David Holtzman - US grants

Several neuronal genes expressed in the CNS, including the amyloid beta protein precursor (APP) have been assigned to human chromosome 21 (HSA 21). Amyloid beta protein is an important component of cerebral vascular amyloid and the neuritic plaques seen in both Alzheimer's disease (AD) and trisomy 21 or Down syndrome (DS). Altered expression of APP or other genes on HSA 21 may contribute to the CNS abnormalities in both AD and DS. An animal model for DS, mouse trisomy 16, provides an opportunity to examine neuronal gene expression and to identify the abnormalities in APP expression induced by the trisomic state. Mouse chromosome 16 (MMU 16) contains at least 5 genes located on HSA 21 including APP. In addition, mice with trisomy 16(Ts 16 have both systemic and nervous system abnormalities similar to DS. To circumvent the problem of death in the late gestational period, chimeras (Ts 16<-->2N) will be used. Nerve growth factor (NGF) is a trophic factor for cholinergic neurons of the basal forebrain. As marked abnormalities of basal forebrain cholinergic neurons are found in both AD and DS, the influence of NGF on gene expression will be investigated. Analysis of mRNA for the genes encoding APP, the growth associated protein (GAP-43) and the prion protein (PrP) will be performed in basal forebrain and in several brain regions. These measurements will include analysis via Northern and slot/blot hybridization in normal mice and in chimeras. A number of different developmental stages will be assessed. The same procedures will be used to examine the response to exogenous NGF. In situ hybridization histochemistry (ISHH) will be used to localize and quantitate trisomic neurons in chimeras. Depending on results of initial studies, ISHH may be used to study mRNA levels in individual normal and trisomic cholinergic basal forebrain neurons. Studies of gene expression in the mouse model for DS may give fresh insights into the molecular pathogenesis of both AD and DS.

pain; enkephalins; gene expression; dynorphins; steroid hormone; messenger RNA; spinal cord; hypothalamus; in situ hybridization; laboratory rat;

DESCRIPTION (Applicant's Abstract): Genetic studies have shown that the E4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease (AD). Both in vitro and more recently in vivo data in both humans and mice strongly suggest a major factor underlying this increased risk relates in some way to the ability of apoE to enhance amyloid Beta (ABeta) deposition. The mechanisms underlying this effect of apoE as well as its isoform specific effects remain unclear. In the brain, apoE is primarily synthesized by astrocytes that secrete apoE in high density lipoprotein (HDL)-like particles. Recent data suggest that the properties of apoE-containing lipoproteins produced in the brain may be unique in regard to their potential function and interactions with other proteins. The multi-ligand apoE receptor known as LRP is expressed at high levels by neural cells. Recent genetic studies suggest that certain alleles of another LRP ligand, alpha2M, as well as specific alleles of LRP itself may also act as AD risk factors. This suggests that clearance of LRP ligands may in some way be involved in the pathogenesis of AD. There is now compelling data that apoE is necessary for fibrillar ABeta deposition in vivo. APPV717F +/+ transgenic mice which develop fibrillar ABeta deposition by 6 months of age were found to have no fibrillar Abeta deposits and a marked decreased in ABeta deposition. This suggests that mouse apoE-containing lipoproteins produced in the brain be in some way required for ABeta deposition. Two non-mutually exclusive possibilities seem likely to explain the influence of apoE on ABeta deposition: (1) apoE/ABeta interactions facilitates conversion of soluble ABeta to fibrillar ABeta and (2) apoE/ABeta interactions influence ABeta clearance. The goal of these studies is to study the interaction of astrocyte-secreted apoE isoforms and ABeta and to determine how these interactions influence ABeta deposition. It is hypothesized that the level of astrocyte-secreted apoE/lipoproteins will determine the amount of ABeta deposition in vivo. Sub-hypotheses are that (1) apoE4 will result in greater ABeta deposition through enhancing ABeta fibrillogenesis more than other apoE isoforms and (2)apoE influences the amount of fibrillar ABeta by affecting the clearance of apoE/ABeta complexes via LRP. These hypotheses will be tested in the following specific alms: 1. To determine the amount of Abeta deposition and other AD neuropathology in 2 lines of APP transgenic mice which express human apoE2, E3, E4 or mouse apoE. 2. To characterize astrocyte-secreted apoE/lipoproteins and determine their influence on ABeta fibril formation utilizing several techniques. 3. To examine the nature of the association between astrocyte-secreted apoE/lipoproteins and ABeta and determine whether cellular uptake and degradation of apoE/ABeta complexes is modulated via LRP. 4. To determine whether altering expression of LRP or LRP domains influences ABeta levels and deposition in vivo. These studies with physiological preparations of apoE as well as both in vivo and in vitro models should provide new insights into mechanisms underlying the link between apoE, LRP, ABeta, and Alzheimer's disease.

A major cause of brain damage in the perinatal period is hypoxic-ischemic (H-I) injury. This type of injury often leads to static encephalopathy with permanent motor deficits (cerebral palsy) as well as mental impairment and seizures. There is currently no treatment for this significant clinical problem. Recent data indicate that endogenous neurotrophic factors, specifically the neurotrophins (NTs) may play a role in H-I injury. Recent work also suggests that NTs may be particularly important to the developing brain where NT actions are much more robust than in the adult and where the mode of cell death following H-I and other insults appears to favor apoptosis. Recently, we have found that certain NTs, probably acting through both direct and indirect mechanisms, appear to he markedly protective to the neonatal rat brain in a model of H-I injury. In this proposal, we will test the hypothesis that NTs are protective against neonatal H-I induced neuronal death and cognitive changes when given before or after an insult. A-corollary to this hypothesis is that alterations in NT signaling or its downstream mediators will impact on neuronal death and alter outcome in neonatal H-I injury. In specific aim l, we will characterize the activity of endogenous and exogenous NTs and their receptors during brain development and following both hypoxia and hypoxia-ischemia in vivo. In aim 2, we determine the extent and timecourse of apoptotic cellular changes following a neonatal H-I insult. In aim 3, we will determine whether NGF, BDNF and NT-3 protect against brain injury in vivo if given either before or after H-I induced injury. In aim 4, we will determine if the absence of the NT receptor trkB will worsen H-I injury and whether absence of the pro-apoptotic gene bax will prevent H-I induced injury. In aim 5, we will determine whether H-I injury in neonatal rats results in alterations in spatial memory and whether NTs can prevent these changes. These studies should provide new insights into differences between the response of the neonatal and adult brain to hypoxia-ischemia and better define the role of NTs and their receptors in this process. They will also further define the potential of NTs as treatments for neonatal H-I brain injury.

clusterin; amyloid proteins; pathologic process; Alzheimer's disease; protein protein interaction; apolipoprotein E; protein localization; atomic force microscopy; human tissue; laboratory mouse; genetically modified animals; tissue /cell culture;

DESCRIPTION (provided by the applicant): Amyloid-beta (Abeta) is a 39-43 amino acid peptide derived from a large precursor protein known as the amyloid precursor protein (APP). Abundant evidence suggests that the conversion of Abeta from soluble to insoluble forms and its buildup in the brain is a key step in the pathogenesis of Alzheimer's disease (AD). There is a strong likelihood that prevention/reversal of this process may serve as a treatment for AD. Utilization of transgenic (Tg) mouse models of AD which develop age-dependent Abeta deposition have provided useful models to test whether different manipulations can influence this and other AD-related pathology. Recent studies have shown that active immunization in Tg mouse models of AD can decrease Abeta deposition and that passive parenteral administration of certain anti-Abeta antibodies appear to mimic this effect. The mechanism as to how anti-Abeta antibodies result in prevention/reversal of Abeta deposition has not yet been clarified. In exploring factors which alter soluble Abeta clearance, we found that a monoclonal antibody (m266) directed against the central domain of Abeta was able to bind and completely sequester all plasma Abeta in a mouse model of APPV717F Tg mice. Peripheral administration of m266 to APPV717F Tg mice, in which Abeta is generated specifically within the CNS, resulted in a rapid 1000-fold increase in plasma Abeta and altered dynamics of extracellular Abeta metabolism in the CNS. Peripheral administration of m266 to APPV717F Tg mice also markedly reduced Abeta deposition in the brain when administered chronically. Thus, our findings have led us to hypothesize that certain anti-Abeta antibodies alter Abeta clearance and diminish AD pathology in large part by increasing net transport of Abeta from CNS to plasma. We will test this and other ideas in the following Aims: Aim 1: We have developed an in vivo assay to measure the net rate of Abeta entering the plasma of APPV717F Tg mice. Utilizing this assay, we will test the hypothesis that the ability of specific anti-Abeta antibodies to result in net "efflux" of Abeta from CNS to plasma will best correlate with their ability to prevent/reverse Abeta deposition chronically. Aim 2: Our preliminary data suggest that the rate of Abeta entry from CNS to plasma is influenced by the presence or absence of Abeta deposits in plaques. We will test this idea in APP V717F 00Tg mice with our in vivo Abeta efflux assay. Aim 3: Abeta is transported from CNS to plasma and plasma to CNS across the blood brain barrier. We will use direct methods to determine the effect of anti-Abeta antibodies on bi-directional transport rates of Abeta from brain to plasma and from plasma to brain. This will determine how anti-Abeta antibodies influence CNS/plasma Abeta transport exchanges.

DESCRIPTION (provided by applicant) The present application seeks continued funding for two training programs at Washington University, one that has been jointly administered by the Departments of Neurology and Neurological Surgery for the past 19 years, and another that has been administered by the Division of Pediatric Neurology for the past eight years. The latter, the Neurological Sciences Academic Development Award, cannot be renewed. We seek to provide a program of extended research training for selected trainees in Neurology, Neurosurgery, or Pediatric Neurology, preparing for academic careers. To take advantage of progress in translational neuroscience research over the last few years, we have increased the emphasis of our training program on patient-oriented research. We thus intend to augment our historical emphasis on bench research by offering our trainees the opportunity to gain a rigorous education in clinical research methodology. We believe that the next advances in clinical neurology and neurosurgery will be brought into practice by investigators who combine clinical expertise with formal education in this methodology. Trainees participating in our training program will be expected to devote at least two years of near full-time effort beyond residency training to the task. This is likely the minimum period necessary to develop the scientifically-trained neurological or neurosurgical clinicians who will be essential for elucidating the pathogenesis of nervous system diseases, and bringing new therapies to patients. Despite accelerating neuroscience research advances, there is presently a serious shortage of neurological or neurosurgical physician-scientists or clinical researchers capable of performing these essential translational functions. Indeed, the American Neurological Association currently lists over 150 open positions in academic neurology alone. Exploiting current strengths, both within the training units themselves (Neurology, Neurosurgery, and Pediatric Neurology), and within the larger neuroscience community at Washington University, the proposed program will continue to focus on two major themes: Nervous System Injury, and Nervous System Development. These twin themes are active fronts in disease-related neuroscience today, relevant to understanding the pathogenesis of many different neurological diseases. These themes are complementary, as well as partially overlapping. Insights gained into the nature of the developing nervous system are highly likely to be relevant to understanding nervous system responses to injury.

Clinical, psychological, and neuropathological investigations have shown that most individuals who develop very mild cognitive impairment (clinical dementia rating 0.5) who meet criteria for mild cognitive impairment-MCI, have Alzheimer's disease (AD). Importantly, pathological studies of elderly individuals who die when they are still cognitively normal as well as persons with Down syndrome demonstrate that the neuropathology of AD (plaques and tangles) begins many years (approximately 10-25) prior to the onset of any clinical symptoms or signs of dementia (i.e. pre-clinical AD). Since cell loss is present even at the earliest clinical stages of AD and promising treatments that can potentially delay the onset or prevent the progression of AD are on the horizon, it will be very important to have biomarkers that 1) will predict development of AD in those that are normal; 2) will be helpful in differentiating subjects with very mild impairment due to AD from those that are clinically normal; and 3) will be useful in monitoring or predicting response to specific treatments. The hypothesis of this proposal is that new biomarkers can be developed and improved that will assist in both predicting development and progression of AD as well as improving early diagnosis. We will test this hypothesis, based on our preliminary data, in the following aims: 1. To assess the ability of a high ratio of Abeta40/42 in CSF lipoproteins in cognitively normal eldedy individuals to predict progression to mild cognitive impairment (MCI) and dementia. In addition, we will determine if repeat CSF assessment of the same parameters 4-5 years after the first analysis is useful in assessing dementia dsk and progression. 2. To assess the sensitivity and specificity of the CSF sulfatide (ST)/phosphatidylinositol (PI) ratio as a biomarker for AD and test the hypotheses that 1) low CSF ST/PI ratio in cognitively normal elderly individuals predicts progression to very mild dementia (MCI) and 2) low CSF ST/PI ratio predicts progression from very mild dementia (MCI) to mild dementia. 3. To determine whether other established AD biomarkers (e.g. Abeta42, tau, p-tau231) alone or in combination with the CSF Abeta40/42 ratio in lipoproteins and the ST/PI ratio best predicts progression of cognitive change. We will also begin to assess possible AD biomarkers in middle-aged adults (ages 45-60) who are children of parents with or without AD.

Epidemiological studies have shown that the _4 allele of apolipoprotein E (apoE) is associated with an increased risk for Alzheimef's disease (AD) while the _2 allele is associated with a decreased risk. ApoE has 3 common isoforms in humans, E2, E3, and E4. Genetic, biochemical, and animal studies strongly suggest that apoE is likely to influence AD pathogenesis via effects on the metabolism of the 38-43 amino acid amyloid-[3 (Al3) peptide. By crossing amyloid precursor protein (APP) transgenic mice that develop AD-like pathology with Apoe / mice and transgenic mice that express human apoE isoforms, we have found that the presence ofapoE is critical in the process by which A[3 converts from soluble to fibrillar forms (amyloid) with neuritic plaque formation and cerebral amyloid angiopathy (CAA). The expression of human apoE (as compared to murine apoE or no apoE) markedly delays Al3 deposition suggesting that apoE also plays an important role in A_ clearance. New data suggest that the LDL receptor (LDLR) should be re- explored as a potential modulator of apoE/A_ clearance in vivo. Determining the effects of altering the levels of different apoE isoforms on At3 in vivo as well as the mechanism(s) underlying these effects is likely to lead to importanl insights into AD and CAA pathogenesis and treatment. Thus, we hypothesize that the level and composition of human apoE isoforms will alter both the timecourse and amount of AI3 deposition in an isoform-specific fashion via effects on AI3 clearance and that human apoE isoforms play a role in CNS A[3 clearance in part via LDLR. These hypotheses will be tested in the following aims: Aim 1: To determine if the level of expression of the human apoE isoforms (via different techniques) influences A]3-related pathology in APP transgenic mice. Aim 2: To assess the mechanism(s) by which apoE influences CNS A[3 metabolism in vivo we will use a novel CNS to blood A[3 effiux assay and a new brain A[3 microdialysis technique. Aim 3: To investigate the role of the apoE receptor, LDLR, in A_3 metabolism and related pathology in vitro and in APP transgenic mice. Aim 4: To determine the role of the cholesterol effiux pump, ABCA 1, in modulating the levels and metabolism of apoE, cholesterol, phospholipid, and A[3 in the brain.

genetic susceptibility; apolipoprotein E; genotype; protein isoforms; amyloid proteins; lipid metabolism; brain metabolism; cholesterol; astrocytes; steroid metabolism; clinical research; human subject;

DESCRIPTION (provided by applicant): The overarching goal of this grant, entitled "Washington University Center for Translational Neuroscience- WUCTN" is to support centralized resources, facilities, and expertise shared by neuroscience investigators at Washington University in order to facilitate discovery of fundamental mechanisms of disorders of the nervous system and to translate this understanding into treatments and cures. Core A, Administration, will organize and facilitate interaction among the 6 other cores: Core B, Biospecimen and Clinical Data Acquisition; Core C, Molecular Analysis; Core D, Viral Vectors; Core E, Optical imaging; Core F, Functional Assessment; and Core G, Informatics. By pooling and organizing resources and expertise, the enormous breadth and collaborative spirit of the Washington University neuroscience community that spans virtually every department, can take advantage of economies of scale, confront challenges too large for individual investigators, and develop an infrastructure that will serve the user group of over 120 investigators. The WUCTN will capitalize on the development and resources of 3 new initiatives at Washington University: Biomed 21, the Hope Center for Neurological Disorders, and the Neurological Clinical Research Unit as well as utilize the support and expertise of the long established McDonnell Neuroscience Centers to assist in supporting the personnel and infrastructure being requested in this proposal. In addition, we will work together with several ongoing NIH funded centers at Washington University including the Alzheimer's Disease Research Center, the Conte Center for research on the neurobiology of schizophrenia, and the NINDS Center Core for Brain Imaging to add "extra value" to this proposal. The increasing complexity and cost of the technologies required to characterize biological phenomenon are often beyond the expertise and resources of an individual laboratory. This makes it very difficult to establish and validate key new discoveries in a cohesive, timely, and cost-efficient manner without access to facilities specializing in these enabling technologies. The proposed Core infrastructure, situated within the collaborative environment that exists between the basic neurosciences and the neurobiology of disease community at Washington University, will help catalyze a strong translational effort to convert basic discoveries into diagnostic and therapeutic advances. This will be manifested as: 1) greater efficiency in utilizing enabling technologies by individual investigators; 2) greater collaboration aimed at pre-clinical development of potential therapeutic strategies; and, 3) faster translation of initial discoveries into useful treatments for disorders of the nervous system. Relevance to public health: This Core grant will establish enabling technology resources and technical expertise to facilitate the transition of basic neuroscience discoveries relevant to neurologic and psychiatric diseases into novel diagnostic and therapeutic opportunities.

Project 3 - Pathogenesis of CAA-induced Neurovascular dysfunction. Cerebral amyloid angiopathy (CAA) is a common cause of cerebral hemorrhage in the elderly and is likely to contribute to arteriolar dysfunction and ischemic brain injury. The mechanisms underlying CAA formation and the extent to which CAA contributes to neurovascular dysfunction, dementia, and stroke are not clear. The major constituent of CAA in most individuals is the amyloid-p (A[3)peptide, which also forms amyloid plaques in the brain parenchyma in Alzheimer's disease (AD). CAA is present in most cases of AD and also occurs independently. Ap produced by neurons may also be linked to neurovascular dysfunction and CAA. We have found that in amyloid precursor protein (APP) transgenic mouse models that develop CAA, apoE is required for CAA formation and APPsw/apoE4 mice develop almost exclusively CAA with almost no parenchymal deposits. It has also been shown that anti-Ap antibodies can decrease Ap deposition and improve both neuronal structural abnormalities as well as cognitive deficits in APP transgenic mice. Several positive effects of anti-Ap antibodies on cognition in APP transgenic mice appear to be due their ability to neutralize the toxicity of soluble Ap and are independent of plaque removal. Thus, understanding the effects of soluble Ap and CAA and different forms of Ap on vascular dysfunction may provide new insights into the effects of CAA and new treatment strategies for ischemic brain injury and dementia linked to Ap and CAA. The overall hypothesis of this proposal is that soluble Ap and CAA lead to ongoing arteriolar damage that contributes to neurovascular dysfunction and ischemic injury. In addition, we hypothesize that blocking effects of soluble forms of Ap can inhibit Ap induced neurovascular dysfunction and ischemic injury. In Aim 1, we will utilize in vivo imaging (video and multiphoton microscopy) in APPsw/apoE4, apoE4, and control mice to examine arteriolar vasoreactivity and to determine effects of different forms of Ap and CAA on vessel dysfunction. In Aim 2, we will utilize a well characterized isolated arteriolar microvessel preparation to determine whether vasoreactivity abnormalities linked with Ap and CAA require an intact neurovascular unit and Ap production, or are present specifically in microvessels. In Aim 3, we will determine whether and how Ap and CAA contribute to cerebral blood flow (CBF) abnormalities and ischemia by assessing quantitative CBF and damage in an ischemia model in the presence and absence of agents that target different forms of Ap. RELEVANCE TO PUBLIC HEALTH: AD and stroke are the two most common causes of dementia. Understanding how CAA leads to changes in brain blood vessel function may provide important insights and potentially lead to new treatments for stroke and dementia.

(hydroxymethylglutaryl-CoA reductase (NADPH)) kinase; 21+ years old; 3,4',5-stilbenetriol; 3,4-dihydroxycinnamic acid; 3,5,4'-trihydroxystilbene; 5'-AMP-activated protein kinase; AMP Kinase; AMP-activated kinase; AMP-activated protein kinase; AMPK enzyme; ATP-AMP Phosphotransferase; ATP-AMP Transphosphorylase; Acquired brain injury; Active Oxygen; Acute; Adenylokinase; Adult; Animal Model; Animal Models and Related Studies; Apoptosis; Apoptosis Pathway; Apoptotic; Behavioral; Biochemical; Birth; Body Tissues; Brain; Brain Hypoxia-Ischemia; Brain Injuries; Caffeic Acids; Cardiac Abnormalities; Cardiac Defects, Congenital; Cardiac Malformation; Cardiac defect; Caspase Inhibitor; Cell Death; Cell Death, Programmed; Cell-Death Protease; Clinical; Closure by Ligation; Common Rat Strains; Congenital Heart Defects; Deacetylase; Development; Encephalon; Encephalons; Ensure; Esters; Exposure to; Fetal Age; Fetal Maturity, Chronologic; Fetus; Food; Fruit; GFAC; Generations; Genes, p53; Gestational Age; Gravid; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; HDAC; HDAC Proteins; HMG CoA reductase (NADPH) kinase; HMG CoA reductase kinase; HMG coenzyme A reductase (NADPH) kinase; Heart Abnormalities; Heart Defects, Congenital; Heart Malformation; Histone Deacetylase; Human; Human, Adult; Human, General; Hypoxia; Hypoxia-Ischemia, Brain; Hypoxic; ICE-like protease; Infant; Infant, Premature; Injury; Investigators; Ipsilateral; Ligation; MR Imaging; MR Tomography; MRI; Magnetic Resonance Imaging; Magnetic Resonance Imaging Scan; Mammalia; Mammals; Mammals, General; Mammals, Mice; Mammals, Rats; Man (Taxonomy); Man, Modern; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; Methods; Mice; Mice, Transgenic; Modeling; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Mothers; Murine; Mus; Myokinase; NMR Imaging; NMR Tomography; Necrosis; Necrotic; Neonatal; Neonatal Brain Injury; Nervous System, Brain; Neurologic; Neurological; Nuclear Magnetic Resonance Imaging; Numbers; Operation; Operative Procedures; Operative Surgical Procedures; Outcome; Overexpression; Oxygen Deficiency; Oxygen Radicals; P53; Parturition; Pathway interactions; Patient currently pregnant; Patient pregnant NOS; Pomegranate Juice; Pregnancy not delivered; Pregnancy, gravid; Premature Infant; Pro-Oxidants; Procedures; Protein Overexpression; Proteins; Public Health; Rat; Rattus; Reactive Oxygen Species; Recovery; Research; Research Personnel; Researchers; Resveratrol; Risk; Role; Seizures; Signal Pathway; Surgical; Surgical Interventions; Surgical Procedure; TP53; TP53 gene; TRP53; Testing; Tissues; Transgenic Mice; Tumor Protein p53 Gene; Week; Zeugmatography; adenylate kinase; adult human (21+); base; brain damage; brain lesion (from injury); caffeic acid; caspase; cystein protease; cystein proteinase; cysteine endopeptidase; dietary fruit; feeding; gene product; heart defect; hydroxymethylglutaryl-CoA-reductase kinase; hypoxia ischemia; model organism; motor impairment; mouse model; necrocytosis; neuroprotection; overexpress; pathway; polyphenol; pregnant; premature baby; premature infant human; preterm baby; preterm infant; preterm infant human; preterm neonate; prevent; preventing; protective effect; public health medicine (field); social role; surgery

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span.

Accumulating evidence suggests that Alzheimer's disease (AD) pathology, such as plaques and tangles, begins 10-20 years prior to the earliest signs and symptoms of cognitive decline. This period, during which pathology is developing but individuals remain cognitively normal, has been referred to as 'pre-clinical AD'. One pathological feature that appears to distinguish individuals with pre-clinical AD from those with very early AD (very mildly demented with plaques and tangles) is that the earliest clinical symptoms coincide with neuronal and synaptic loss and/or dysfunction in certain brain regions. Given this observation and that promising disease-modifying treatments are on the horizon, it will be critically important to have biomarkers that: 1) correlate with the presence of AD pathology in the brain regardless of clinical status; 2) predict with high likelihood the development of cognitive decline in individuals who are still cognitively normal but developing AD pathology (antecedent biomarkers); and 3) differentiate those individuals with very mild or uncertain dementia (mild cognitive impairment) who are most likely to experience cognitive decline. Over the last five years, we have found that: 1) low CSF A(342 is very sensitive and specific for determining the presence or absence of amyloid in the brain as assessed by imaging with Pittsburgh Compound B (PIB), regardless of clinical status; 2) ratios of CSF tau/A(342 and ptau181/A(342 are highly predictive of progression from CDR 0 to CDR > 0.5 over an average 3-4 year period; and 3) new potential biomarkers for AD (ATIII, ZAG, CNDP1, ACT) can be identified in CSF by unbiased proteomic techniques. We hypothesize that an assessment of the CSFand plasma proteome, including markers such as A042, tau, ptau181, and other proteins, can be combined to develop an accurate determination of dementia risk in cognitively normal elderly individuals. To test this hypothesis, we propose the following aims: Aim 1:To determine the ability of CSF AB42, tau, ptaul81, ATIII, ACT, ZAG, and CNDP1, both alone and in combination, to predict clinical progression from CDR 0 to CDR > 0 and progression from CDR 0.5 (very mild dementia) to CDR 1 (mild dementia); Aim 2: To determine whether CSF levels of Ap42, tau, ptaul81, ATIII, ACT, ZAG, and CNDP1, both alone and in combination, correlate with brain amyloid load as measured with PiB, brain volume as assessed by structural MRI, and neuropsychological test scores; Aim 3: To determine, in collaboration with the Wyss-Coray lab, whether a group of 18 plasma signaling proteins that was recently shown to classify AD vs. control subjects correlates with CSF biomarkers of AD studied in Aims 1 and 2, as well as whether these 18 proteins predict progression from CDR 0 to CDR > 0 or from CDR 0.5 to CDR 1; and Aim 4: To identify novel CSF biomarkers of AD utilizing a new quantitative and highly sensitive proteomic technique called targeted label free LC-MS/MS analysis.

DESCRIPTION (provided by applicant): This project is under the broad challenge area of 05: Comparative Effectiveness Research. The specific challenge topic is 05-AG-103, Imaging and Fluid Biomarkers for Early Diagnosis and Progression of Aging-related Diseases and Conditions including Neurodegenerative Diseases. Evidence suggests that the pathology of Alzheimer's disease (AD) begins to accrue as many as 10- 15 years prior to the earliest signs and symptoms of cognitive decline characteristic of AD. This period, during which pathology is developing but individuals remain cognitively normal, has been referred to as "pre-clinical AD". Given that neurodegeneration is present even at the earliest clinical stages of AD and that promising treatments could potentially delay the onset or prevent progression of AD, it will be important to have antecedent biomarkers that: predict with high likelihood the development of cognitive decline in individuals who are still cognitively normal but developing AD pathology. Using a combination of CSF biomarkers and both structural and amyloid imaging, we have found that CSF amyloid-[unreadable]42 (A[unreadable]42) and tau are useful biomarkers for both detecting A[unreadable] and tau deposition and for predicting progression from cognitively normal to very mild dementia. However, there is a great need for finding additional biomarkers that when used together are even stronger in predicting the prognosis in individuals who have preclinical AD. It is likely that unbiased biomarker discovery approaches utilized in combination with current biomarker and imaging techniques will provide additive predictive value. Using a new quantitative mass spectrometry technique termed targeted label free LC-MS/MS analysis, we have preliminary data showing that we can assess several thousand individual proteins accurately and quantitatively in individual CSF samples as well as differentiate subjects with very mild AD from controls by using unsupervised hierarchical clustering analyses. By applying this technique to our current large collection of CSF obtained from longitudinally followed research volunteers who have already been assessed clinically, neuropsychologically, with structural MRI and amyloid imaging, we hypothesize that we can distinguish novel patterns in the CSF proteome that will enhance our ability to diagnose the preclinical stage of AD, and identify subjects who will soon progress to dementia. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is a major public health problem. The most effective therapies are likely going to be those that that are implemented before there is irreversible cell and synaptic loss. This means that there is a great need to identify antecedent biomarkers for AD so that therapies can be initiated as early as possible in the disease course to delay or even prevent the disease.

PROJECT SUMMARY (See insti-uctions): Most neurodegenerative diseases, including Alzheimer's disease (AD), are disorders of protein aggregation in which specific proteins that are normally soluble, aggregate in the intra or extracellular space of the brain. In AD, a key factor that regulates whether an aggregation prone protein goes on to misfold is its concentration. Studies have shown that synaptic activity due to both presynaptic arid postsynaptic factors is coupled with the neuronal release of amyloid-B (AB). This suggests that understanding how integrated synaptic and network activity regulate both AB and potentially other proteins involved in neurodegenerative disease, may provide novel insights into pathogenesis. We found that in the brain interstitial fluid of mice (ISF), AP levels are dynamically and positively associated with the number of minutes awake per hour. Pharmacological studies showed that exogenous and endogenous orexin modulates both wakefulness and AB levels. Chronic sleep deprivation markedly increased and an orexin-receptor antagonist decreased AB deposition in APP Tg mouse models. Our preliminary data show that in ISF AB levels as well as measures of brain metabolic/synaptic activity predict how much AB deposition will occur in specific brain regions. Based on these results, we hypothesize that differences in brain metabolic/synaptic activity between brain regions regulate ISF AB levels and that the sleep/wake state regulates both soluble AB levels and ultimately AB deposition via effects on synaptic activity. The specific aims are: 1) To dynamically measure the levels of ISF AB, molecules linked with synaptic activity such as lactate, molecules controlling wakefulness (orexin), and sleep (growth hormone releasing hormone-GHRH) by in vivo microdialysis in different brain regions with aging. 2) To determine the effects of genetic and pharmacological manipulation of orexin and GHRH signaling as well as effects of endogenous NMDA receptor activation, ERK signaling, and LRP1 on ISF AB fluctuations and the sleep/wake cycle in collaboration with projects 2 and 3. 3) To determine the relationship between mean sleep/wake time, other sleep features, and AD biomarkers (amyloid imaging, CSF AB and tau) in an ongoing study of cognitively normal humans (the adult children study) 45-75 years of age.

PROJECT SUMMARY (See instructions): The Administrative Core (Core A) will provide coordination of scientific activities for the 3 projects and 3 cores. It will also facilitate daily operations and longer term planning for the individual and overall scientific program. Statistical consultation will also be available from the Core via Chengjie Xiong, a biostatistician. Core A has 2 aims. Specific Aim 1. To facilitate communication, data sharing, and organize meefings between project leaders and our internal and external advisory boards. Dr. Xiong, as biostatistician, will provide statistical consultation for the projects and cores. Specific Aim 2: To assist in sharing PPG generated research with the general scientific and lay community as well as within and among PPG investigators.

instnjctions): The Synaptic Aß Microdialysis Core will serve projects within this PPG to assess dynamic changes in extracellular, or interstitial fluid (ISF), Aß levels in living mice. Microdialysis enables us to measure Aß levels in the context of a living brain with intact neural networks, a functioning blood-brain barrier, and a normal extracellular milieau. This means that studies are performed in a physiologic setting. Having a core facility organize and conduct these studies, particularly for an intricate technique such as microdialysis, ensures experiments and data across all projects can be combined and compared. RELEVANCE (See instructions): Alzheimer's disease is characterized and likely caused by accumulation of the Aß peptide within the bran extracellular space. Understanding how extracellular Aß changes over time will be critical for determining what factors contribute to disease risk as well as for developing therapeutic interventions. Microdialysis enables us to measure how genes, proteins, or behaviors rapidly change the levels of extracellular Aß.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia. There is compelling data that the amyloid- beta (Abeta) peptide plays a key early role in initiating disease pathogenesis. The progressive buildup of toxic forms of Abeta in the brain appears to ultimately lead to downstream events culminating in dementia. Since the concentration of soluble Abeta peptide is directly related to the probability that it will aggregate, determining what normally regulates Abeta levels in the brain will likely provide critical insights ino factors that initiate the AD pathological cascade. The investigators on this PPG proposal have found that synaptic activity is dynamically coupled with the release of the Abeta peptide in the extracellular space of the brain. Our labs have utilized mouse models of AD to discover some of the cellular mechanisms that link synaptic transmission and dynamic changes in Abeta levels in awake, behaving mice with confirmation in human studies. Abeta is dynamically regulated by the sleep-wake cycle and this regulation appears important in determining Abeta deposition later in life. The regulation of Abeta by the sleep-wake cycle may be tied to synaptic activity as brain interstitial fluid (ISF) levels of Abeta are directly coupled with synaptic activity both pre- and post-synapticall. A molecule likely involved in this coupling is LRP1, since APP endocytosis is required for a large component of Abeta generation and LRP1 influences APP endocytosis and Abeta generation. Our hypothesis is that synaptic activity influences both Abeta production and clearance in the brain and that over time this activity influences whether, where, and when Abeta aggregates into toxic species in the brain. In addition, we hypothesize that synaptic activity-mediated Abeta generation and release 1) is influenced by the sleep/wake cycle and molecules that regulate that cycle; 2) occurs in part via post-synaptic stimulation of NMDA receptors via ERK signaling; and 3) is influenced by the LDL-receptor related protein-1 (LRP1) via its interactions with APP. We will combine unique techniques including in vivo protein microdialysis, 13C-labeled amino acid pulse chase labeling combined with mass spectrometry, and focal viral-mediated gene delivery with approaches that assess systems level network function, synaptic and molecular signaling, and cell biology.

The aggregation of normally soluble amyloid-? (A?) into toxic forms, such as amyloid plaques and oligomers, first begins approximately 10-15 years prior to the onset of cognitive decline associated with Alzheimer's disease (AD). In humans, there is a unique susceptibility of specific brain regions to A? deposition. These regions markedly overlap with the brain's default mode network (DMN). The DMN is a series of brain regions that display high functional connectivity (fc) and neuronal activity while an individual is not engaged in a goal directed task. These regions also demonstrate increased glycolysis, which for the purposes of this PPG refers to glucose uptake in excess of that used for oxidative phosphorylation despite sufficient oxygen to completely metabolize glucose to carbon dioxide and water. It is unclear whether increased glucose/insulin levels can alter glycolysis within the DMN and whether this influences A? levels, synaptic function, or exacerbates AD pathology. This is of interest given the increased risk of AD in patients with diabetes. To understand the relationship between brain glucose/insulin utilization, A? deposition, behavior, fc, and sleep, we have been studying APP/PS1 transgenic (Tg) mice that develop A? plaques in a region-specific pattern similar to that in humans. We have also developed techniques to assess fc in mice using optical imaging (fcOIS). Our published and preliminary data suggest that endogenous synaptic activity and the sleep/wake cycle regulate levels of ISF A? and lactate acutely and A? accumulation chronically. Our preliminary data show that fc in young APP/PS1 Tg mice is normal; however, at 12 months of age, APP/PS1 mice have significant A? deposition and decreased fc as well as disrupted sleep. In this project, we will investigate the mechanisms linking glycolysis, hyperglycemia/ hyperinsulinemia, and sleep deprivation to changes in the default mode network, fc, behavior, and A? deposition. Hypothesis: Hyperglycemia, chronic hyperinsulinemia, and sleep deprivation all increase glycolysis and synaptic A? release, which chronically leads to increased A? deposition in the brain as well as synaptic dysfunction, accelerating the pathogenesis of AD. These ideas will be tested by combining microdialysis, fcOIS, behavior and neuropathology.

DESCRIPTION (provided by applicant): APOE genotype is by orders of magnitude the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The ?4 allele increases risk of AD by ~ 3.7 fold and 2 copies ~ 12 fold; the ?2 allele decreases risk by ~ 50%. Evidence strongly suggests that a major reason underlying these effects is related to the ability of the apoE protein to interact with the amyloid-ß (Aß) peptide and in an isoform-dependent fashion influence Aß clearance and aggregation. ApoE may also influence brain function and dysfunction via additional mechanisms such as influencing synaptic/network activity and lipid metabolism. It is not yet clear how to target apoE biology to develop therapeutic strategies. Our preliminary data suggest the hypothesis that apoE4, when present in the brain interstitial fluid (ISF), reduces Aß clearance and enhances Aß oligomerization/fibrillization, as well as synaptic damage. Increasing apoE2, E3, and E4 via gene delivery methods decreases, is neutral, or increases Aß aggregation and its associated toxicity. Decreasing the amount of toxic apoE/Aß complexes might serve as a therapeutic approach. In fact, our preliminary data utilizing monoclonal antibodies to apoE shows strong effects of decreasing Aß pathology and improving brain network function possibly via microglial-mediated clearance of Aß aggregates. We hypothesize that 1) decreasing Aß aggregation and toxicity may be possible by increasing apoE2 levels in the ISF of the brain; 2) targeting apoE/Aß aggregates with anti-apoE antibodies may serve as a potential therapeutic approach; and 3) that apoE in the ISF and at the synapse may play important non-Aß related functions, which will be critical to understand in the context of any therapeutics based on an apoE mechanism. The specific aims are: 1) To determine whether altering apoE isoform level in specific compartments in the brain influences Aß pathology and associated Aß-dependent brain dysfunction in an isoform-specific and Aß-dependent manner. 2) To explore the effects of anti-apoE antibodies and their mechanism of action in human APP transgenic (Tg) mice expressing human apoE isoforms. 3) To explore potential effects of apoE isoforms on synaptic structure/network function in human apoE knockin mice, wild-type, and apoE knockout mice +/- Aß.

DESCRIPTION (provided by applicant): APOE genotype is the strongest genetic risk factor for Alzheimer's disease (AD); the ¿4 allele increases risk in a dose-dependent fashion and the ¿2 allele decreases risk. APOE appears to influence AD at least in part via isoform-specific effects of apoE on A¿ clearance and aggregation; however, there are likely additional mechanisms by which apoE influences AD. Interactions between apoE and tau, whose aggregation is linked with neurodegeneration, may be an important mechanism as to how apoE influences AD. Certain forms of apoE can bind to tau in an isoform-specific fashion and tau pathology is increased in the brain of ¿4 carriers with AD. We have preliminary data validating a recent paper which shows that a mouse model of tauopathy, (P301S), develops significantly more tauopathy in the absence of apoE. Data from many labs including our own strongly suggests that an important mechanism in tau pathogenesis in tauopathies including AD is that aggregated forms of tau escape the cytoplasm and spread to both adjacent and synaptically connected cells to induce transcellular seeding of tau and disease progression. Tau is present in the interstitial fluid space of the brain normally and this is likely also the case for tau aggregates. Since apoE is an abundantly secreted protein from glial cells, can bind to tau, and both apoE and tau aggregates strongly bind to HSPGs, we hypothesize that apoE interacts with tau in the brain extracellular space to influence its metabolism, aggregation, and effects on neurodegeneration. Our primary hypothesis is that human APOE isoforms dose-dependently influences tau pathology both directly as well as via effects on A¿. Our secondary hypothesis is that human apoE-containing lipoproteins bind to extracellular tau aggregates and influence their ability to induce tau seeding/spreading. The specific aims are: 1) To determine the effect of human APOE isoforms on tau pathology in the presence and absence of human A¿. 2) To determine the effects of human APOE isoforms on the mouse models in Aim 1 on brain interstitial fluid (ISF) tau by microdialysis as well as tau seeding activity in ISF, CSF, and brain lysates. 3) To assess the binding of apoE-containing lipoproteins to both monomeric and aggregated tau and assess their effect on cellular binding, uptake, and tau seeding activity

Project 2: Project Summary/Abstract There is a window of time in humans, hypothesized as preclinical Alzheimer's disease (AD) during which amyloid-? (A?) deposition and tauopathy accrues prior to the detection of clinical symptoms and signs of cognitive impairment. By the time of clinical detection of symptomatic AD, there is already significant cell and synaptic loss. An important goal is to determine whether there are changes not only in imaging and fluid biomarkers, but also in brain function, that are associated with preclinical AD that are 1) quantitative; 2) predict prognosis; and 3) respond to therapeutic intervention. Sleep in an essential biological function that becomes significantly abnormal during the course of dementia due to AD. We have shown that soluble, monomeric A? in both mice and humans is regulated by the sleep/wake cycle. We have also found that as APP transgenic mice develop A? deposition, sleep, particularly non-REM sleep, is markedly disrupted and that this is secondary to A? accumulation. We have also assessed sleep in a mouse model of tauopathy (P301S Tau transgenic mice) and have noted a marked decline in delta power during non-REM sleep. In recent studies, we have assessed sleep in a cohort of late-middle aged individuals with actigraphy, and found that those with A? deposition have significantly decreased sleep efficiency (% of time sleeping while in bed). Very few of the individuals in our first study had developed neurodegeneration (e.g. increased CSF tau) and we did not assess sleep stages directly by EEG. Herein, we will assess a cohort of individuals who are somewhat older (with mean age ~75); many are cognitively normal with and without different stages of preclinical AD and some have very mild dementia. We hypothesize that decreases in non-REM sleep and delta power will begin during the initial phases of A? deposition and worsen with biomarker evidence of neurodegeneration and tauopathy during preclinical AD and mild cognitive impairment (MCI). In addition, we hypothesize that changes in sleep detected initially and longitudinally will be quantitative diagnostic and prognostic markers of brain injury that have potential to respond to therapeutic intervention (theranostic markers). We predict that progressive changes in sleep will correlate with brain atrophy and dysfunction as assessed by structural MRI, functional connectivity MRI, and certain aspects of longitudinal cognitive performance such as the practice effect.

instnjctions): The Synaptic Aß Microdialysis Core will serve projects within this PPG to assess dynamic changes in extracellular, or interstitial fluid (ISF), Aß levels in living mice. Microdialysis enables us to measure Aß levels in the context of a living brain with intact neural networks, a functioning blood-brain barrier, and a normal extracellular milieau. This means that studies are performed in a physiologic setting. Having a core facility organize and conduct these studies, particularly for an intricate technique such as microdialysis, ensures experiments and data across all projects can be combined and compared. RELEVANCE (See instructions): Alzheimer's disease is characterized and likely caused by accumulation of the Aß peptide within the bran extracellular space. Understanding how extracellular Aß changes over time will be critical for determining what factors contribute to disease risk as well as for developing therapeutic interventions. Microdialysis enables us to measure how genes, proteins, or behaviors rapidly change the levels of extracellular Aß.

The aggregation of normally soluble amyloid-ß (Aß) into toxic forms, such as amyloid plaques and oligomers, first begins approximately 10-15 years prior to the onset of cognitive decline associated with Alzheimer's disease (AD). In humans, there is a unique susceptibility of specific brain regions to Aß deposition. These regions markedly overlap with the brain's default mode network (DMN). The DMN is a series of brain regions that display high functional connectivity (fc) and neuronal activity while an individual is not engaged in a goal directed task. These regions also demonstrate increased glycolysis, which for the purposes of this PPG refers to glucose uptake in excess of that used for oxidative phosphorylation despite sufficient oxygen to completely metabolize glucose to carbon dioxide and water. It is unclear whether increased glucose/insulin levels can alter glycolysis within the DMN and whether this influences Aß levels, synaptic function, or exacerbates AD pathology. This is of interest given the increased risk of AD in patients with diabetes. To understand the relationship between brain glucose/insulin utilization, Aß deposition, behavior, fc, and sleep, we have been studying APP/PS1 transgenic (Tg) mice that develop Aß plaques in a region-specific pattern similar to that in humans. We have also developed techniques to assess fc in mice using optical imaging (fcOIS). Our published and preliminary data suggest that endogenous synaptic activity and the sleep/wake cycle regulate levels of ISF Aß and lactate acutely and Aß accumulation chronically. Our preliminary data show that fc in young APP/PS1 Tg mice is normal; however, at 12 months of age, APP/PS1 mice have significant Aß deposition and decreased fc as well as disrupted sleep. In this project, we will investigate the mechanisms linking glycolysis, hyperglycemia/ hyperinsulinemia, and sleep deprivation to changes in the default mode network, fc, behavior, and Aß deposition. Hypothesis: Hyperglycemia, chronic hyperinsulinemia, and sleep deprivation all increase glycolysis and synaptic Aß release, which chronically leads to increased Aß deposition in the brain as well as synaptic dysfunction, accelerating the pathogenesis of AD. These ideas will be tested by combining microdialysis, fcOIS, behavior and neuropathology.

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is the most common cause of dementia and a major public health problem. Data from genetic, biochemical, animal, and human studies suggest that the amyloid-? (A?) peptide plays a key early role in initiating disease pathogenesis and that the microtubule associated protein tau plays a critical role in neurodegeneration and disease progression. Progressive accumulation of A? in the brain appears to ultimately lead to and exacerbate downstream events directly linked to cognitive decline and dementia such as inflammation and tau aggregation. Prior to this PPG proposal, we found that synaptic and network activity is tightly coupled with the release of the A? peptide in the extracellular space of the brain as part of a normal biological process. Our labs discovered some of the cellular mechanisms that link synaptic transmission and network activity with dynamic changes in A? levels in awake, behaving mice with confirmation in human studies. This collaborative work led to the submission and funding of the current PPG which has been funded from 4/1/12 to the present. We have made substantial progress over the last 4 years. Some key findings are that the sleep/wake cycle regulates A? levels dynamically with A? release being higher during wake and lower during sleep. This effect, at least in part, is via neuronal activity differences between wake and sleep. We also found that A? and tau release by neurons is controlled by synaptic activity and can be monitored dynamically. It was also found that A? levels, clearance, and aggregation can be strongly influenced by neuronal LRP1 and heparan sulfate proteoglycans (HSPG). In addition to our findings, increasing evidence indicates that once key proteins involved in neurodegenerative diseases aggregate in the brain (e.g. A? and tau), they appear to spread from one region to others within neuronal networks that are synaptically connected. There is also growing evidence that in AD, A? aggregation in some way drives the progression and spread of tauopathy. We believe that new studies are now warranted to understand the relationship between synaptic and network activity, the sleep/wake cycle, and the impact of the apoE/HSPG/LRP1 on A?, tau, and the spreading of these protein aggregates in the brain. The overall hypothesis of this PPG renewal is that the sleep-wake cycle and brain network activity modulates both A? and tau aggregation and the effect of A? on tau spreading. We further hypothesize that apoE/LRP1/HSPG pathways influence these effects. We will utilize innovative techniques and approaches to study these hypotheses such as the use of DREADDs, in vivo microdialysis, and microimmunoelectrodes as well as a variety of genetically modified mouse models and viral vectors. The specific projects and Cores are listed here. Project 1, D. Holtzman, PI: Effects of the sleep/wake cycle on A?, tau, and spreading. Project 2, J. Cirrito, PI: Neuronal Network Regulation in A? and Tau Conformation and Spreading. Project 3, G. Bu, PI: Neuronal LRP1 and HSPG in pathological spreading of A? and tau. Core A: Administration (D. Holtzman, PI); Core B: Viral Vectors Core (B. J. Snider, PI).

MAYO CLINIC JACKSONVILLE - Subcontract PROJECT SUMMARY The amyloid plaques composed of amyloid-? (A?) and neurofibrillary tangles containing hyperphosphorylated tau are pathological hallmarks of Alzheimer?s disease (AD). Recent studies have shown that these pathological lesions progressively spread from one brain region to another likely through a trans-synaptic mechanism. The major goal of Project 3 is to dissect the molecular and cellular pathways that either promote or inhibit A?/tau spreading with a specific focus on neuronal receptors LRP1 and heparan sulfate proteoglycan (HSPG). As the ?4 allele of the apolipoprotein E gene (APOE4) is the strongest genetic risk factor for late-onset AD, we will also evaluate the apoE isoform-specific effects on A?/tau spreading and whether such effects depend on the presence of neuronal LRP1/HSPG. During the previous funding cycle, Project 3 has defined the roles of apoE isoforms and apoE receptor LRP1 and HSPG in brain A? metabolism, establishing the opposing roles of LRP1 (beneficial) and HSPG (harmful) in brain A? metabolism, as well as the modulatory effects of apoE isoforms. HSPG has been suggested to promote pathological spreading of tau. Importantly, our preliminary studies in collaboration with Project 1 (Holtzman, PI) have also demonstrated a role of LRP1 in cellular uptake of tau monomer and aggregates. Further, we have detected an apoE isoform-dependent effects on tau-mediated behavioral deficits in mice, thus linking A?, tau, apoE, LRP1 and HSPG in related pathogenic pathways. Thus, we hypothesize that LRP1 inhibits and HSPG promotes the speed and extent of pathological spreading of A? and tau aggregates, and that apoE further modulates these events in an isoform-specific manner. We propose three specific aims to address our hypothesis. In Aim 1, we will examine how neuronal LRP1 and HSPG modulate pathological spreading of A? and tau aggregates in vivo using conditional mouse models. We will also address how the presence of amyloid pathology impacts tau pathology and spreading in an LRP1- or HSPG-dependent manner, In Aim 2, we will analyze how apoE isoforms modulate pathological spreading of tau aggregates and whether any apoE effects depend on the presence of LRP1, HSPG, or amyloid pathology. Conditional mouse models as well as viral mediated expression of apoE isoforms will be employed in these studies. In Aim 3, we will dissect the mechanisms by which LRP1, HSPG and apoE isoforms regulate cellular trafficking, degradation or accumulation of various aggregated forms of A? and tau. Mouse primary neurons, induced pluripotent stem cell (iPSC)-derived human neurons, as well as microfluidic platform will be used for these studies. Interactions with other PPG projects will include evaluating the effects of neuronal activities and sleep/wake cycle on LRP1/HSPG/apoE-dependent A?/tau spreading. This project will use Viral Vector Core for the production of AAV and lentiviral vectors for in vivo and in vitro studies. Together, these studies should generate critical insights into the molecular mechanism underlying A?/tau spreading and inform strategies for mechanism-based therapy to combat AD and other neurodegenerative diseases with tauopathies.

PROJECT SUMMARY/ABSTRACT In most neurodegenerative diseases including Alzheimer's disease (AD), specific proteins that are normally soluble, aggregate in either the intra- or extracellular space of the brain. In AD, the aggregation of amyloid-? (A?) appears to initiate disease pathogenesis and the aggregation of tau in specific brain regions is associated with neurodegeneration. Understanding factors that lead to protein aggregation and their spread through specific neural networks will likely provide important insights for development of new treatments. One factor that influences the likelihood that A? or tau will aggregate is concentration. Prior to and over the first 4 years of this PPG, project 1, working with the other PPG investigators, has produced strong evidence that something associated with the sleep wake cycle regulates interstitial fluid (ISF) A? levels at least in part via influencing synaptic activity. Further, manipulations that influence the sleep wake cycle that are linked with increasing or decreasing ISF A? acutely also increase or decrease A? deposition chronically if such changes occur over longer periods of time. While tau is a predominantly a cytosolic protein, we also found that it is present in the ISF and that its levels there can be regulated by excitatory synaptic activity. A key concept that has emerged in neurodegenerative diseases is that certain proteins that aggregate, such as A? and tau, appear to spread within the brain. Once aggregation occurs in one region, protein aggregates will often next appear in another brain region that is in a synaptically connected network. There is strong evidence in AD and in animal models that A? aggregation in some way drives the progression and spread of tauopathy within brain networks. This spread of protein aggregates may occur via a prion-like mechanism. In prior studies of the sleep/wake cycle, we performed manipulations that affect more than just sleep (e.g. stress) and did not specifically affect slow wave sleep. Some important questions remain. Does direct neural manipulation of wakefulness and slow wave sleep have the same effects we have previously seen in regard to A?? Is ISF tau, tau pathology, and tau spreading acutely and chronically affected by the sleep/wake cycle? How does A? influence ISF tau, tau pathology, and tau spreading in the context of changes in the sleep/wake cycle? We hypothesize that ISF A? and A? pathology is strongly affected by the sleep wake cycle and that the ability of A? to drive tauopathy occurs in part via effects of the sleep wake cycle influencing trans synaptic spread of tau aggregates. This hypothesis will be tested in these aims. Aim 1: To directly manipulate slow wave sleep and wakefulness via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and determine the acute effects on ISF A? and tau. Aim 2: To determine the effects of modulating the sleep/wake cycle chronically via DREADDs and other methods on A? pathology, tau pathology synaptic integrity, network function, sleep, and behavior in APP/PS1?E9 mice +/- human tau. Aim 3: To determine the effects of modulating the sleep/wake cycle via DREADDs and other methods in a tau spreading model in the presence and absence of A?.

PROJECT SUMMARY Sleep and Circadian Rhythms in Alzhiemer?s Disease: Potential bi-directional relationship with tau Sleep and circadian rhythm disturbances have long been described in symptomatic Alzheimer?s Disease (AD). Recent studies by our group and others show that these disturbances are detectable years before the onset of cognitive impairment, during the preclinical phase of AD. Our group has shown that modulating the amount of sleep in mice has striking effects on amyloid plaque deposition, as sleep deprivation augments plaque burden while sleep enhancement reduces plaques. Moreover, we have found that levels of A? peptide in the interstitial fluid (ISF) exhibit clear diurnal rhythms which are regulated by the sleep/wake cycle and the central circadian clock, and that disruption of the circadian system and promotes amyloid plaque formation. While amyloid plaque deposition is the first known biomarker change in AD, it appears to be the ability of amyloid plaques to augment tau aggregation and spreading that is directly linked to neurodegeneration and cognitive decline in AD. Tau spreading though the brain, and the effect of A? pathology on tau aggregation, can be modeled by injection of tau-enriched AD brain lysate into the brain of A? plaque-bearing APP knock-in mice. Based on our preliminary data, we hypothesize that sleep disturbance and circadian rhythm disruption may promote tau spreading and aggregation by increasing the release of tau seeding species from neurons. We propose to examine the impact of chronically restricting or increasing sleep on neuronal tau spreading and plaque-induced tau aggregation in mice. Because apolipoprotein E (apoE) strongly influence A? and tau pathology and interacts with sleep, we will elucidate the interaction between APOE genotype, sleep deprivation, and tau spreading and aggregation. Using both genetic and environmental circadian disruption models, we will perform similar experiments to determine the effects of circadian disruption on tau spreading and A?-induced tau aggregation, and explore the interplay between circadian disruption, sleep, and apoE on A? and tau pathology. Finally, we will examine the longitudinal relationship between sleep disturbance, circadian fragmentation, and preclinical A? and tau pathology in humans. We hypothesize a bidirectional relationship between AD pathology and sleep/circadian rhythms, in which AD pathology disrupts sleep/circadian function, while sleep/circadian disruption promotes AD pathology. We will if sleep or circadian rhythm changes are associated with increased future risk of plaque deposition, tau aggregation, or cognitive decline in humans. These studies will elucidate the interaction between sleep, circadian rhythms, and tau aggregation in mice and humans, as well as the role of apoE in that process.