Title: Novel strategies for studying proteostasis in the aging brain Problem or question being addressed: Aging is the largest risk factor for a majority of neurodegenerative disorders, including Alzheimer’s disease (AD) and vascular dementia[1]. Despite growing incidence for such diseases, there are currently no therapeutics capable of reversing progression of aging-related cognitive decline. Through the use of heterochronic parabiosis however, or the surgical joining of circulatory systems between young and old mice, our group and others have demonstrated that blood-borne factors (generated by cells in the periphery) from young animals are capable of reversing many deleterious effects seen in the brain as a result of aging[2-6]. Understanding the molecular mechanisms underlying functional improvement in the brain post-parabiosis may help to identify novel therapeutic targets for neurodegeneration seen in disease or normal healthy aging. Complicating this, heterochronic parabiosis is an extraordinarily complex experimental paradigm, involving intricate cell-cell communication networks across functionally distinct cell types within and between the organisms. Recent attempts to profile the “rejuvenatory” effects of parabiosis via single cell RNA sequencing (scRNAseq) experiments have demonstrated that many circulating blood-borne factors act directly on brain endothelial cells (BECs) and other cell types of the blood-brain-barrier (BBB) to influence brain health[7,8]. Importantly, BECs represent critical barrier cells of the blood-brain-barrier, regulating communication between systemic circulating factors and cells throughout the brain. At the same time, studies now point to the contribution of defects in vasculature to cognitive decline seen in AD pathology, with many patients presenting with arterio- and atherosclerosis, as well as altered BBB functionality (summarized in Figure 1). Collectively, this body of work suggests that cells of the brain vasculature and specifically BECs, may constitute major mediators of neurodegeneration in normal aging and disease; however, despite these observations, brain vasculature remains an understudied therapeutic target for aging-related dementia. Figure 1. Heterochronic parabiosis is a novel experimental paradigm capable of elucidating communication between peripheral tissue and the brain in the context of aging. Work by our group and others have identified a number of functional changes occurring in numerous cell types throughout the aging mouse brain (top). These include decreased chaperone expression, increased protein aggregation, increased stress-inducible heat shock protein (HSP) expression, and decreased functionality of the blood-brain-barrier (BBB) and associated cells of the neurovascular unit (NVU). Heterochronic parabiosis (bottom) demonstrates that circulating factors from the young mouse can elicit positive changes in the aged brain through a variety of mechanisms (e.g. transversing the BBB to directly interact with brain cells, interacting with BECs to influence surrounding cell types, etc.). Rationale: One hallmark of aging, linked to the pathogenesis of many neurodegenerative disorders, is the collapse of protein homeostasis (proteostasis) networks which regulate the folding of newly synthesized proteins via molecular chaperones and sequester or degrade toxic aggregates of misfolded proteins[9,10]. During aging, cellular proteostasis becomes impaired, leading to increased abundance of protein aggregates with physiological consequences including activation of stress response pathways in cell types throughout the organism. Through our group’s recent efforts profiling the effects of aging and parabiosis on the murine brain via scRNAseq, we identified numerous proteostasis network components, including molecular chaperones (e.g. Clusterin), that decrease in several cell types, including BECs of the BBB with age. In parallel, transcription of several stress-inducible genes known to increase upon sensing misfolded proteins (e.g. Hspa1a, Hsp90aa1, Hsp40), were elevated in the same cell types – signatures which reversed after heterochronic parabiosis. These observations led us to ask the question, can systemic circulating factors produced by cells in the periphery influence the proteostatic environment of the brain? Taken together, our data demonstrate that in the aging brain, (1) molecular chaperones decrease, while (2) stress-inducible heat shock proteins (HSPs) are elevated, suggesting increased burden on proteostasis machinery throughout the aged brain. Since decreased levels of chaperone proteins and increased activation of stress-responsive machinery are indicative of an environment containing protein aggregates, we first seek to identify and quantify proteins that aggregate in the brain during aging as well as assess their levels post-parabiosis. Second, since these experiments will identify proteins which aggregate during normal healthy aging (i.e. in wild-type mice), we will then modulate levels of identified aging-associated, destabilized proteins in AD mutation-containing induced pluripotent stem cell (iPSC)-derived cell types and determine their effects on cell function. Details of approach: The overall goals of this project are: 1. to identify proteins which aggregate in the brain in an aging-associated yet reversible manner and 2. determine their effect on cell function. These goals will be accomplished through the following aims: 1. Profile the soluble and insoluble proteomic changes occurring in the cortex and hippocampus during aging and heterochronic parabiosis. In this aim we ask, can we identify proteins which aggregate in the brain during aging and become attenuated post-parabiosis? To accomplish this, hippocampus and cortex will be isolated from young and old C57/BL6 wild-type mice as well as old mice post-heterochronic parabiosis and old mice post isochronic parabiosis (as a control for the parabiosis surgery). Soluble and insoluble (i.e. aggregate containing, urea soluble) protein fractions will be biochemically separated and subsequently subjected to tandem mass tag-based mass spectrometry (TMT-MS). Proteins of interest in this dataset will be selected for follow-up study based on the following criteria: Enrichment in the old insoluble protein fraction compared to the young insoluble protein fraction Proteins which decrease in the aged insoluble fraction post-parabiosis Proteins specifically found to be expressed in cells of the neurovascular unit (e.g. endothelial cells, astrocytes, pericytes, vascular smooth muscle cells) via our scRNAseq datasets[7,8] Proteins known to be involved in protein folding disorders In a preliminary set of experiments (Figure 2), we have dissected the hippocampus and cortex from young (8 week old) and old (80 week old) C57/BL6 mice, and biochemically separated the soluble and insoluble protein fractions. After performing TMT-MS on these samples, we identified roughly 150 proteins specific to the old, insoluble fraction in both the hippocampus and cortex. We are now utilizing our scRNAseq atlas of the aging mouse brain to identify cell types that are producing identified aging-associated, aggregating proteins via immunohistochemistry. Figure 2. TMT-MS identifies aging-associated protein aggregates in the cortex and hippocampus of wild-type mice. (A) In this pilot experiment, hippocampus and cortex were dissected from young (8 week old) and old (80 week old) mice (n=3) and separated into soluble and insoluble protein fractions and interrogated via TMT-MS. (B, C) Samples group together by PCA, with differences between young and old samples being greatest in the insoluble fractions. (D, E) Heatmaps depicting relative levels of proteins across age and protein fraction. Black boxes depict proteins specific to old insoluble fractions (i.e. putative aging-associated protein aggregates). 2. Assess the effects of aging-associated protein aggregates on AD mutation-containing iPSC-derived cell types. Here, we seek to ask the question, do levels of aging-associated destabilized proteins exacerbate AD-related phenotypes and in turn accelerate functional decline? In order to study the effects of aging-associated stressors on AD pathogenesis, our group has generated syngeneic human induced pluripotent stem cell (iPSC) lines containing a variety of protective or pathogenic mutations in the amyloid precursor protein (APP) gene associated with AD pathology via CRISPR/Cas9. Importantly, said iPSCs have undergone appropriate quality control including the ability to differentiate into neuronal and glial cell lineages as well as contain normal karyotype. After confirming cell type-specific expression of aging-associated, aggregation-prone proteins as described above, we will then generate lentiviral constructs engineered to encode destabilized, disease-associated versions of each protein. We will then infect APP mutation-containing iPSCs with said destabilized protein viruses and confirm overexpression of our protein of interest via qRT-PCR and Western blot. Aggregation-prone protein expressing iPSCs will subsequently be differentiated into the cell type expressing the protein in vivo as determined in Aim 1. Successful differentiation will be assessed via qRT-PCR, immunofluorescence (IF), and Western blot for expression of cell type-specific markers. To assess the functional impact of expression of aging-associated destabilized proteins on iPSC-derived neuronal cell types, we will measure cell type-specific functions (e.g. transwell permeability for BECs) and activation of AD pathology-associated phenotypes including ER stress (e.g. the unfolded protein response) and apoptosis via qRT-PCR, IF, and Western blot. How it will affect the broader field: Aging is the number one risk factor for most neurodegenerative disorders; however, no therapeutics are capable of reversing aging-associated cognitive decline. Through the use of proteomics, heterochronic parabiosis, and induced pluripotent stem cells, this project seeks to understand how circulating factors may affect the proteostatic environment of the aged brain. In doing so, we aim to uncover novel therapeutic avenues for decreasing potentially toxic aggregates of misfolded protein in the brain seen in aging and disease. Through the aims described above, we will generate rich datasets constituting proteomic changes occurring throughout the aged brain and parabiosis, while at the same time identify proteins which aggregate in a wild-type mouse brain with age. Lastly, by manipulating levels of aging-associated aggregation-prone proteins in iPSC-derived cell types, we will gain insight into novel modifiers of AD pathology. 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