Berislav V. Zlokovic - US grants

Lens transparency is essential for proper visual function. Glutathione (GSH) plays an important role in preventing lens opacification (cataract) by maintaining SH-groups of the lens proteins (membrane components, enzymes and crystallins) in reduced state. The lens is rich in GSH, and the current concept states that low levels of GSH are not consistent with lens clarity, but themselves are cataract inducing. Uptake of GSH by the lens and GSH lens synthesis de novo from sulfur amino acids (SAA) have been demonstrated in vitro, but direct evidence is lacking to conclude that this approximates the situation invivo. A new invivo vascular eye perfusion (VEP) model in the guinea-pig has been developed in our laboratory. The use of this species as an animal model for proposed studies has been justified during the course of our preliminary work. Biochemical characterization of guinea-pig lens suggested that metabolic scheme for GSH homeostasis is similar to that in other mammalian lenses. Using present VEP model, we obtained strong preliminary evidence indicating significant insitu rapid cellular uptake of newly secreted plasma-derived [35S]-GSH by the lens. A rapid in situ GSH lens synthesis from newly secreted blood-borne (35S)-cysteine was shown by radio HPLC analysis. The focus of this proposal is directed at plasma-derived GSH and SAA in the lens. The proposal is designed to test in a comprehensive fashion the hypothesis that blood-to-lens transport of circulating GSH and SAA is essential for the regulation of GSH levels in normal lenses. Towards this end, several experiments are proposed to test the following two hypotheses: I. Plasma-derived GSH is rapidly taken up at the lenticular epithelium by a specific transport system. II. GSH lens synthesis de novo is dependent on blood-to-lens transport of SAA. All transport and metabolic studies in normal guinea-pigs will use the VEP model, and consider for kinetic analysis four eye compartments including plasma, aqueous humor, lens capsule/epithelium and lens/cortex. Molecular forms of uptake during compartmental blood-to-lens transfer will be determined by radio-HPLC. The specificity and kinetic properties of GSH transport system in the lens will be characterized in situ, and GSH de novo synthesis from circulating SAA precursors will be estimated. These studies will help us understand the significance of blood-to-lens transport of GSH and SAA for normal lens function. Defining the role of plasma-derived GSH and SAA in normal lenses may be important in designing therapeutic strategies to decelerate cataract-inducing processes and/or to prevent formation of cataract.

Deposition of amyloid fibrils in cerebral blood vessels and brain is a histopathologic hallmark of Alzheimer's disease (AD), and amyloid beta (a(beta), the principal component of amyloid fibrils, has been implicated in the neuropathogenesis of AD. A (beta) has been identified also as a soluble peptide (sA(beta) normally present in body fluids. With respect to the origin of A(beta) in cerebral vasculature and brain it has been suggested that A(beta) can be produced locally in brain and/or sA(beta) that normally circulates in plasma and cerebrospinal fluid (CSF) may represent an immediate precursor of A(beta). Recent work indicated blood-brain barrier (BBB) in young/adult rodents may have predominantly anti-amyloidogneic function by favoring degradation of blood borne sA(beta) vs. cerebrovascular sequestration and transport, as well as by participating in the clearance from brain and CSF of CSF-derived peptide. It is no known, however whether the BBB exerts the same role in humans, and whether the BBB transport mechanism to sA(beta) become disregulated by the aging process and in AD patients, as well as in senescent non- human primates that are considered as useful models for studying factors that contribute to the development of cerebral Beta-amyloidosis (rhesus monkey) and/or cerebral amyloid angiopathy, CAA (squirrel monkey). Our pilot data using in vitro transport model of human BBB indicate that adult human brain microvascular endothelial cells (HBMEC) from AD patients retained and transported about 2-fold more intact peptide in comparison to adults, possibly as a result of enhanced endothelial uptake and transcellular transport and/or reduced metabolism of sA(beta) at the BBB. In vivo pharmakinetic-BBB permeability analysis in 1 aged squirrel monkey indicated only moderate plasma clearance and metabolism of circulating sA(beta), significant cortical and leptomeningeal microvascular sequestration of intact peptide, and significant and prefereital cortical BBB uptake of sA(Beta). Present proposal will examine in greater detail the effects of aging BBB mechanisms to A(beta) peptides Studies will be initiated with sA(beta) and sA(beta) by using i) human BBB in vitro transport model from adult, geriatric vs. AD population ; ii) in vivo pharmacokinetic-BBB permeability analysis in adult and aged rhesus (model for cerebral amyloidosis) and squirrel (model for CAA) monkeys. The hypothesis is that during aging, in AD and in senescent non human primates with cerebral amyloidosis and/or CAA, a the BBB is transformed into pro-amyloidgenic transporting membrane predisposing to vascular and/or possibly parenchymal deposition of sA(beta). An ultimate goal is to develop strategies that may reduce vascular and/or parenchymal accumulation of sA(beta) in aged brain and in AD, thereby decelerating and preventing potential cytotoxic effects of sA(beta) and amyloid formation.

DESCRIPTION (provided by applicant): Tissue-type plasminogen activator (tPA) is the approved therapy for acute thrombotic stroke. tPA promotes intravascular fibrinolysis, but increases risk for hemorrhage and is neurotoxic. Activated protein C (APC) has anti-thrombotic activity and is neuroprotective in a mouse model of stroke. In human hypoxic brain endothelium and in mice in vivo, we have shown that APC activates anti-apoptotic pathway through endothelial protein C receptor (EPCR)-dependent activation of protease activated receptor 1 (PAR1) and blockade of p53-dependent apoptosis. Our preliminary data suggest that APC acting via PAR1 and PAR3 directly protects mouse cortical cells from N-methyl-D-aspartate (NMDA)-induced apoptosis by blocking p53 and caspase-3 signaling and that APC limits NMDA excitotoxicity in vivo. Our central hypothesis is that APC and certain of its functional mutants protect the brain from ischemic/thrombotic events after stroke by combined anti-thrombotic activity and direct anti-apoptic effects on brain cells through PAR1, PAR3 and EPCR by acting on both neurons and brain endothelium. APC/tPA combined therapy for stroke should exert synergistic neuroprotective effects through the combination of anti-thrombotic and fibrinolytic intravascular activities of APC and tPA, respectively, while APC should limit tPA's direct neurotoxicity by activating anti-apoptotic mechanisms in perturbed brain cells. Recombinant human and mouse tPA and mouse wild-type and mutant APCs with reduced anticoagulant activity but normal cytoprotective activity (termed "APC functional mutants") will be studied. The research design proposes to test the hypothesis, first, in an in vivo mouse model of focal ischemic stroke with secondary brain microvascular thrombosis (aim 1); second, in an in vivo mouse model of NMDA-induced and tPA-induced excitotoxic brain injury to isolate APC/tPA systemic effects from their direct effects on brain cells (aim 2); and third, in an in vitro cell culture model of NMDA induced apoptosis in mouse cortical neurons (aim 3). We will study PAR1, PAR3 and PAR4 null mice and severely EPCR-deficient mice. Studies proposed in this application will help to determine the safety and efficacy of new APC/tPA combined therapies for thrombotic stroke and will provide new mechanistic insights into the anti-thrombotic and anti-apoptotic activities of APC and its functional mutants.

This proposal represents investigators from the University of Southern California, Columbia University and State University of New York at Stony Brook Schools of Medicine who participate in a multi-disciplinary program on Cerebrovascular Mechanisms in the Aging Brain. The goal of the program is to advance current knowledge regarding the role of vasculature in the aging brain and major CNS disorders in elderly that predispose to cerebrovascular amyloidosis (e.g., Alzheimer's Disease and related amyloid-beta-peptide (Abeta) disorders, such as hereditary cerebral hemorrhage with amyloidosis Dutch type), Abeta-related vascular injury, brain damage and stroke. We will apply concepts and techniques developed in cerebrovascular biology, blood-brain barrier (BBB) and cerebrospinal fluid physiology, molecular biology, molecular genetics, transgene mice with age-dependent vascular risk factors, and tissues and cell cultures from patients diagnosed with AD. The program consists of five Research Projects and three Core resources. Project 1, Dr. Zlokovic will study the role of BBB and brain clearance in regulating Abeta concentrations in cerebral vessel wall and brain. Project the role of BBB and brain clearance in regulating Abeta concentrations in cerebral vessel wall and brain. Project 2, Dr. Van Nostrand will study Abeta production by cerebrovascular smooth muscle cells in relation to amyloidosis. Project, Dr. Stern will study the role of receptor for advanced glycation and end products in acute and chronic cerebrovascular perturbation caused by Abeta and stroke-risk factors. Project 44, Drs. Schreiber and Zlokovic will delineate the roles of Abeta dn amyloid in vascular hemostasis in relation to ischemic or hemorrhagic stroke. Project 5, Drs. Kalra and Rhodin will study the role of Abeta in migration of monocytes across the BBB and vascular wall. Core A is the administrative facility. Core B, Dr. Mackic and Kim will provide animal and cell culture facility. Core C, Dr. Miller will provide neuropathologic analysis. The integrated and complementary scientific research projects will provide a molecular and therapeutic rationale to prevent accumulation of Abeta and formation of amyloid in cerebral blood vessels and brain, and counteract age-dependent mechanisms responsible for abnormal vascular responses, injury and brain damage.

Deposition of amyloid beta peptide (Abeta) in vascular CNS tissues and the choroid plexus (CP) occurs during normal aging and is accelerated by Alzheimer's disease (AD). Recent studies suggest major roles for the blood-brain barrier (BBB) and the cerebrospinal fluid (CSF) clearance in regulating the concentrations of Abeta in the CNS. The CP has important functions in regulating the levels of several proteins in the CSF and brain, and in cleansing the CSF of waste and potentially cytotoxic substances for brain. The role of CP in Abeta CNS homeostasis is largely unknown. Our preliminary in vivo data in guinea pigs indicate that glycoprotein 330 and possibly some other members of the low density lipoprotein receptor family participate in CP epithelial internalization of lipid-free Abeta/1-40 complexed to apolipoproteins (apo) J and E4, respectively. The CP uptake of blood-borne lipidated apoE3 and apoE4 was up to 4.5-fold greater compared to their respective dilapidated monomers. Circulating unbound free Abeta/1-40 is prevented from crossing the blood-CSF barrier, in contrast to its rapid transport out of the CSF, remarkable accumulation of CNS-derived Abeta by the CP epithelium and significant uptake by leptomeningeal vessels. Preliminary attempts to develop an in vitro transport model of the blood-CSF barrier constructed from cultured human CP epithelial cells confirmed asymmetrical Abeta transcytosis across human CP epithelium consistent with in vivo data. We propose to use in vivo vascular brain/CP and ventriculo-cisternal perfusions in guinea-pigs and an in vitro transport model of human blood-CSF barrier from control and AD subjects to test the hypothesis that the CP regulates Abeta CNS homeostasis by controlling transport in and out of the CSF and brain of lipid-free and lipid-bound apoJ and different apoE isoforms free and complexed to Abeta. We will determine in vivo basolateral CP uptake, metabolism and transport across the blood-CSF barrier of circulating apoJ, E2, E3 and E4, delipidated, lipidated, free and complexed to Abeta (aim 1), and their clearance from the CSF, uptake at the apical side of the CP and sequestration by leptomeningeal and parenchymal microvessels (aim 2). In aims 3 and 4 we will study the CP epithelial basolateral-to-apical and apical-to-basolateral Abeta/apolipoprotein binding, endocytosis and transcytosis using human in vitro model. The role of different lipoprotein and Abeta receptors will be defined. The studies will provide the molecular basis to understand the CP functions involved in the regulation of the CSF and CNS levels of Abeta to minimize its CNS accumulation and pathogenic effects.

DESCRIPTION: Deposition of amyloid-B peptide (AB) in the CNS occurs during normal aging and is accelerated by Alzheimer's Disease. AB is implicated in the neuropathology of AD and related disorders. Apolipoproteins J and E (apoJ, apoE) co-localize with the senile plaques and congophilic angiopathy of AD. ApoJ is the major protein carrier of AB in body fluids and across biological membranes. ApoE4 is a risk factor for AD, and the amount of AB deposition in AD brains is dependent on the number of copies of the e4 allele. Recent studies suggest a major role of the blood-brain barrier (BBB) in determining the concentrations of AB in the CNS. The BBB has a dual role: 1) to control the entry of plasma derived AB and the proteins to which it binds into the CNS, and 2) to regulate the levels of brain-derived AB via clearance mechanisms. AB putative receptors (e.g., RAGE) and lipoprotein receptors (e.g., gp330/megalin, LRP-1) in cerebral endothelial cells and in brain mediate the BBB and CNS transport of free AB and/or AB complexed to apoJ and apoE. Our hypothesis is that apoJ and apoE differentially regulate the BBB and CNS transport of brain-derived and plasma-derived Aft, and that aging predisposes to CNS AJ3 accumulation, formation of amyloid lesions and AB-related cytotoxic effects by upsetting the balance in apolipoprotein-mediated BBB and CNS transport of Aft, and this is further enhanced by AD, in transgenic (Tg) models of brain amvloidosis - Alzheimer's type and by the E4/E4 genotype. We will study the BBB and CNS transport of AB/apolipoproteins during normal aging (aim 1), in Tg mice expressing human AL precursor protein (APP) (aim 2) and in TgAPP mice expressing human apoE2, E3 and E4 on a mouse null apoE background (aim 3). Since apoJ, apoE and AB receptors/transporters are potential drug targets, understanding their function in vivo in different animal models should help developing strategies to prevent and/or decelerate brain accumulation of AB, amyloid formation and associated cytotoxic effects.

Cerebrovascular deposition of amyloid-13 (AI3) is a pathological hallmark of Alzheimer's Disease (AD). AI3 is neurotoxic and mutations within AI3 primarily manifest as cerebrovascular 13-amyloidoses, for example Dutch and Iowa type. AI3 within the intravascular space is linked to AI3 deposited in the brain and AI3 transport between the CNS, blood and cerebrospinal fluid, and across the blood-brain barrier (BBB), regulates brain AI3. At the BBB, LRP-1 (Low-density Lipoprotein Receptor-Related Protein 1) and RAGE (Receptor for Advanced Glycation Endproducts) transport AI3 out and into the CNS, respectively. RAGE mediates AI3 suppression of cerebral blood flow, neurovascular stress and development of cerebral 13-amyloidosis. Treatment of Tg PD hAPP mice with soluble RAGE (sRAGE) decreases the CNS amyloid load and AI3 levels, and increases AI340/42 in plasma. Our pilot data provide evidence for a direct LRP-1/AI3 interaction and demonstrate this interaction regulates AI3 clearance. We show high affinity binding of Ap40 to LRP-1, but greatly reduced binding of Ap42 and mutant AI3, by surface plasmon resonance analysis. LRP-1- mediated brain capillary uptake of AI3 and transcytosis across the mouse BBB in vivo required RAP gene and were vastly diminished by the high I_-sheet content in AI3, loss of negative charges caused by Dutch/Iowa AI3 mutations and reduced LRP-1 BBB activity/expression. Transgenic APP SwDI mice producing Dutch/Iowa Ap with high content of 13-sheets/low LRP-1 clearance develop amyloid/AI3-pathology much earlier than Tg-2576 mice, despite 24-fold lower neuronal levels of human APP. RAGE null mice do not transport circulating AI340 into the CNS. Transport of wild-type and Dutch/Iowa AI340 into the CNS is accelerated in Tg-2576 mice and APP SwDI mice consistent with high expression of RAGE at the BBB. Transgenic Tie-2 LRP-1 mice express human LRP-1 transgene in brain endothelium at functionally high enough levels. We hypothesize that LRP-1 and RAGE can be manipulated at the BBB to control p- amyloidosis in mouse models relevant to AD and familial p-amyloidoses. We will study clearance from the CNS, transport across the BBB and the effects on cerebral blood flow and neurovascular stress of wild type and mutant Ap40 and 42 in controls, APP SwDI, Tg-2576,Tie-2-LRP-1 and RAGE null mice (aim 1), the effects of human LRP-1 transgene at the BBB (aim 2) and deletion of RAGE gene (aim 3) on pathology in Tg-2576 and APP SwDI mice, and the effects of treatment with sRAGE and sLRP-1 fragments in Tg-2576 and APP SwDI mice (aim 4). These studies will provide new therapeutic insights to lower AI3 and prevent development of _-amyloidosis in AD and familial AI3-disorders by controlling AI3 CNS transport pathways.

[unreadable] DESCRIPTION (provided by applicant): Tissue plasminogen activator (tPA) thrombolysis is beneficial for thrombotic stroke, but its direct neuronal and vascular toxicities are problematic. Our extensive pilot data suggest that: (1) protein S (PS) has antithrombotic and neuroprotective activities in mouse stroke models; (2) PS protects neurons and brain endothelial cells (BEC) from N-methyl-D-aspartate (NMDA) and oxygen/glucose deprivation (OGD) injuries; (3) PS's cytoprotection requires its C-terminal sex hormone binding globulin-like (rSHBG) module, but not the N-terminal micro-PS [Gla domain, thrombin-sensitive (TSR) region, EGF1 module]; (4) PS activates the PISK-Akt cell survival axis and inhibits the intrinsic apoptotic cascade in NMDA-treated neurons and OGD- treated BEC and the extrinsic apoptotic cascade in tPA/NMDA-treated neurons and tPA/OGD-treated BEC; and (5) PS's cytoprotection may require the rse/Tyro-3 receptor tyrosine kinase (RTK), but not the Axl/Tyro 7 or Mer/Tyro 12 RTKs. Thus, we hypothesize that PS and certain of its variants may protect brain from ischemic/thrombotic events by antithrombotic activity involving Gla, TSR and EGF1 modules, and by direct cytoprotection involving the SHBG domain. We further hypothesize that combined therapies for stroke with tPA and PS, rSHBG and the GlaFII PS mutant will directly protect brain cells because PS and its variants with intact SHBG domain can activate the Tyro 3-Akt cell survival signaling pathway, and because this cytoprotective action can compensate for tPA's cytotoxicities. The research design proposes to test the hypothesis: first, using mechanical and embolic mouse stroke models (aim 1); second, using an in vivo mouse model of NMDA and tPA excitotoxic brain lesions to isolate intravascular PS and tPA effects from their direct effects on brain cells (aim 2); and third, using in vitro models of NMDA and tPA/NMDA neuronal injuries (aim 3) and OGD and tPA/OGD BEC injuries (aim 4). Expertise and reagents provided by collaborators/consultants will be Dr. J.H. Griffin (Scripps) (PS reagents, mouse tPA), Dr. G. Lemke (Salk) (Tyro 3 RTK mutants), Dr. R. Freeman (Rochester) (various Akt, Bel and FKHRL1 mutants), Dr. M. Moskowitz (Harvard) (intrinsic/extrinsic apoptotic signaling), Dr. B. Berk (Rochester) (Tyro 3 RTK-Akt signaling) and Dr. M. Chopp (Henry Ford) (rat stroke model). The results will provide mechanistic insights and test therapeutic agents that may be translated to improved therapy for ischemic stroke. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Amyloid [unreadable]-peptide (A[unreadable]) accumulation in brain and its neuronal toxicity contribute to the pathogenesis and progression of Alzheimer's disease (AD). A[unreadable] clearance has a key role in determining A[unreadable] concentration in the CNS. We hypothesize that the interactions between apolipoprotein (apo) E and the low density lipoprotein receptor (LDLR) and the interactions between apoJ and LDLR-related protein 2 (LRP2) regulate A[unreadable] clearance from brain and its retention in the CNS. We hypothesize that these interactions control (1) soluble A[unreadable] efflux across the blood-brain barrier (BBB) and from cerebrospinal fluid (CSF) and soluble A[unreadable] clearance by brain endothelial cells (BEC) and astrocytes; and (2) degradation of A[unreadable] deposits by BEC and astrocytes. The research design proposes to test these hypotheses (1) in an in vivo murine clearance model using exogenous human unlabeled A(3/apolipoproteins in wild type and LDLR-/- mice and in mice with blockade of LRP2 pathway; (2) in an in vivo model of endogenous soluble A[unreadable] clearance using microdialysis in APPsw mice, APPsw/apoE-/- mice, APPsw mice expressing apoE2, apoES and apoE4, and APPsw/apoJ-/- mice; and (3) in in vitro A[unreadable] deposition/clearance models using A[unreadable] coated surfaces and brain tissue sections from APPsw transgenic mice. Using these deposition/clearance models, we will access BEC and astrocytes that are wild type, express apoES or apoE4, or lack expression of apoJ, apoE, both apoE and apoJ, and LDLR. Aim 1 will determine effects of apoE on retention, BBB and CSF-to-blood efflux and cellular clearance of soluble A[unreadable]. Aim 2 will determine apoE-assisted removal of A[unreadable] deposits in vitro and from APPsw mice. Aim 3 will determine effects of apoJ on BBB and CSF-to-blood efflux and cellular clearance of soluble A[unreadable]. Aim 4 will determine effects of apoJ on A? removal in vitro and from APPsw mice. Since apoE, apoJ and the LDLR and LRP2 are potential drug targets for lowering brain A[unreadable], understanding their functions in vivo in different animal models and in vitro on BEC and astrocytes may help developing strategies to prevent and/or decelerate A[unreadable] brain accumulation, dissolve pre-existing A[unreadable] deposits and control A[unreadable]-associated cytotoxicity. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Pericytes are essential cells of the neurovascular unit. They are embedded within the vascular membrane of brain capillaries making direct focal contacts with the endothelium. The existence and role of pericytes has been neglected for a long time. Interactions between endothelial cells and pericytes are important for normal functions of the capillary vessel wall. In the embryonic CNS, pericytes play a key role in the development of the microcirculation. Still, the field is at the beginning of a journey to fully understand and appreciate the biology of brain pericytes and its implications for neurological disorders. The major goals of the proposed research are to determine how pericyte deficiency in the adult and the aging brain affects key neurovascular functions and neuronal structure and function. Our central hypothesis is that pericytes maintain critical neurovascular functions which are essential for normal brain performance. We hypothesize that pericyte loss in the adult brain leads to a progressive age-dependent vascular damage by two parallel pathways: (1) reductions in brain microcirculation causing diminished capillary perfusion, reduced local CBF and hypoxic tissue damage; and (2) BBB disruption leading to brain accumulation of several neurotoxic and vasculotoxic macromolecules. We next hypothesize that pericyte loss from the adult brain leads to microvascular degeneration and vascular-mediated secondary neurodegenerative changes followed by a general inflammatory response. To test our hypothesis we propose to use 3 models of cerebrovascular hypoplasia mediated by (1) an inherited embryonic loss of CNS pericytes and PDGFR¿ global deficiency (i.e., F7 mutants), (2) an inducible pericyte loss in the adult CNS with intact signaling pathways in pericytes (i.e., NG2-Cre;Pdgfr¿DTR mice) and (3) a primary cerebral endothelial hypoplasia in which pericytes remain intact (i.e., Meox2+/- mice) as a non-pericyte deficiency hypoplasia model with genetically intact PDGFR¿ signaling. Several state-of-the art techniques will be used including in vivo multiphoton microscopy, high resolution confocal microscopy, quantitative autoradiography, mathematical modeling of CBF and BBB permeability, methods to study neuronal structure and function (e.g., electrophysiology), behavioral tests and neuroinflammation. The proposed application will fill in the gap of our knowledge regarding the role of pericytes in the CNS and will likely have important implications for our understanding of a neurodegenerative process and treatment of it. We expect to generate definitive data showing that pericytes control key neurovascular functions necessary for normal structure and function of neurons and that loss of pericytes from the adult CNS has a key role in the development of microvascular and neuronal degeneration. This data should establish brain pericytes as a major new therapeutic target for neurodegenerative disorders.

DESCRIPTION (provided by applicant): This research will be done primarily in Serbia at the Institute for Biological Research University of Belgrade (LMIC site) in collaboration with Selma Kanazir with the companion grant being R37AG023084, 9/15/2004 to 8/31/2014. We propose to extend aim 4 of the companion grant to study the effects of caloric dietary restriction (DR) on brain and systemic clearance of Alzheimer's disease (AD) neurotoxin amyloid ß-peptide (Aß) and on cognitive functions using a transgenic model of AD-like cerebral ß-amyloidosis. The proposed research will significantly enhance the neuroscience research capacity at the LMIC site which is a major biomedical research institution in Serbia and the capabilities of both the LMIC collaborator and the next generation of Serbian researchers to study brain disorders. The LMIC site has experience in DR models. DR reduces Aß pathology and improves behavior in mice with AD-like cerebral pathology, but the molecular and cellular mechanisms of this improvement remain elusive. The major goal of this proposal is to determine the effects of DR on (i) brain and systemic Aß clearance mediated by the sterol response element binding protein 2 (SREBP2)/low density lipoprotein receptor related protein 1 (LRP1) and (ii) cognitive functions. LRP1-mediated Aß clearance in the cerebrovascular system, blood and liver is the major mechanism for removal of Aß toxin. SREBP2, a key gene regulating cholesterol metabolism, is a major transcriptional suppressor of LRP1. DR or 24 h fasting downregulates SREBP2 in the liver and brain and increases LRP1 expression as shown by Kanazir's pilot data. Our central hypothesis is that DR downregulates SREBP2 in brain vasculature and liver which in turn increases LRP1 activity promoting brain and systemic Aß clearance thereby improving cognitive functions. We will study APPsw/0 mice on normal diet or DR (aim 1) and with forced SREBP2 expression induced by myocardin gene transfer to brain blood vessels (aim 2). APPsw/0 mice have been transferred to Belgrade in Nov 2010 to allow Kanazir to start her own colony. This is the first transgenic colony established at the Belgrade University. Members of the Kanazir group are versed with the proposed methods to be performed at the LMIC site and will continue to be trained by Zlokovic's group in new state-of-the art methods as needed. Prior to the FIRCA proposal, Zlokovic visited Kanazir and members of her team in Belgrade during January and June 2010, and Kanazir spent 4 months in Zlokovic's former lab in Rochester from Aug 4 to Dec 7, 2010. Kanazir provided creative and important scientific input to the research plan of the FIRCA. This proposal will contribute to building much needed research capacity at the University of Belgrade by enabling the LMIC site to expand research in brain disorders, to introduce new technological approaches, and develop existing research facilities. It will enable short exchange visits of junior researchers between Belgrade and Los Angeles and help improve the productivity and build enthusiasm among young Serbian researchers. We expect the proposal will establish a long-standing collaboration between the Zlokovic group and the LMIC site/PI's group.

DESCRIPTION (provided by applicant): For this competing renewal project (R01HL63290-16), we propose to continue collaborative studies between the Zlokovic and Griffin labs aimed at advancing basic knowledge on protein C/activated protein C (APC) pathways in the central nervous system (CNS) with an overall goal of advancing from basic knowledge to translation to the clinic. Previous results funded by this R01 project led to discovery of direct vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective and anti-inflammatory activities of APC and its cytoprotective-selective mutants using rodent models of stroke, brain trauma and chronic neuronal degeneration. This project identified APC receptors and downstream pathways in the CNS that mediate cytoprotective APC signaling in neurons, brain endothelium and at the BBB and showed that protease activated receptor-1 (PAR1) has a major role in protection. Our progress was translated in 2014 into clinical Phase II trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective APC. For extending this project, our goals include: 1) providing proof of concept for hypothesized mechanisms for neuroprotective activities of APC, 2) characterizing novel 3rd generation APC-mimetic agents, and 3) discovering improved biologics for ischemic stroke with potential for treating other neurologic diseases. Our exciting new findings and preliminary data show that activation of PAR1 by APC involves a novel cleavage of this receptor's N-terminal domain at Arg46 which reveals a novel tethered ligand ending at residue Asn47 that causes APC's biased, ß-arrestin-dependent cytoprotective signaling. In contrast, classical activation of PAR1 by thrombin involves cleavage at Arg41 resulting in G-protein signaling and cellular toxicity. APC can cleave PAR1 at both Arg41 and Arg46, and in vivo proof of concept is lacking for the attractive hypothesis that APC's neuroprotection is based on PAR1 cleavage at Arg46. We hypothesize that: i) APC cleavage of PAR1 at Arg46 protects CNS, BBB and neurons (AIM 1); ii) an APC-mimetic peptide with the sequence of PAR1 residues 47-66 (TR47) (i.e., the tethered agonist generated after Arg46 cleavage) elicits ß-arrestin 2-dependent cytoprotective signaling in brain endothelium and neurons in vitro and in vivo after ischemic stroke (AIM 2); and iii) APC mutants with enhanced ability to cleave PAR1 at Arg46 may provide 3rd generation neuroprotective biologics. PRELIMINARY DATA include: i) generation of new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 to prevent cleavages at either Arg41 or Arg46; ii) proof for feasibility of studying the neuroprotective actions of the TR47 peptide; and iii) success at engineering novel APC variants with mutations that enhance PAR1 cleavage at Arg46. Neuroprotection will be assessed using in vitro models of BBB and neuronal injury, stroke models in mice and rats, magnetic resonance imaging, neuropathological analysis, confocal microscopy and behavioral testing. We expect that new knowledge generated from this project will likely have significant, direct impact for translation to the clinic for ischemic stroke and, by extension, implications for therapies for othr neurological disorders.

DESCRIPTION (provided by applicant): In this competing renewal, we propose to continue our studies aimed at understanding the role of amyloid ?- peptide (A?) trans-vascular clearance in the development and prevention of cerebral ?-amyloidosis, neuronal dysfunction and neurodegeneration. Previous work on this project has identified that A? binds directly to the ectodomain of LRP1 (low density lipoprotein receptor related protein 1), and that endocytotic LRP1 receptor is a key receptor for A? clearance at the blood-brain barrier (BBB), by vascular cells and neurons, as shown by us and others. These findings contributed to development of A? clearance approaches for Alzheimer's disease (AD). Here, we focus on PICALM, the gene encoding phosphatidylinositol binding clathrin assembly protein that plays a key role in controlling endocytosis and the function of cell receptors. PICALM is a significant risk factor for AD, but its role in disease pathogenesis remains elusive. AD-associated SNPs in PICALM (~20) are all located upstream to the coding region of the gene. Our preliminary data show that PICALM could have a central role in A? trans-vascular clearance, development of cerebral ?-amyloidosis, neuronal dysfunction and neurodegeneration. Based on our pilot data, we hypothesize that i) PICALM deficiency in endothelium and neurons results in A? accumulation and increased neuronal vulnerability to A? species causing neuronal dysfunction and neurodegeneration, respectively; ii) PICALM regulates endocytosis of A?-LRP1 complex and A? transcytosis leading to clearance of A? across the BBB; and iii) PICALM allelic variants (rs3851179, rs541458) affect A? BBB clearance and neuronal toxicity by influencing PICALM expression. To test our hypothesis we will cross our novel Picalmlox/lox mouse line with Tie2-Cre mice and Camk2a-Cre mice for endothelium-specific (AIM1) and neuron-specific (AIM2) PICALM deletions, respectively, and will cross these mice with APPsw/0 mice. We will use intrahippocampal microdialysis to study clearance of soluble A?, voltage- sensitive dye imaging to study neuronal function, confocal and light microscopy to study A?, BBB and neuronal pathology, MRI to study microstructural and connectivity changes and BBB permeability changes, and behavioral tests. We will use an in vitro model of human BBB to identify the role of PICALM in A? endocytosis and transcytosis (AIM3); and, CRISPR genome editing to generate human iPSCs with rs3851179 (protective) and rs541458 (pathogenic) allelic variants in PICALM followed by direct lineage conversion to generate human endothelial cells and cortical neurons for studies on PICALM expression, A? BBB clearance and neuronal toxicity (AIM4). To achieve our goals, we propose interdisciplinary studies by a team of Investigators with expertise in A? transport and pathology, BBB, neurodegeneration, behavior, vascular biology, cellular reprogramming and stem cell technology, small rodent neuroimaging and high-resolution connectivity brain mapping. We expect to provide unique novel insights into PICALM biology that will have important implications for our understanding of the disease pathogenesis and may guide the development of new therapies for AD.

Summary. Vascular disease is a common contributor to dementia, occurring in up to 40% of dementia cases, and is particularly common in patients with Alzheimer's disease (AD). It is also well established that vascular risk factors, such as hypertension, high LDL cholesterol, and type 2 diabetes, are risk factors for Alzheimer's dementia. More recently, specific vascular risk factors have been linked to cerebral spinal fluid-based biological markers of AD, even among cognitively normal adults. These findings could suggest that vascular risk factors could play an important role in the earliest stages of AD, which is thought to begin as much as 25 years prior to the onset of clinically significant symptoms. It has been hypothesized that vascular injury may precede and precipitate the development of neurodegeneration in AD due to blood-brain-barrier dysfunction and reduced cerebral blood flow, leading to brain leakage of neurotoxic blood products and chronic deprivation of oxygen and nutrient supply. Thus, vascular risk factors may be influencing the onset and course of AD through their effects on brain vascular function. The current proposal seeks to further examine this hypothesis by investigating whether cerebral spinal fluid- and magnetic resonance imaging-based measures of blood- brain-barrier damage and cerebral blood flow exhibit abnormalities during the earliest stages of cognitive decline in those with varying degrees of vascular risk, and whether these changes predict future decline and development of neuronal injury and structural brain changes consistent with AD. The proposed study will employ novel measures of cerebral spinal fluid proteins allowing for comprehensive investigation of brain vascular and neuronal function, as well as innovative magnetic resonance imaging technologies capable of identifying brain regions exhibiting increased blood-brain-barrier permeability and reduced cerebral blood flow. The application of these cutting-edge methods to the investigation of early vascular injury in those exhibiting vascular risk factors will provide unique insights into the earliest stages of AD, a time when preventative efforts are most likely to have an impact. Findings are likely to have major implications for early identification of vulnerable patient subgroups for prevention trials since vascular risk factors are often amenable to treatment.

The Biomarker Core (Core B) of the P01 ?Vascular Contributions to Dementia and Genetic Risk Factors for Alzheimer?s Disease? will provide services, resources and expertise on biomarkers of the neurovascular unit (NVU) for Projects 1-3 and all P01 investigators. Since the overall design of this program includes individuals with genetic risk for late-onset AD (APOE4 carriers) and early, autosomal dominant AD (ADAD) (PSEN1 mutation carriers) and a novel rat model of AD with early vascular changes (line TgF344-AD) and controls during aging and/or different experimental conditions, this core will provide the essential, quantified molecular measures relatable to the imaging and cognitive measures collected as part of this program. The Biomarker Core has developed a novel panel of multiple molecular biomarkers that integrate NVU cell- and system-specific biomarkers with established AD biomarkers (A?, tau, pTau) to simultaneously determine biomarkers of vascular/blood-brain barrier (BBB) injury in relation to inflammatory, neuronal injury, and A? and tau biomarkers in both human and rat cerebrospinal fluid (CSF) and plasma (Projects 1 and 3). Existing strengths of our core include decades of experience and knowledge in studying the NVU, BBB and cerebrovascular system, and a more recent experience over the past two years working closely with the Meso Scale Discovery (MSD) scientists to develop reliable and simultaneous measurements of multiple NVU biomarkers in human and rat biofluid for all pilot data analyses in Projects 1 and 3 using the ultrasensitive electrochemiluminescent MSD multiplex platform that is up to 2-3 orders of magnitude more sensitive than ELISA or other multiplex platforms. Our strengths also include existing and established collaborations with clinical site investigators participating in the P01 including: 1) USC ADRC (Chui, Schneider, Law); 2) Washington University Knight ADRC (Morris, Fagan, Benzinger); 3) Huntington Medical Research Institutes (HMRI) (Harrington); and 4) Dominantly Inherited Alzheimer?s Network (DIAN) including Washington University site (Morris, Fagan) and USC site (Ringman, former UCLA site moved to USC 5/15); as well as with Project 1, 2, and 3 investigators - laboratories of Drs. Zlokovic, Nation, Pa, Toga, Town, Thompson, Dong and Jacobs (CalTech). Core B has provided simultaneous measurements of different NVU biomarker categories in CSF for all pilot data analyses in APOE4 and PSEN1 mutation carriers and non-carriers and TgF344-AD rats and controls, and has determined subjects? APOE genotype. Under the leadership of Dr. Zlokovic and the resources available at USC Zilkha Neurogenetic Institute, Core B will 1) Coordinate and standardize collection, processing, and delivery of biofluid samples at different sites; 2) Assay CSF and plasma simultaneously for NVU biomarkers and conduct APOE genotyping; 3) provide further technological developments, and 4) facilitate data management, requests and distribution to projects to support the scientific goals of this program project, all investigators and research Projects 1-3.

OVERALL ? PROJECT SUMMARY/ABSTRACT Age related diseases causing dementia are an increasing global, social and economic catastrophe that mandates broad and aggressive research. Alzheimer?s disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Vascular contributions to dementia and AD are increasingly recognized. However, the role of the cerebrovascular system in the pathogenesis of dementia and AD, and the underlying neurovascular mechanisms remain, to date, largely unknown and under- researched, representing a critical barrier in the field. The overall goals of this program are to advance current knowledge on the vascular contributions to dementia and AD, and establish whether the neurovasculature plays a major role in cognitive decline, and therefore is a key new therapeutic target to treat dementia and AD. This is a program project application with multiple projects, cores, institutions and investigators. It represents an integrated whole far greater than the sum of its parts. Each project and core complements the others so that a synergistic relationship among them is achieved with a common focus on goals of the program, namely to test the neurovascular hypothesis of AD. This hypothesis holds that cerebrovascular dysfunction and disruption in the neurovascular integrity underlies and contributes to the onset and progression of cognitive decline. We have enlisted established clinical and translational research groups that collectively bring significant expertise in all aspects of the research plan and each have contributed productively over many years to the study of dementia and AD. To test the ?neurovascular hypothesis?, the participating investigators will apply cutting-edge molecular and imaging methods. We will perform parallel studies with analogous measures in humans and rats in two AD genetic risk groups with the major genetic risk factors for late-onset AD, i.e., apolipoprotein E-?4 (APOE4) gene and early-onset autosomal dominant AD (ADAD), i.e., presenilin 1 (PSEN1) mutations that both develop early vascular dysfunction and significant cerebrovascular pathology, and in the rat model of AD (line TgF344-AD) that faithfully recapitulates the rich clinico-pathological spectrum of human AD including the presence of early vascular dysfunction and cerebrovascular pathology. Central to our approach is our commitment to take a new research direction with the overarching goal to provide an answer to the broader question; ?what is the role of the vascular system in the pathogenesis of dementia and AD?, and ?what is the prognostic and diagnostic value of neurovascular molecular and imaging biomarkers in predicting cognitive decline?. The relationship between neurovascular integrity, brain connectivity and cognitive function has not been explored. The collective expertise of the investigators, overall environment, preliminary results, and experimental design for each of the projects and supporting cores hold tremendous promise for the success of this program. We are confident that the proposed studies will have a significant impact on our understanding of pathogenesis, treatment, and early prevention of dementia and AD.

OVERALL ? PROJECT SUMMARY/ABSTRACT Age related diseases causing dementia are an increasing global, social and economic catastrophe that mandates broad and aggressive research. Alzheimer?s disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Vascular contributions to dementia and AD are increasingly recognized. However, the role of the cerebrovascular system in the pathogenesis of dementia and AD, and the underlying neurovascular mechanisms remain, to date, largely unknown and under- researched, representing a critical barrier in the field. The overall goals of this program are to advance current knowledge on the vascular contributions to dementia and AD, and establish whether the neurovasculature plays a major role in cognitive decline, and therefore is a key new therapeutic target to treat dementia and AD. This is a program project application with multiple projects, cores, institutions and investigators. It represents an integrated whole far greater than the sum of its parts. Each project and core complements the others so that a synergistic relationship among them is achieved with a common focus on goals of the program, namely to test the neurovascular hypothesis of AD. This hypothesis holds that cerebrovascular dysfunction and disruption in the neurovascular integrity underlies and contributes to the onset and progression of cognitive decline. We have enlisted established clinical and translational research groups that collectively bring significant expertise in all aspects of the research plan and each have contributed productively over many years to the study of dementia and AD. To test the ?neurovascular hypothesis?, the participating investigators will apply cutting-edge molecular and imaging methods. We will perform parallel studies with analogous measures in humans and rats in two AD genetic risk groups with the major genetic risk factors for late-onset AD, i.e., apolipoprotein E-?4 (APOE4) gene and early-onset autosomal dominant AD (ADAD), i.e., presenilin 1 (PSEN1) mutations that both develop early vascular dysfunction and significant cerebrovascular pathology, and in the rat model of AD (line TgF344-AD) that faithfully recapitulates the rich clinico-pathological spectrum of human AD including the presence of early vascular dysfunction and cerebrovascular pathology. Central to our approach is our commitment to take a new research direction with the overarching goal to provide an answer to the broader question; ?what is the role of the vascular system in the pathogenesis of dementia and AD?, and ?what is the prognostic and diagnostic value of neurovascular molecular and imaging biomarkers in predicting cognitive decline?. The relationship between neurovascular integrity, brain connectivity and cognitive function has not been explored. The collective expertise of the investigators, overall environment, preliminary results, and experimental design for each of the projects and supporting cores hold tremendous promise for the success of this program. We are confident that the proposed studies will have a significant impact on our understanding of pathogenesis, treatment, and early prevention of dementia and AD.

Alzheimer?s disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Individuals who carry the apolipoprotein E-?4 (APOE4) gene are at heightened risk for developing late onset AD while individuals with the presenilin 1 (PSEN1) gene mutation will develop autosomal-dominant AD. Project 2 of the proposed P01 ?Vascular Contributions to Dementia and Genetic Risk Factors for Alzheimer?s Disease? will provide critical advances towards discovering how changes in brain connectivity, structure and function relate to neurovascular integrity and ultimately confer cognitive impairment in AD genetic risk groups. Across 5 sites, Project 2 will recruit 722 participants, including 294 APOE4 carriers and 340 non-carriers, and 44 PSEN1 mutation carriers and 44 non-carriers, followed longitudinally to evaluate changes in brain structure and function. Participants with NCI or early MCI will receive our imaging protocol every 2 years: 1) multi-shell DTI for white matter network connectivity; 2) resting fMRI for functional network connectivity; 3) structural MRI for gray matter shape, volume; and 4) DCE-MRI, ASL perfusion (from Project 1); in addition to Uniform Data Sets (UDS) cognitive tests. 150 participants (50 APOE4 carriers, 100 non-carriers) will also complete an amyloid PET scan to examine the effect of amyloid deposition on brain function and structure. We will address aims directed at assessing differences in structural and functional connectivity, examining the temporal association between brain connectivity changes over time, understanding how brain connectivity predicts future cognitive decline, and evaluating how blood-brain barrier integrity impacts brain connectivity. The relationship between structural connectivity, functional connectivity, and neurovascular integrity has not been explored. Using advanced neuroimaging methodology, this project will apply a hypothesis-driven approach to understand how brain structure and function change in individuals with high genetic risk for AD, and the impact of neurovascular integrity on these changes.

OVERALL ? PROJECT SUMMARY/ABSTRACT Age related diseases causing dementia are an increasing global, social and economic catastrophe that mandates broad and aggressive research. Alzheimer?s disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Vascular contributions to dementia and AD are increasingly recognized. However, the role of the cerebrovascular system in the pathogenesis of dementia and AD, and the underlying neurovascular mechanisms remain, to date, largely unknown and under- researched, representing a critical barrier in the field. The overall goals of this program are to advance current knowledge on the vascular contributions to dementia and AD, and establish whether the neurovasculature plays a major role in cognitive decline, and therefore is a key new therapeutic target to treat dementia and AD. This is a program project application with multiple projects, cores, institutions and investigators. It represents an integrated whole far greater than the sum of its parts. Each project and core complements the others so that a synergistic relationship among them is achieved with a common focus on goals of the program, namely to test the neurovascular hypothesis of AD. This hypothesis holds that cerebrovascular dysfunction and disruption in the neurovascular integrity underlies and contributes to the onset and progression of cognitive decline. We have enlisted established clinical and translational research groups that collectively bring significant expertise in all aspects of the research plan and each have contributed productively over many years to the study of dementia and AD. To test the ?neurovascular hypothesis?, the participating investigators will apply cutting-edge molecular and imaging methods. We will perform parallel studies with analogous measures in humans and rats in two AD genetic risk groups with the major genetic risk factors for late-onset AD, i.e., apolipoprotein E-?4 (APOE4) gene and early-onset autosomal dominant AD (ADAD), i.e., presenilin 1 (PSEN1) mutations that both develop early vascular dysfunction and significant cerebrovascular pathology, and in the rat model of AD (line TgF344-AD) that faithfully recapitulates the rich clinico-pathological spectrum of human AD including the presence of early vascular dysfunction and cerebrovascular pathology. Central to our approach is our commitment to take a new research direction with the overarching goal to provide an answer to the broader question; ?what is the role of the vascular system in the pathogenesis of dementia and AD?, and ?what is the prognostic and diagnostic value of neurovascular molecular and imaging biomarkers in predicting cognitive decline?. The relationship between neurovascular integrity, brain connectivity and cognitive function has not been explored. The collective expertise of the investigators, overall environment, preliminary results, and experimental design for each of the projects and supporting cores hold tremendous promise for the success of this program. We are confident that the proposed studies will have a significant impact on our understanding of pathogenesis, treatment, and early prevention of dementia and AD.

OVERALL ? PROJECT SUMMARY/ABSTRACT Age related diseases causing dementia are an increasing global, social and economic catastrophe that mandates broad and aggressive research. Alzheimer?s disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Vascular contributions to dementia and AD are increasingly recognized. However, the role of the cerebrovascular system in the pathogenesis of dementia and AD, and the underlying neurovascular mechanisms remain, to date, largely unknown and under- researched, representing a critical barrier in the field. The overall goals of this program are to advance current knowledge on the vascular contributions to dementia and AD, and establish whether the neurovasculature plays a major role in cognitive decline, and therefore is a key new therapeutic target to treat dementia and AD. This is a program project application with multiple projects, cores, institutions and investigators. It represents an integrated whole far greater than the sum of its parts. Each project and core complements the others so that a synergistic relationship among them is achieved with a common focus on goals of the program, namely to test the neurovascular hypothesis of AD. This hypothesis holds that cerebrovascular dysfunction and disruption in the neurovascular integrity underlies and contributes to the onset and progression of cognitive decline. We have enlisted established clinical and translational research groups that collectively bring significant expertise in all aspects of the research plan and each have contributed productively over many years to the study of dementia and AD. To test the ?neurovascular hypothesis?, the participating investigators will apply cutting-edge molecular and imaging methods. We will perform parallel studies with analogous measures in humans and rats in two AD genetic risk groups with the major genetic risk factors for late-onset AD, i.e., apolipoprotein E-?4 (APOE4) gene and early-onset autosomal dominant AD (ADAD), i.e., presenilin 1 (PSEN1) mutations that both develop early vascular dysfunction and significant cerebrovascular pathology, and in the rat model of AD (line TgF344-AD) that faithfully recapitulates the rich clinico-pathological spectrum of human AD including the presence of early vascular dysfunction and cerebrovascular pathology. Central to our approach is our commitment to take a new research direction with the overarching goal to provide an answer to the broader question; ?what is the role of the vascular system in the pathogenesis of dementia and AD?, and ?what is the prognostic and diagnostic value of neurovascular molecular and imaging biomarkers in predicting cognitive decline?. The relationship between neurovascular integrity, brain connectivity and cognitive function has not been explored. The collective expertise of the investigators, overall environment, preliminary results, and experimental design for each of the projects and supporting cores hold tremendous promise for the success of this program. We are confident that the proposed studies will have a significant impact on our understanding of pathogenesis, treatment, and early prevention of dementia and AD.

In response to the RFA-NS-16-021 to address some of the gaps in our knowledge of the biologic mechanisms of the commonly occurring cerebrovascular disease and age-related white matter (WM) disease at the molecular, cellular and brain circuit level, we propose to study the role of pericytes in WM disease using new pericyte-specific animal models and advanced molecular, cellular and neuroimaging methods, and circuit level analysis. Up to 45% of all dementias worldwide are estimated to be wholly or partly due to age-related small vessel disease (SVD) of the brain. Pericytes are vascular mural cells embedded in the wall of small blood vessels such as capillaries, pre-capillary arterioles, and post-capillary venules. In the brain, they control key neurovascular functions such as blood-brain barrier (BBB) integrity and cerebral blood flow (CBF). Pericyte degeneration leads to BBB breakdown and impaired hemodynamic responses, and is found in neurologic disorders exhibiting SVD, WM disease and cognitive impairment such as Alzheimer's, stroke and CADASIL - the most common genetic cause of ischemic SVD and VCID; yet, the role of pericytes in the pathophysiology of SVD and WM disease is largely not known. Based on our pilot data obtained in pericyte-deficient platelet- derived growth factor receptor-? (Pdgfr?F7/F7) mice and published studies in TgNotch3R169C mice expressing Notch3 CADASIL mutant in vascular smooth muscle cells and pericytes, we hypothesize that WM pericyte loss leads to BBB breakdown and CBF reductions causing loss of oligodendrocytes, demyelination, axon damage, disrupted connectivity and disintegration of CNS circuits, which leads to functional deficits and neuron loss. Since currently available pericyte-deficient Pdgfb/Pdgfr? lines and TgNotch3R169C mice are not pericyte specific, to test our hypothesis we have generated new pericyte-specific lines such as Pdgfr?-Flp; Cspg4-FSF-CreER; iDTR mice with inducible pericyte ablation, and will develop a new mouse line with inducible expression of Notch3R169C mutation only in pericytes. We will use i) cutting-edge longitudinal dynamic contrast-enhanced magnetic resonance imaging (MRI) of regional BBB integrity, dynamic susceptibility contrast MRI of CBF, diffusion tensor imaging (DTI) and DTI-based tractography for structural/connectivity changes, and tract-tracing based connectomics for CNS circuit level analysis; ii) behavior tests; and iii) immunohistology, neuropathology, flow cytometry, and electron microscopy tissue analyses. We will determine the effects of global inducible pericyte ablation (20-70%) (AIM 1), focal inducible pericyte loss in the anterior cingulum of the corticolimbic ciruit (AIM 2) and pericyte-specific inducible Notch3R169C expression (AIM 3) on BBB integrity, CBF reductions, WM integrity, disruption of CNS circuits and functional deficits (behavior). We expect that the proposed studies will contribute towards better understanding of the mechanistic basis of WM disease in vascular cognitive impairment and dementia, and will establish pericyte as a new key target for WM disease.

? DESCRIPTION (provided by applicant): Age related diseases causing dementia are an increasing global, social and economic catastrophe that mandates broad and aggressive research. Alzheimer's disease (AD) is the most common cause of cognitive impairment in older adults and affects over 5 million people in the US alone. Vascular contributions to dementia and AD are increasingly recognized. However, the role of the cerebrovascular system in the pathogenesis of dementia and AD, and the underlying neurovascular mechanisms remain, to date, largely unknown and under researched, representing a critical barrier in the field. The overall goals of this program are to advance current knowledge on the vascular contributions to dementia and AD, and establish whether the neurovasculature plays a major role in cognitive decline, and therefore is a key new therapeutic target to treat dementia and AD. This is a program project application with multiple projects, cores, institutions and investigators. It represents an integrated whole far greater than the sum of its parts. Each project and core complements the others so that a synergistic relationship among them is achieved with a common focus on goals of the program, namely to test the neurovascular hypothesis of AD. This hypothesis holds that cerebrovascular dysfunction and disruption in the neurovascular integrity underlies and contributes to the onset and progression of cognitive decline. We have enlisted the established clinical and translational research groups that collectively bring significant expertise in all aspects of the research plan and each have contributed productively over many years to the study of dementia and AD. To test the 'neurovascular hypothesis', the participating investigators will apply cutting-edge molecular and imaging methods. We will perform parallel studies with analogous measures in humans and rats in two AD genetic risk groups with the major genetic risk factors for late-onset AD, i.e., apolipoprotein E-?4 (APOE4) gene and early-onset autosomal dominant AD (ADAD), i.e., presenilin 1 (PSEN1) mutations that both develop early vascular dysfunction and significant cerebrovascular pathology, and in the rat model of AD (line TgF344-AD) that faithfully recapitulates the rich clinico-pathological spectrum of human AD including the presence of early vascular dysfunction and cerebrovascular pathology. Central to our approach is our commitment to take a new research direction with the overarching goal to provide an answer to the broader question; 'what is the role of the vascular system in the pathogenesis of dementia and AD', and 'what is the prognostic and diagnostic value of neurovascular molecular and imaging biomarkers in predicting cognitive decline'. The relationship between neurovascular integrity, brain connectivity and cognitive function has not been explored. The collective expertise of the investigators, overall environment, preliminary results, and experimental design for each of the projects and supporting cores hold tremendous promise for the success of this program. We are confident that the proposed studies will have a significant impact on our understanding of pathogenesis, treatment, and early prevention of dementia and AD.

PROJECT SUMMARY/ABSTRACT In response to FOA PAR-17-029 ?Dynamic interactions between systemic or non-neuronal systems and the brain in aging and in Alzheimer?s disease (AD)?, we propose to continue our studies (R01AG039452-6) aimed at understanding the role of pericytes in the pathogenesis and treatment of AD-like vascular, neuronal, amyloid-? (A?) and tau disorders using new pericyte-specific animal models and human cranial pericytes derived from pluripotent stem cells via a neural crest cells (NCC) intermediate (termed below iPSC-PC) in collaboration with Dr. Ruchi Bajpai. Pericytes are brain capillary mural cells that regulate blood-brain barrier (BBB), cerebral blood flow (CBF) and neuronal functions. Pericyte loss is found in AD; yet, their role in AD pathophysiology and the therapeutic potential of cell-based pericyte therapy for AD remain largely unknown. For this proposal, we generated a novel inducible pericyte-specific Cre line, and pericyte-CreER; iDTR mice carrying Cre-dependent human diphtheria toxin receptor (DTR), which leads to pericyte ablation after DT treatment. After partial (60%) pericyte ablation, we preliminarily observed rapidly evolving ischemia-like CBF reductions, BBB breakdown, and rapid loss of neurons and behavioral deficits. We show preliminarily that iPSC-PC resemble forebrain pericytes in gene expression and function such as maintenance of BBB integrity, homing to and integration with host (mouse) blood vessels, role in A? and tau clearance, and neuronal protective role. Using state-of-the-art molecular and imaging methodology this project will test novel hypothesis that loss of brain pericytes provides a pathogenic link to age-dependent AD-like vascular, neuronal, A? and tau pathology, whereas cell therapy with iPSC-PC will slow down and/or reverse vascular and neurodegenerative changes, A? and tau pathology. We will study CBF and BBB changes (AIM 1) and neuron loss and behavior (AIM 2) in pericyte-CreER; iDTR mice after pericyte ablation with DT, and generate proof-of-concept data for use of iPSC-PC therapy for vascular and neuronal disorders. We will also determine CBF, BBB, A? and/or tau pathology, neuron loss and behavior in 5xFAD (AIM 3) and P301S Tg tau (AIM 4) mice crossed with pericyte-CreER; iDTR mice after pericyte ablation with DT, and generate proof-of-concept data for use of iPSC-PC cell therapy for vascular, neuronal, A? and tau disorders in amyloid and tau models with normal and accelerated pericyte loss. Male and female mice at different ages will be used. Understanding the role of pericytes in the pathogenesis and treatment of vascular, neuronal, A? and tau disorders will have profound implications for our understanding of AD pathophysiology, and development of new, non-neuronal pericyte-based cell therapy for AD and related neurodegenerative disorders.

CORE F ? PROJECT SUMMARY/ABSTRACT The Biomarker Core (Core F) of the ADRC will provide services, resources and expertise in the vascular, blood- brain barrier (BBB), neurovascular unit (NVU) and standard Alzheimer?s disease (AD) amyloid-? (A?) and tau biomarkers for USC ADRC and outside investigators. This will support the central theme of our ADRC to elucidate vascular contributions to cognitive impairment and AD, and the role of cerebrovascular system, vascular and metabolic risk factors (VMRF), and NVU in cognitive decline and the pathogenesis of dementia and AD. The Biomarker Core F has expertise in developing novel vascular, BBB and NVU cell- and system-specific biomarkers in biofluids in addition to AD biomarkers (e.g., A?, tau, pTau). Core F will determine simultaneously in the cerebrospinal fluid (CSF) and plasma in participants of the primary ADRC vascular cohort study (VCS) biomarkers of vascular and BBB injury, other NVU biomarkers (e.g., astrocytes, microglia, inflammatory response, and neuronal injury) and AD A?, tau, and pTau biomarkers. Core F will also conduct A? and tau measurements and APOE genotyping in all ADRC participants that consent to CSF and/or blood collection. Existing strengths of our core include i) decades of experience and knowledge in studying the BBB, cerebrovascular system, and NVU; ii) experience in developing novel vascular/BBB biomarkers, as for example new, sensitive assays for novel analytes of interest including sPDGFR? (pericyte injury marker), pleiotrophin (PTN, a growth factor expressed by brain capillary pericytes), cyclophilin A, ferritin, active TGF-?1, as well as A? and tau oligomer assays.; iii) providing reliable and simultaneous measurements of multiple NVU biomarkers in biofluids for all pilot data analyses of ADRC investigators using the ultrasensitive electrochemiluminescent Meso Scale Discovery (MSD) multiplex platform; and iv) on-going multi-year collaborations with several USC ADRC investigators including Drs. Chui, Toga, Harrington, Ringman, Wang, Nation, Yassine, Braskie, Pa, and others, and several external investigators working in the AD field (resulting in numerous publications). Core F will also generate and maintain a BioBank of peripheral blood mononuclear cells (PBMCs) to be sent and deposited to the NIH National Centralized Repository for Alzheimer?s Disease and Related Dementias (NCRAD), and of induced pluripotent stem cells (iPSC) for ADRC investigators? working on in vitro models of NVU, BBB and/or ?brain on a chip?. Under the leadership of Dr. Zlokovic and the resources available at USC Zilkha Neurogenetic Institute, Core F will 1) coordinate and standardize biofluid collection, processing, storage and distribution; 2) assay CSF and plasma for vascular, BBB, NVU and standard AD biomarkers and conduct APOE genotyping; 3) provide technological developments; 4) generate and maintain a BioBank of PBMCs and iPSCs; and 5) facilitate data management, requests, and distribution to USC ADRC investigators to support our ADRC?s scientific goals. .

ABSTRACT For the competing renewal of our R01NS34467-19 project, we propose to continue collaborative studies between the Zlokovic and the Holtzman labs aimed at understanding at the cellular, molecular and systems level, and in allele-specific and gender-specific manner apolipoprotein E (apoE) effects on the cerebrovascular system, specifically, 1) how they contribute to Alzheimer?s disease (AD) neurovascular dysfunction, neurodegenerative, amyloid-? (A?) and tau disorders; and 2) how targeting apoE-low density lipoprotein receptor-related protein 1 (LRP1) interactions on different cerebrovascular cell types can mitigate and/or block cerebrovascular and brain damage causing dementia in AD. We will focus on APOE4, the strongest genetic risk factor for AD, and APOE3 that carries a significantly lower risk for AD. Neurovascular apoE is produced by astrocytes, pericytes, and vascular smooth muscle cells (VSMCs). Based on published and our pilot data, we hypothesize that apoE isoform-specific interactions with LRP1 on brain vascular cells provide a pathogenic link to apoE-related cerebrovascular disorder by controlling in an apoE-isoform-specific manner: 1) cyclophilin A (CypA)-matrix- metalloproteinase 9 (MMP9) pathway which disrupts blood-brain barrier (BBB) and arterial blood vessels; and 2) Ab and tau clearance on capillary and arterial vessels. Specifically, the apoE4 pathophysiological cerebrovascular effects accelerate cerebrovascular, neurodegenerative, Ab and tau disorders, whereas apoE3 has less disruptive effects. ApoE allele-specific effects on BBB integrity, vascular and neuronal function and structure will be studied in new APOE knockinflox/flox E3F and E4F mice with apoE cell-specific deletion from astrocytes, pericytes, or VSMCs (AIM 1); and, in the context of Ab and tau pathology, in APP/PS1-21 mice (AIM 2) and P301S tau mice (AIM 3), respectively, crossed with our new E3F and E4F mice with apoE cell-specific deletion from astrocytes, pericytes, or VSMCs. We will therapeutically target LRP1 and CypA-MMP9 pathway with intravenous cell-specific adeno-associated virus (AAV)-mediated delivery of LRP1 minigene to endothelium and pericytes, and CypA inhibition with Debio 025. Understanding the effects of astrocyte-derived, pericyte- derived and/or VSMC-derived apoE on cerebrovascular and neuronal functions, Ab and tau clearance and pathology, in an allele-specific and gender-specific manner, will advance our understanding of the pathogenesis and treatment of AD neurovascular dysfunction, neurodegeneration, Ab and tau disorders. If successful, this could lead to new therapeutic approaches targeting LRP1 and/or CypA-MMP9 pathway in different brain vascular cells to mitigate and/or block apoE?s cerebrovascular and brain damage causing dementia in AD.

Abstract The 3 overarching goals of the USC Alzheimer Disease Research Center (ADRC) are to: 1) Elucidate vascular contributions to Alzheimer disease (AD), 2) catalyze basic, clinical, and translational research in AD at USC, and 3) contribute expertise in vascular disease, biomarkers, and imaging to national collaborative initiatives. The ADRC is led by 3 multiple PD/PIs: Chui, Zlokovic, and Toga and comprised of 6 required cores, the required Research Education Component (REC), and 1 optional imaging core. The Administration Core (Chui, Zlokovic, Toga) provides administrative and scientific oversight across USC ADRC, including fostering development projects and supporting ADRC-affiliated studies. The Clinical Core (Schneider, Ringman, Chui) performs standardized evaluations and diagnoses using the NACC Uniform Data Set (UDS), enrolls and follows participants in our 2 primary ADRC cohorts: Vascular Cohort Study (VCS) and Brain Research Study (BRS). The Data Management and Statistical Core (Toga and Chen) oversees the NACC UDS database, provides study- and core-specific databases and curates our large imaging data sets as a local and national resource. The Neuropathology Core (Miller and Hawes) performs standardized neuropathological examinations, stores and distributes biological tissues to research investigators. The Outreach, Recruitment, and Engagement Core (Aranda) works closely with the Clinical Core to recruit and retain the primary ADRC cohort, focusing on under- represented minority groups (especially Latinx), and the development of a participant-caregiver dyad resource database. The Biomarker Core (Zlokovic) uses state of the art methods to determine cell-and system-specific biomarkers related to the neurovascular unit, as well as to measure standard AD biomarkers. The Imaging Core (Toga and Pa) provides high field (3T and 7T) MR imaging, as well as amyloid/tau PET scans. The Research Education Component (Yassine) is dedicated to mentoring post-doctoral students committed to the study of minority issues in Alzheimer disease and related disorders. The USC Health Science Campus is located near high Latinx catchment areas. Treatable vascular-metabolic risk factors (VMRF) are particularly prevalent among the Latinx population and dovetail with the research focus of the USC ADRC.

ABSTRACT Stroke in small brain vessels in subcortical white matter (WM) regions account for 25% of all strokes. It leads to vascular cognitive impairment and dementia (VCID), and is the second leading cause of dementia overall. Despite such clinical importance, the pathophysiology of ischemic WM injury (WMI) and VCID is still poorly understood. Moreover, there is no yet an approved therapy for prevention and/or treatment of WM strokes and VCID. Here, we propose collaborative studies between the Zlokovic and Griffin labs on activated protein C (APC) pathways in the WM, and to evaluate therapeutic potential of APC-based therapies for ischemic WMI using a model of vasoconstriction of small brain vessels in the WM. Our previous studies using models of large artery infracts, brain trauma and neurodegeneration led to discovery of vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective, and anti-inflammatory activities of APC and its cytoprotective-selective mutants. In 2019, these findings have been translated into successfully completed phase 2 trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective-selective APC analog with >90% loss of anticoagulant activity. However, whether activation of APC pathways in the WM is beneficial or not during ischemic WMI, remains unknown. Our goals include: 1) providing proof of concept for hypothesized mechanisms for protective activities of APC in the WM; and 2) characterizing novel protease activated receptor 1 (PAR1)-related P1-47 and PAR3-related P3-42 APC-mimetic peptides, and 3) testing improved 3rd generation APC R-46-selective biologics for treating and preventing ischemic WMI and WM stroke. Our pilot data support our hypotheses that: i) APC will be beneficial for ischemic WMI via PAR1 cleavage at Arg46 to protect WM fiber tracts, oligodendrocytes and BBB from ischemic WMI (AIM 1); ii) APC-mimetic peptides derived from PAR1 and PAR3 sequences (e.g,, P1-47 and P3-42, (i.e., the tethered PAR agonists created by APC cleavages) exhibit synergistic biased agonism, and will elicit ?-arrestin 2-dependent cytoprotective signaling in brain endothelium and oligodendrocytes in vitro and in vivo after WM stroke (AIM 2); and iii) E56K-APC and D180E-APC newly engineered APC mutants have enhanced ability to cleave PAR1 at Arg46 and will provide improved APC biologics for WM stroke therapy (AIM 3). To address our hypotheses, we will use i) WM model of stroke; ii) new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 point mutations, and ?-arrestin 2-/- and G?12-/- mice; iii) new APC-mimetic PAR1- and PAR3-related peptides with the respective PAR1 and PAR3 tethered-ligand amino acid sequences; iv) new APC R46-cleavage site selective biologics; v) in vivo mutiparametric longitudinal MRI of WM lesion volume, BBB integrity, blood flow, structural and connectivity changes, and tract-tracing based connectomics for circuit level analysis; vi) behavior tests; vii) immunohistology, neuropathology; and viii) oligodendrocyte cultures and in vitro BBB model. If successful, new knowledge generated from this project could translate to the clinic as new therapies for WM stroke and VCID.