Plant Fund: Administrative Supplement: Strategies for Targeting Astrocyte Reactivity in Alzheimer's Disease and Related Dementias: Core B Equipment

CORE A – ABSTRACT Core A (Administrative Core) will provide leadership, promote cohesion, and ensure fiscal oversight of the overall P01. A strong Administrative Core is essential to the success of the P01. En route to stimulating and fostering interactions among the P01 team, Core A will provide common resources, manage budgetary issues, and fulfill administrative obligations to the University and to the NIH/NIA. These services will allow the Projects and Cores to focus on the Science and reconceptualize the role of reactive astrocytes in the pathophysiology of Alzheimer’s disease and related disorders (ADRDs). Specific Aim will provide coordinated and coherent infrastructure that enhances scientific activities and promotes communication across the Projects and Cores. Aim 2 will oversee animal colonies and production/characterization of AAV reagents. And Aim 3 will provide fiscal management and administrative support. CORE B – ABSTRACT Astrocytes support brain function by integrating their roles with several cell types. They support neurons and regulate blood flow to supply and preserve active brain region. Astrocytes become activated in Alzheimer’s disease (AD) and their functions are not fully understood. Our group at the Sanders-Brown Center on Aging including Drs. Wilcock, Thibault, Norris, and Nelson investigate roles of astrocytes, neurons and vascular in aging and AD brain. These research topics are inter-connected and the successful of this program project will move our understanding of activated astrocyte beyond neuroinflammation constraints. Our core will provide multiple assays to characterize functional phenotypes of activated astrocytes and their neuronal and vascular partners. Moreover, results uniformly generated from our core will benefit comparison across projects. The central purpose of our Core B is to support the proposed projects by providing our reliable routine service on functional assays in living animals. We will do so through these specific aims: (1) Coordinate for small animal surgeries and intravital imaging techniques, including training and oversight. (2) Coordinate all brain slice electrophysiology experiments. (3) Coordinate and provide service for microelectrode arrays experiments. (4) Coordinate and provide service for Magnetic Resonance Imaging experiments. Furthermore, our Core will be coordinating with the data core (Core D), where the results will be stored, processed, and easily accessed from our collaborative groups both in UK and beyond. CORE C – ABSTRACT The Human Studies Core functions to serve and complement the P01 Projects. Variables of human data and mouse model projects are not always comparable even when the similar-seeming tests were used. There are specific challenges for incorporating relevant research on human data, and clinical biosamples – brain tissue, neuropathology, biofluids, and neuroimages —that require clinical research expertise to liaise with the Project leaders. Human Core C will bridge those critical gaps. Core C will work through the following Specific Aims: Specific Aim 1: Optimize clinically-relevant study design and manage an ongoing data streams incorporating human biomarker and autopsy data. The main goals of this Aim are to foster Integration of molecular, metabolic, and network-wide domains in animal models, human endophenotypes, and human preclinical and clinical endpoints. There will be regular meetings between clinicians researchers and mouse modelers; interactions between Projects and longitudinal clinical research data: UK-ADRC and MARK-VCID; and, a data management system for organizing data that are then sent on to Core D Specific Aim 2: From existing autopsy material and data, generate a set of samples from human cases from the UK-ADRC that encompasses the ADRD phenotypes studied in all 4 Projects (AD, VCID, LATE+HS, and controls) and provides a common basis for downstream biochemical and neuropathological endpoint assessments relevant to the Projects. We will analyze a panel of 100 cases for this Aim. Aim 2a: Biochemistry. We will perform multi-level protein extracts and run western blots with quantitative measurements on each sample with the following: GFAP, AQP4, Kir4.1, MMP9, SLC1A2, IR-B2, SUR2B, SUR2(total), A?, Tau, TDP-43, and ?-Synuclein. Aim 2b: Neuropathology. Neuropathologic endpoints will be characterized in a quantitative manner: Astrocytosis (GFAP+), A? plaques, and Tau tangles. We also will perform highly quantitative assessments of blood vessel morphology. Double-label immunofluorescence will characterize IR and SUR2 proteins’ cellular distributions Specific Aim 3: From existing clinical material, generate a set of biomarker (in vivo) data from human subjects that represents a common basis for downstream biofluids and neuroimaging endpoint assessments relevant to the projects. Aim 3A: Neuroimaging: Spectroscopy, blood flow, and cerebrovascular reactivity data from existing human imaging on a 3T MRI scanner will be generated and provided to Projects and Core D. Drs. Powell and Bahrani in Core C are also performing parallel neuroimaging experiments in mice as a part of Core B. Aim 3B: Fluid Biomarkers: Cerebrospinal fluid (CSF) and plasma that are banked from UK-ADRC and UK-MarkVCID participants wil be assayed for MMP9 and GFAP using Quanterix Simoa assays and AQP4, Kir4.1 and Dp71 will be assayed using a standard, colorimetric ELISA method. Additional candidate fluid biomarkers identified by the projects can also be assayed by Core C. CORE D – ABSTRACT The Data Management and Biostatistics (DMB) Core is designed to provide critical support for the P01 investigators to manage and analyze data. Given that interdisciplinary research requires researchers to use methods and data from a range of disciplines, the role of the DMB Core is vital to the success of the P01 project. Data management efforts will focus on collecting, storing, and sharing high quality data. This focus begins with the leadership and vision and attention to detail provided by the DMB Core. The DMB Core will provide statistical analyses individually tailored to the P01 projects and Cores’ needs, and develop and apply statistical methodology for multidimensional data generated across the P01 projects and Cores. Further, the DMB Core will actively interact with the P01 investigators, understand their research hypotheses, plan and suggest appropriate statistical analysis methods and models, build tailored analysis pipelines, interpret and discuss results, create graphs and tables for presentations and manuscripts, and write a statistical analysis section in manuscripts. The DMB Core will host an in-person (or Zoom) data quality and management workshops with the entire P01 team. The materials developed and used for workshops will be documented and shared with to the P01 team members. The DMB Core will continue these critical responsibilities through the following specific aims. Aim 1: Maintain and expand an integrated data warehouse and suite of Browser-based data collection applications and reporting platforms for the P01 Cores and projects. Aim 2: Provide expertise on tailored and integrated statistical analyses. Aim 3: Host training in data management practices for the research team PROJECT 1 – ABSTRACT Astrocytes are integral cellular components of the neurovascular unit; the specialized unit that sees vascular and brain parenchymal cell types come together to precisely match cerebral blood flow with neuronal activity in discreet regions of the brain. In order to link the cerebral vasculature with neuronal activity, astrocytes project processes to the vasculature where they form astrocytic end-feet that almost entirely sheathe the cerebral vasculature. These end-feet are highly specialized to their function; namely neurovascular coupling and homeostasis. Such functions are critical to maintaining an optimal environment in which neurons can function appropriately. Astrocytic end-feet are physically anchored to the vascular basement membranes via dystroglycan complexes. At the end-feet are potassium and water channels Kir4.1 and aquaporin 4 (AQP4), as well as monocarboxylic acid transporters (MCTs). AQP4 and Kir4.1 channels are polarized at the end-foot and are co- anchored via a α-syntrophin-dystrophin 1 complex. We have found that cerebral amyloid angiopathy (CAA) and cerebral small vessel disease (cSVD) induced by hyperhomocysteinemia (HHcy) both resulted in degeneration and dissociation of the astrocytic end-feet and an associated loss of AQP4 and Kir4.1 from the end-feet. This suggests that the end-feet are particularly susceptible to vascular injury and as such may mediate neuronal dysfunction and neurodegeneration associated with dementia. We have observed increased activity of matrix metalloproteinases (MMPs), in particular MMP9, coincident with astrocytic end-foto degeneration. We therefore hypothesize that MMP9 is necessary and sufficient for astrocytic end-foot degeneration and associated neurovascular coupling, and metabolic and ionic dyshomeostasis. To test this hypothesis we propose three distinct aims. • Specific Aim 1: MMP9 mediated astrocytic end-foot degeneration will result in cerebrovascular dysfunction impaired neurovascular coupling. • Specific Aim 2: MMP9 mediated astrocytic end-foot degeneration will result in metabolic and ionic dyshomeostasis. • Specific Aim 3: Astrocytic end-foot degeneration results in profound changes in gene and protein expression and end-foot proteins can be used as biomarkers of end-foot degeneration. PROJECT 2 – ABSTRACT In alignment with the overall proposal and the goal of this program project, we believe astrocytes, because of theβ close physical proximity to other cell types and essential role in brain metabolism, can be targeted to impact a multitude of functions that are known to be dysregulated in AD. It is our intention to use an astrocyte- centric approach and discover new pathways and signals previously uncharacterized in vivo to better address future therapies targeting the health of astrocytes in AD. Here, we will test the hypothesis that elevating insulin signaling - specifically in astrocytes - in mouse models of AD-like pathology, will normalize cerebrovascular, metabolic, and neuronal Ca2+ network changes commonly associated with AD and AD-related disorders (ADRD). We will combine our expertise using intranasal insulin (INI) delivery in combination with 2P microscopy in the 5XFAD mouse model of AD-like pathology, to investigate astrocyte-specific overexpression and knockdown of insulin receptors (IR) in the brain, and characterize the impact of the manipulation on: (Aim 1) Vascular control of the neurovascular unit (NVU) using red blood cell (RBC) flow measures; (Aim 2) Astrocyte and neuronal bioenergetics using fluorescent measures of ATP:ADP ratios, glucose imaging using 2- NBDG, and lactate measures; and (Aim 3) Neuronal/ astrocytic Ca2+ networks using G-CaMP6 fluorescent measures of Ca2+. Results in 5XFAD males and females will be compared to age-matched WT littermates. All imaging experiments will be conducted in the somatosensory cortex (S1) at rest and during ambulation in head-fixed mice and we will corroborate some of our results with hippocampal investigations using electrophysiology and Ca2+ imaging while also measuring spatial and memory processes. Our work will investigate networks of neurons, astrocytes, and blood vessels as targets of amyloidogenesis and vascular derangement across sex, it will also test the hypothesis that astrocytes represent the target of the impact of INI on the brain and can help regulate the onset of Ca2+ dysregulation. Together, a conceptually novel and experimentally rich set of data will be extracted and investigated in order to address new potential therapeutic targets in AD and ADRDs, and will elucidate how particular elements of the NVU integrate to maintain function in health and in disease states. Based on our prior track record of colloborations and sustained interactions with the other 3 projects under the leadership of Core A and in combination with the animal and statistical Cores will provide a unique opportunity to share experimental protocols, data results and interpretation across the groups, and will produce a thoughtful, and unprecedented characterization of astrocytic reactivity through the lens of experienced leaders in the field. This project within the context of the P01, could lead to a paradigm shift such that astrocyte health and reactivity are considered with other biological manifestations of AD in the clinic 1, perhaps switching from the ATN classification (amyloid/ tau/ neuron degeneration) to the ATAN (amyloid/ tau/ reactive astrocytes/ neuron degeneration) classification. PROJECT 3 – ABSTRACT Alzheimer’s disease (AD) is associated with hyperexcitable circuits and excitotoxic damage. A fundamental mechanism for safeguarding against excitotoxicity is glutamate uptake through astrocytic excitatory amino acid transporters (EAATs). The EAAT2 isoform (gene name SLC1A2), which accounts for the bulk of glutamate uptake, is downregulated in reactive astrocytes with AD. Loss of SLC1A2 emerges with AD-related cognitive decline, and is seen in many AD-like mouse models. Though the loss of SLC1A2 is frequently cited as a mechanism for excitotoxic neuronal damage, few studies have investigated more nuanced effects of impaired glutamate uptake in AD, including: disruption of neuronal energy status and/or high-fidelity responsivity to stimulation; disruption of local blood flow and neurovascular coupling (NVC) due to Ca2+ dysregulation and/or excitotoxic damage of astrocyte endfeet; and perturbations in the lactate shuttle. These knowledge gaps will be addressed in this Project. Our overarching hypothesis is that loss of astrocytic SLC1A2 underlies key AD pathophysiologic phenotypes including impaired vascular function/integrity, hypometabolism, and disrupted neural network activity. We will test our hypothesis using an astrocyte-specific intervention strategy, similar to our published work***, combined with intravital approaches, carried out by Core B and in collaboration with other Projects. To model the mixed AD and cerebrovascular pathology found in most individuals with dementia, we will investigate 5xFAD amyloid mice with induced hyperhomocysteinemia (HHcy) and cerebrovascular pathology (as characterized by Project 1 leader, Wilcock). Aim 1 will use MRI and two photon microscopy to determine the extent to which cerebrovascular deficits (impaired blood flow, neurovascular coupling, astrocyte endfoot Ca2+ dysregulation) and pathology (blood vessel leakiness, endfeet deterioration, vessel degeneration) are ameliorated by AAV-mediated upregulation of SLC1A2 in astrocytes. Aim 2 will use glutamate, glucose, and lactate sensing microelectrode arrays to determine if metabolic deficits are prevented in 5xFAD/HHcy mice by overexpression of astrocytic SL1CA2. And Aim 3 will determine if SLCA2 overexpression in astrocytes normalizes synaptic function and neuronal network fidelity in 5xFAD/HHcy mice. Additional studies in each Aim will determine if astrocyte specific knock-down of SLC1CA2 recapitulates AD-like phenotypes in otherwise healthy adult mice. Mouse results will be validated using archived human biospecimens (Core C). All data will flow through Core D to quantitatively assess the impact of astrocytic SLC1A2 across biomeasures within this project and across P01 projects. PROJECT 4 – ABSTRACT We propose to study an astrocyte mechanism that may be targeted to prevent a common subtype of dementia. Limbic-predominant age-related TDP-43 encephalopathy (LATE) and hippocampal sclerosis (HS) pathologies are present at autopsy in approximately 25% of “Alzheimer’s-type dementia” cases. We discovered that a specific ABCC9 gene variant is associated with increased risk of developing LATE+HS. We hypothesize that dysregulation of ABCC9 in astrocytes causes or exacerbates LATE+HS via impaired gliovascular function, hypometabolism (with ionic dyshomeostasis), and neuronal hyperexcitability, aligning perfectly with themes of our P01. This astrocyte pathway can be targeted by a well-tolerated drug (nicorandil) that may be clinically safe and effective, with high potential public health impact. The research will be conducted with these Specific Aims: Specific Aim 1: Characterize ABCC9/SUR2 transcripts and proteins in human astrocytes. We will test the hypothesis that the ABCC9 genetic variant associated with LATE+HS risk is also associated with decreased ABCC9 expression in astrocytes. Astrocyte ABCC9 transcripts are mostly unknown. In humans samples, we will evaluate the repertoire of mRNA transcripts and splicing in astrocytes. We will characterize ABCC9 gene expression with RNA-Seq data across cell types. In genotyped human brains from Core C (LATE+HS and controls), we will evaluate ABCC9 proteins immunohistochemically. Specific Aim 2: Test the hypothesis that modulation of astrocytic ABCC9//KATP leads to improved neurovascular and metabolic function in LATE+HS model mice. A focalpoint is the novel (TetR-TDP) mice, an excellent animal model that has proven hippocampal cell loss, astrocytosis, TDP-43 proteinopathy, and memory impairment. In TetR-TDP and control mice, we will test the impact of astrocyte specific ABCC9 knockin on glioascular function (multiphoton to look at neurovascular coupling and vessel leakiness; and MRI to look at cerebral blood flow and other endpoints). We also will test the impact of astrocyte specific knockin of SUR2A and SUR2B on brain metabolism (endpoints: MRS to look at brain metabolites; and, secondary assessments of glutamate uptake in astrocytes and Ca2+ dynamics;). Specific Aim 3: Test the hypothesis that pharmacological modulation with nicorandil (a well-tolerated KATP channel agonist) leads to improved neuronal and astrocyte function and neurobehavioral assessment outcomes in TetR-TDP mice. These outcomes include readouts of neurobehavioral and physiological assessments focusing on astrocytes. Follow-up studies will be performed with detailed neuropathology (assessing impact of nicorandil on astrocytosis, HS, TDP-43 proteinopathy, and small vessel morphology). In summary, while studying the roles of astrocyte metabolic and neurovascular pathophysiology on the progression of LATE+HS, we hope to test a novel therapeutic strategy for preventing dementia in millions.