will focus on one of the central themes of our ADRC correlating postmortem neuropathology data with pre- mortem and post-mortem imaging data, genotype information, presence of psychosis, and neuroinflammation. 5) Brain bank cases will be utilized to educate neuropathology fellows, residents, medical students and investigators about the neuropathologic features of neurodegenerative diseases. The PITT-ADRC NP Core is committed to participate in multi-institutional collaborative research studies by providing neuropathology data and tissue resources, to continue its efforts to improve our understanding of neuropathologic correlates of psychosis in AD and PET imaging findings and to expand our knowledge of neuroinflammatory and neuropathologic phenotypes of AD risk genes.

PROJECT SUMMARY Psychosis is an often debilitating syndrome occurring in 40-60% of people with Alzheimer's disease (AD). Psychosis in AD (AD+P) is associated with a more severe disease course, mortality, hospitalization, carer burden and faster decline in cognition and function. Atypical antipsychotics ? first developed for schizophrenia - are widely used off license to treat these symptoms, with minimal benefits and considerable harm, including a 1.5- to 1.8-fold increase in mortality and a 3- fold increase in stroke. The development of safe and effective therapies for AD+P is an urgent priority, which has to start with a better understanding of disease mechanisms. There is abundant evidence from post-mortem, neuroimaging and genetic studies that AD-P is associated with a distinct profile of neurobiological changes but little is known about the molecular processes driving etiology. Moreover, one of the most robust clinical correlates of AD+P is a more rapid cognitive decline the trajectory of which appears to diverge before the onset of symptoms, suggests that stratification of individuals early in the disease by biomarkers that suggest the individual will develop psychosis could bring clinical benefits by identifying individuals at risk of AD+P and targeting interventions to them before the onset of symptoms. Thus, in this project we will test our overall hypothesis that AD+P is characterized by specific molecular changes in both the brain and blood that cut across multiple layers of genomic regulation. The main aim of this project is to identify novel disease mechanisms and biomarkers of AD+P, via the following specific aims; 1: Identify novel disease mechanisms implicated in AD+P; 2: Identify specific signatures of psychotic symptoms in blood samples of individuals with AD+P and evaluate their potential to predict whether individuals with MCI are more likely to develop AD; 3: Elucidate the extent to which psychotic symptoms in AD are mechanistically linked to schizophrenia The study brings together unique sample cohorts, cutting-edge methodologies and world-leading experts in genome regulation, clinical neuropsychiatry and AD neuropathology. This project builds on the state-of-the-art research methods utilized in our previously successful NIH R01 grants. The project will provide a major step forward in identifying 1) novel drug targets for AD+P, 2) better treatment of AD+P with existing medications and 3) novel peripheral biomarkers to predict which individuals with MCI will develop AD+P and are thus more likely to have a rapidly progressive disease course.

PROJECT SUMMARY: Psychotic symptoms occur in ~ 40-60% of individuals with Alzheimer Disease (AD with psychosis, AD+P). Numerous studies have found that the AD+P phenotype is associated with more rapid cognitive decline than AD subjects without psychosis (AD-P). Current, empirically developed, treatments for psychosis in AD have limited efficacy, do not alter the more rapid disease progression, and are associated with substantial toxicity, including excess mortality. Because the annual incidence of psychosis in AD is only ~ 10%, there is a window of opportunity to intervene to prevent psychosis onset if resilience factors can be identified. Multiple brain imaging studies have shown that relative to AD+P, subjects with AD-P have preserved indices of cortical synaptic function, especially in the dorsolateral prefrontal cortex (DLPFC). Our recent genetic and proteomic findings in patients and model systems have converged on a possible mechanism to explain this synaptic resilience in AD-P: Preservation of postsynaptic density (PSD) protein levels in DLPFC. First, using targeted mass spectrometry (MS) in DLPFC grey matter homogenates from mild to moderate AD subjects, we found a robust increase in homogenate levels of canonical PSD proteins in AD-P subjects relative to both AD+P and Control subjects. Second, we identified and independently confirmed a polygenic protection against psychosis in AD which included an allele associated with reduced DLPFC expression of TOM1L2. TOM1L2 is an adaptor protein that facilitates degradation of synaptic proteins via actin-based endocytic trafficking. Finally, in the APPswe/PSEN1dE9 mouse model of A? overproduction, we found that reduction of Kalrn, a Rac1/RhoA guanine nucleotide exchange factor that regulates endocytic trafficking, elevated canonical PSD protein levels in cortical homogenates, preserved these proteins' levels in PSD enrichments, and protected against psychosis-associated behaviors. We thus hypothesize: resilience to psychosis onset in AD is conferred by preservation of protein levels in PSD enrichments, due to reduced trafficking of PSD proteins for degradation, and can be used to identify novel therapeutics. We will test this hypothesis in three Aims: Aim 1) To determine if PSD proteome alterations and gene-protein interactions are associated with resilience to AD+P; Aim 2) To test the effect of reduction in Tom1l2 on the synaptic proteome in a mouse model, and; Aim 3) To use computational chemogenomics to identify drugs that induce synaptic proteome compensations which confer resilience to AD+P, providing for rational prevention and/or treatment. The above aims benefit from the tight integration and leveraging of Multiple PIs with expertise in the synaptic pathology of psychosis (Sweet), the neuropathology of AD (Kofler), and the use of computation for novel therapeutic discovery (Wang). Upon completion, we will have delineated the synaptic protein compensations associated with resilience to psychosis in AD and discovered leads to compounds that generate synaptic resilience for future testing in future studies.

Core D: Neuropathology Core. Summary/Abstract: The Neuropathology (NP) Core fulfills several important roles within the University of Pittsburgh ADRC (PITT-ADRC). Postmortem neuropathological confirmation of the clinical diagnosis of Alzheimer?s disease (AD) and related disorders and the assessment of co-existing neurodegenerative and vascular disease processes is essential to promote progress in diagnosis, treatment and prevention of AD. Since its inception, the NP Core has collected and characterized >739 brains from PITT-ADRC subjects at various disease stages. This resource has been extensively utilized for teaching purposes and for research studies by local and national investigators to develop amyloid PET imaging agents and correlate in vivo amyloid imaging results with postmortem findings, improve our understanding of genetic factors contributing to AD risk and advance our knowledge regarding the neurobiology of psychiatric comorbidities and neuroinflammatory processes. Our proposed approach to fulfill the diagnostic, tissue banking, research and educational roles of the NP Core is outlined in five specific aims: 1) The NP Core will continue to maintain and expand a well-catalogued bank of brain tissue samples and fibroblasts of ADRC participants and subjects from affiliated cohorts. A special emphasis will be placed on harvesting brains from subjects with premortem PiB and tau PET imaging studies. 2) All banked cases will undergo detailed diagnostic evaluation using the latest consensus criteria and detailed characterization of comorbid and aging-related pathologies. Results will be shared with ADRC clinical staff, families and uploaded to local and national databases (NACC). 3) The NP Core will provide neuropathological skills and expertise to researchers and will expand its efforts to utilize digital microscopy technologies and image analysis tools for identification and quantification of neurodegenerative pathologies. The generated pathology endophenotypes will be made available for association studies with genetic polymorphisms and clinical phenotypes such as the presence of psychiatric comorbidities, a major area of research at the PITT-ADRC. 4) The NP Core will continue to participate in local and national research efforts by providing brain bank tissue materials and neuropathology data from diagnostic evaluations. The NP core will closely collaborate with other PITT-ADRC cores and projects in joint research studies and will engage in NP Core-driven research projects. 5) In collaboration with the Research Education Component, the NP core will leverage brain bank materials and expertise to educate neuropathology fellows, residents, medical students and investigators about the neuropathologic features of neurodegenerative diseases. Through these aims, the NP core will provide precise diagnostic classifications of ADRC research participants, enable studies of phenotype variations and broadly support efforts to improve our understanding of the underlying disease processes.

PROJECT SUMMARY: Although Alzheimer's disease (AD) is typically defined by the accumulation of beta- amyloid and hyperphosphorylated tau proteins, synaptic loss and neuronal degeneration, the disease is not restricted to the gray matter. Neuroimaging and neuropathological studies have documented a significant loss of white matter in AD, which begins early in the disease course and is correlated with cognitive decline. In addition to contributions of hypoperfusion-related ischemic injury and neurodegeneration-associated axonal loss, emerging evidence indicates a decline and dysfunction of oligodendrocyte populations as additional factors in this multifactorial white matter disease process. Oligodendrocytes are the most abundant glial cell type in the brain, but are the least studied cell population in the context of neurodegeneration despite their vital role for myelin maintenance and neuronal support. With the increasing recognition of the role of myelin in AD, it becomes important to understand the genetic and molecular factors that link oligodendrocytes to the AD process. Our knowledge about genetic variants contributing to overall AD risk and influencing AD-associated endophenotypes is accelerating. Our proposal is designed to bring these two lines of investigation together and begin to explore genetic modifiers of oligodendrocyte and myelin abnormalities in AD and underlying molecular mechanisms using a quantitative trait approach of neuropathologically defined myelin endophenotypes. The central hypothesis of our proposal is that loss of myelin integrity and oligodendrocyte dysfunction in AD are associated with genetic variants and molecular changes. We will test this hypothesis by first performing genome-wide association studies (GWAS) of white and gray matter neuropathological endophenotypes in human postmortem brain tissue samples and will then conduct bulk and spatially defined gene expression studies to explore underlying molecular mechanisms. Our experiments are divided into two specific aims: Aim 1) To determine genetic modifiers of myelin and oligodendrocyte pathologies in AD. Aim 2) To determine associations between white matter gene expression changes and white matter pathologies in AD. The above aims benefit from the tight integration and leveraging of a diverse group of investigators with expertise in the neuropathology of AD and digitally quantified pathology endophenotypes (PI Kofler), AD- associated oligodendrocyte pathology (Co-I Herrup), GWAS data analysis (Co-Is Kamboh and Fan), biostatistical analysis of transcriptomics datasets (Co-I Ding) and digital image analysis and machine learning (Co-I Pearce). Upon completion of our proposed studies, we will have identified novel candidate genes as mediators of myelin pathology in AD, increased our understanding about the biology underlying their linkage to AD and revealed novel targets for therapeutic interventions. As our study design includes separate analyses of gray and white matter regions and stratification by sex, we will have further delineated regional and sex- specific differences in myelin and oligodendrocyte pathobiology in the context of AD.