Frank LaFerla, Ph.D. - US grants

Despite significant progress in understanding the genetic basis of familial Alzheimer's disease (AD), the causes of sporadic cases have not been well-defined. Many environmental factors (including heavy metals, Infectious agents, and head injury) have been proposed to underlie this neurodegenerative disorder but no plausible mechanisms have been clearly identified. With regard to infectious agents, speculation has often centered on the neurotropic herpesviruses, with herpes simplex virus 1 (HSV1) considered a likely candidate. We found that an internal sequence of the HSV1 glycoprotein B (gB) bears striking sequence identity/homology to the carboxyl-terminal region of the Beta-amyloid (ABeta) peptide. Our preliminary data indicate that the physical and biological properties of this gB fragment are comparable to ABeta and we demonstrate that the gB peptide forms beta- pleated sheets, self-assembles into fibrils that are thioflavin 5- positive and ultrastructurally indistinguishable from ABeta, and is toxic to primary cortical neurons at doses comparable to ABeta. Notably, this same viral protein also shares homology to apolipoprotein E. Therefore, within the same viral protein exists homology to two of the most Important proteins correlated with the pathology of AD. These intriguing findings may suggest a possible role for an infectious agent in the pathophysiology of sporadic cases of AD. With this knowledge, the proposed experiments are designed to evaluate a possible role for a viral agent as a cofactor in AD. The specific aims of this proposal are [1] to determine whether the gB protein is processed under physiological conditions to yield the ABeta-like region, [2] to determine if the gB protein exists in the AD brain, [3] to determine if high levels of gB expression in the brains of transgenic mice leads to AD-like neuropathology, Establishing a correlation between herpesvirus and AD would clearly provide a novel approach to therapeutic intervention and would warrant clinical investigations into the efficacy of antiviral agents in the treatment of some AD cases. Further, it would indicate that it might eventually be possible to develop effective vaccines to prevent or delay the onset of some sporadic AD cases. The funds provided by this RO3 grant will allow us to obtain the necessary preliminary data that should make it possible to obtain funding through an RO1 grant in the near future.

Inclusion body myositis (IBM) is the most common muscle disease in persons over 50 years of age. Although the cause of this disease remains to be determined, surprisingly it has recently been observed that many of the biochemical features that occur in brains afflicted with AIzheimer's disease also occur in IBM. The Alzheimer's disease brain is characterized by diffuse and neuritic plaques, the hallmark structures of this insidious disease. The principal constituent of these plaques is a small peptide called beta-amyloid. Various histochemical and immunological reagents have been used to show that Beta-amyloid deposits accumulate in affected muscle fibers in IBM. Consequently, IBM represents the first disease, other than those disorders related to Alzheimer's disease such as Down syndrome and vascular dementia, where pathological accumulation of the Beta- amyloid peptide occurs outside the central nervous system. Notably, the accumulation of Beta-amyloid appears to be a specific component of IBM since this peptide is not present in other muscle disorders. This finding is significant and suggests a pathophysiological role for Beta-amyloid in this common, age-related myopathy. The observation that Beta-amyloid accumulates in inclusion body myositis represents an important opportunity to study the role that this peptide plays in this muscular disorder. In this application, we propose to investigate the pathophysiological role of Beta-amyloid accumulation in skeletal muscle tissue by deriving transgenic mice that selectively overproduce this peptide. The muscle creatine kinase promoter, which has been used by other investigators to direct expression to skeletal muscle of transgenic mice, will be used to specifically target transgenes to these tissues. Two sets of transgenic mice will be derived. The first will express only the Beta-amyloid peptide and will test the hypothesis that Beta-amyloid accumulation in muscle leads to pathological changes resembling IBM and provide insights into the pathophysiological role of this peptide in muscle. Another transgenic mouse model will also be derived in which the Beta-amyloid precursor protein, the molecule from which Beta-amyloid is derived, will be overproduced in muscle; these mice will allow us to test the hypothesis that increased expression of the precursor molecule leads to unmanageable levels of Beta-amyloid. Not only would such animal models be useful for studying an age-related muscle disease like IBM, but they may also provide relevant insights into the pathophysiological role these proteins play in Alzheimer's disease as well.

DESCRIPTION (From the Applicant's Abstract): Dysregulation of intracellular calcium signaling process has been critically implicated in the pathogenesis of Alzheimer's disease (AD). Notably, disrupting calcium homeostasis can markedly affect formation of each of the hallmark pathological lesions of this insidious disorder: b-amyloid, neurofibrillary tangles, and neuronal cell death. Moreover, calcium dyshomeostasis is an early and highly consistent alteration that occurs in certain earlyonset, autosomal dominant familial AD cases that are caused by mutations in the presenilin (PS1, PS2) genes. Mutations in the presenilin genes cause gain-of-function effects that lead to AD. Thus far, the two most consistent and pathologically significant disturbances associated with mutant presenilin molecules are alterations of proteolysis of proteins such as the b-amyloid precursor protein (APP) and disruption of intracellular calcium signaling processes. Surprisingly, the relationship between APP mismetabolism and calcium dyshomeostasis mediated by mutant presenilins has not been thoroughly investigated, which is the overarching objective of this research application. Toward this end, we propose 5 specific aims that will address the relationship between presenilin-mediated alterations of intracellular calcium signaling and APP proteolysis. Aim 1 will establish if Ab is necessary for presenilin-mediated effects on calcium signaling, by studying cells that contain mutant PS1 but in which the APP gene is eliminated. Aim 2 investigates whether increased A13 formation is sufficient to reproduce the effects of presenilin mutations on calcium signaling by studying primary neurons and cultured cells overexpressing APP. Aim 3 uses cells derived from presenilin knock-out mice to establish the role of endogenous presenilins on calcium signaling. Aim 4 will determine if y-secretase activity is required for the presenilin-mediated effects on calcium signaling by studying cells expressing dominant-negative presenilin mutations (i.e., mutants of the highly conserved transmembrane aspartate residues). Lastly, by manipulating calcium signaling and determining its effects on AB production, aim 5 will address whether the effects of presenilin mutations on calcium signaling are necessary and/or sufficient to increase Ab formation.

DESCRIPTION (Adapted from applicant's abstract): The investigator proposes to develop and characterize novel transgenic mouse models to study the molecular pathogenesis of inclusion body myositis (IBM). Although the primary cause of this disease remains unknown, surprisingly many of the pathobiological features that occur in IBM are known to be long-standing alterations that occur in the brains of those afflicted with Alzheimer's disease (AD). Histochemical and immunological reagents have conclusively demonstrated that Abeta deposits accumulate in muscle fibers in IBM. Notably, Abeta accumulation does not occur in other muscle disorders. These findings are significant and point to Abeta and its precursor molecule, beta-amyloid precursor protein (betaAPP), as playing a critical role in the pathogenesis of this common, age-related myopathy. By targeting betaAPP and Abeta over expression to muscle, the potential effects of these molecules in the pathogenesis of IBM can be evaluated. The investigator has derived transgenic mice in which the muscle creatine kinase (MCK) promoter drives betaAPP expression in skeletal muscle. Transgenic over expression of betaAPP in skeletal muscle induces an IBM-like phenotype, including intracellular Abeta deposits, centric nuclei, cellular inflammation, and an age-related deficit in motor function. The Aims of this application focus on determining factors that can modulate the IBM phenotype in these transgenic mice. In Aim 1, the MCK-betaAPPtransgenic mice will be crossed to mutant presenilin-l knock-in mice. Since mutations in presenilins augment Abeta formation in every tissue that has been analyzed, these double transgenic mice are expected to develop muscle pathology at an earlier age or exhibit an exacerbated pathology. In Aim 2, attempts will be made to attenuate the IBM-like pathology either through the use of an Abeta vaccine or through the use of pharmacological inhibitors (gamma-secretase inhibitors) that can prevent Abeta production. Aim 3 will focus on characterizing a novel transgenic mouse model that over expresses only the Abeta peptide; this will indicate if Abeta is sufficient to act as the molecular trigger that induces IBM or whether another derivative product of betaAPP is the trigger.

DESCRIPTION (provided by applicant): My lab utilized a novel strategy to produce a triple transgenic model of Alzheimer's disease (AD). Rather than using a crossbreeding approach, we directly inserted two transgenes (human -beta APP/Swe and -tau P301L) into the genome of homozygous PS1M146V knocking mice. This novel strategy produces several unique and significant advantages compared to other approaches: (i) Because the tau P301L and beta APPSwe transgenes were microinjected into embryos from PS1M146V mice, the triple transgenic are on the same genetic background, thereby eliminating genetic heterogeneity. (ii) Both transgenes integrated at the same genetic locus and, therefore, will not independently assort in subsequent generations; likewise the M146V mutation is knocked-in to the PS1 gene, and this allele will also not assort in future generations. Consequently, our triple transgenic mice breed as easily as "single" transgenic mice, a significant advantage for colony management and for introducing additional transgenes into this line. (iii) A large colony of triple transgenic mice can be easily and efficiently generated. In contrast, crossbreeding strategies for generating multi-transgenic genotypes are labor intensive, require extensive genotyping, and result in a low proportion of mice with the desired genotype. (iv) The triple transgenic mice have been bred to homozygosity, which doubles the expression levels, and also further facilitates colony management and breeding. Our triple transgenic mice develop a progressive, age-related AD-like phenotype that includes both both Abeta and tau pathology, synaptic dysfunction, deficits in long term potentiation, and impaired memory-related performance in the Morris water maze, a hippocampal-dependent task of spatial memory. The broad goal of this proposal is to identify and correlate genetic interactions that underlie neuronal and synaptic dysfunction in our triple transgenic mice. In aim 1, we will perform a detailed molecular and neuropathological characterization of the triple transgenic mice and determine how various gene interactions (e.g., tau alone, tau + APP, etc.) influence the development of AD neuropathology. In aim 2, we will determine if the synaptic dysfunction is age-related, identify molecular determinants (e.g., soluble vs. insoluble Abeta, tau hyperphosphorylation) and gene interactions (tau, PS1, or APP) that underlie the synaptic dysfunction. In aim 3, we will determine if calcium dyshomeostasis underlies the synaptic dysfunction and propose to develop a novel transgenic mouse the expresses a calcium indicator protein (inverse pericam) in neurons.

One of the most fundamental and unresolved questions in the Alzheimer's disease (AD) field is whether therapies aimed at clearing Abeta will suffice to stop the progression of this insidious disease. In other words, will removing Abeta plaques impact the subsequent development of AD neuropathology, including the progression of the neurofibrillary pathology? This question, to date, has been intractable in humans. Because my lab developed the first transgenic mouse model (herein referred to as the 3xTg-AD mice) in which both amyloid plaques and neurofibrillary pathology develop in AD-relevant brain regions, in a progressive and age-related fashion, we are uniquely positioned to determine whether treatments targeted specifically at Abeta can also ameliorate the tau pathology. The approach we utilized involved the administration of anti-Abeta antibodies into the hippocampus of the 3xTg-AD mice. Our preliminary findings show that Abeta immunotherapy effectively clears early tau pathology in the brains of the 3xTg-AD mice, although hyperphosphorylated tau aggregates are resistant to clearance. Our results have direct application for the clinical treatment of AD and provide an opportunity to investigate the mechanisms by which Abeta and tau interact. To study the underlying mechanisms by which Abeta and tau lesions are affected by each other the following aims are proposed. Aim 1: Determine the impact of clearing Abeta deposits on the onset and progression of tau pathology. Aim 2: Determine if phospho-tau specific antibodies can clear hyperphosphorylated tau aggregates, Aim 3: Determine the effect of Abeta and tau clearance on behavior: intraventricular delivery of Abeta and tau antibodies. Aim 4. Determine the impact of lowering Ap42 levels on the onset and progression of tau pathology. In summary, the implications for AD and other tauopathies is highly significant as it suggests that interventions such as vaccinations will work provided that it is administered early in the disease course, prior to tau hyperphosphorylation. Moreover, these data provide strong evidence for a link between Abeta and tau in an in vivo, physiologically relevant system.

One of the most fundamental and unresolved questions in the Alzheimer's disease (AD) field is whether therapies aimed at clearing Abeta will suffice to stop the progression of this insidious disease. In other words, will removing Abeta plaques impact the subsequent development of AD neuropathology, including the progression of the neurofibrillary pathology? This question, to date, has been intractable in humans. Because my lab developed the first transgenic mouse model (herein referred to as the 3xTg-AD mice) in which both amyloid plaques and neurofibrillary pathology develop in AD-relevant brain regions, in a progressive and age-related fashion, we are uniquely positioned to determine whether treatments targeted specifically at Abeta can also ameliorate the tau pathology. The approach we utilized involved the administration of anti-Abeta antibodies into the hippocampus of the 3xTg-AD mice. Our preliminary findings show that Abeta immunotherapy effectively clears early tau pathology in the brains of the 3xTg-AD mice, although hyperphosphorylated tau aggregates are resistant to clearance. Our results have direct application for the clinical treatment of AD and provide an opportunity to investigate the mechanisms by which Abeta and tau interact. To study the underlying mechanisms by which Abeta and tau lesions are affected by each other the following aims are proposed. Aim 1: Determine the impact of clearing Abeta deposits on the onset and progression of tau pathology. Aim 2: Determine if phospho-tau specific antibodies can clear hyperphosphorylated tau aggregates, Aim 3: Determine the effect of Abeta and tau clearance on behavior: intraventricular delivery of Abeta and tau antibodies. Aim 4. Determine the impact of lowering Ap42 levels on the onset and progression of tau pathology. In summary, the implications for AD and other tauopathies is highly significant as it suggests that interventions such as vaccinations will work provided that it is administered early in the disease course, prior to tau hyperphosphorylation. Moreover, these data provide strong evidence for a link between Abeta and tau in an in vivo, physiologically relevant system.

DESCRIPTION (provided by applicant): Transgenic mouse models of Alzheimer disease (AD), such as the 3xTg-AD mice, are instrumental for elucidating genetic, pharmacologic, environmental, and behavioral factors that affect the cognitive phenotype. Here we present the novel findings that longitudinal water-maze spatial training produces a significant, albeit transient, improvement in subsequent learning performance and reduces A[unreadable] and tau neuropathology. The 3xTg-AD mice were trained and tested at 3 month intervals from 2 to 18 months. Separate groups of naive mice were also tested at each age. The improvement in performance seen at 6 to 12 months is dependent on spatial training, as animals that were similarly handled and exposed to swimming without a learning contingency failed to show improved performance. Training prior to the development of overt neuropathology is required for full expression of the training effect as we have found it delays A[unreadable] redistribution to extracellular plaques and reduces A[unreadable] oligomers associated with cognitive decline. In addition learning leads to decreased GSK3p activity which likely underlies the reduced tau pathology. The prior-training effects on both maze performance and neuropathology are attenuated at 15 and 18 months. These findings indicate that in young and middle-aged 3xTg-AD mice, repeated spatial training can significantly retard the development of neuropathology and cognitive decline. Aim 1: analyze the cognitive phenotype and determine how lifelong learning in 3xTg-AD mice affects the development of the neuropathology and learning and memory. Aim 2: determine the effects of ApoE4 on tau pathology and learning and memory. Aim 3: determine the impact of exacerbating tau phosphorylation in the presence or absence of A[unreadable] on learning and memory. These studies will lead to a better understanding of the genesis of learning and memory deficits in the 3xTg-AD mice and the relationship of plaques and tangles to cognitive impairments. This proposal represents an in-depth evaluation of learning and memory deficits in a model with both neuropathological hallmarks. More importantly, the outcome will have significant therapeutic implications, allowing us to elucidate the extent to which cognitive decline can be rescued in the context of a brain with both plaques and tangles, and allow us to begin to dissect the molecular mechanisms by which learning and memory are affected.

Project 3: Role of Inflammation on Induction of Tau Pathology in the Brains of Transgenic Mice The Alzheimer disease (AD) brain is characterized by two types of protein aggregates, neurofibrillary tangles and amyloid plaques, which are found predominantly in the hippocampus, amygdala, and cortex. The buildup of Bamyloid (AB) and tau pathology is believed to directly cause or contribute to the progressive cognitive deficits. Because APP and/or presenilin mutant transgenic mice do not develop neurofibrillary pathology despite extensive plaque deposition, the introduction of multiple transgenes into the mouse germline is required to produce both hallmark lesions in mice. My lab recently derived a triple transgenic model of AD (3xTg-AD) in which both plaques and tangles develop in AD-relevant brain regions. The mice were derived by co-injecting human APPswe and tauP301L transgenes, both under the control of the Thyl.2 regulatory element, into single cell embryos harvested from PS1M146V homozygous knockin mice. The mice generated via this strategy are on the same genetic background (thereby avoiding an important confounding variable), breed efficiently (as easily as a single transgenic strain), and exist in both a hemizygous and homozygous genotype (allowing one to assess the effects of doubling gene expression on cognition in mice of the same genetic background). The 3xTg-AD mice represent a critical new resource for understanding the relationship between AB and tau interactions. Preliminary data from our laboratory show that AB can directly affect the tau pathology, as genetic or pharmacological approaches that modulate AB levels have a direct effect on tau pathology. In contrast, modulating tau levels through genetic or pharmacological means does not appear to significantly affect AB pathology. The sum of these studies indicates that AB lies upstream of tau in the pathogenic cascade. The primary focus of this grant application is to better elucidate the pathways by which AB modulates tau pathology in the 3xTg-AD mice. Although evidence from our laboratory indicates that AB likely affects tau pathology through several different mechanisms, one of the key pathways appears to involve microglial-mediated inflammation. Here we propose to elucidate the immune mediated mechanisms by which AB modulates tau pathology. Four specific aims are proposed: Aim 1 will determine the role that microglia play in the induction of the tau pathology. Aim 2 will investigate the role of inflammation in mice with advanced neuropathology and whether it exacerbates or ameliorates the pathology. Aim 3 proposes to use a genetic or pharmacologic approach to selectively increase IL1 levels in the brains of the 3xTg-AD mice to test the hypothesis that IL1 is a potent mediator that increases the severity of plaques and tangles. The last aim proposes to determine the effect that cerebral amyloid angiopathy has on inflammation, and focuses on crossing the 3xTg-AD mice to the ApoE3 and ApoE4 knockin mice.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Alzheimer's disease (AD) is one of a number of age-related neurodegenerative diseases. The cause is unknown, but patients often present with brain pathology of plaques and tangles and, even more importantly, cell death. Vascular defects have been linked to AD development. For example, elderly patients who have undergone a stroke or ischemic episode are 2 times more likely to develop AD than patients who have not. The effects of reduced blood flow on the main pathological peptide in the AD brain, amyloid beta (A[unreadable]), has been documented in vitro and been linked to increased production. However, little is known about the vascular reactivity in AD, a more subtle defect which could contribute to cell death as the AD brain struggles to compensate for hypoxic episodes. In this work we employ spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) for non-contact intrinsic signal in vivo optical imaging of brain tissue composition and function. In SFDI, intrinsic chromophore concentrations of oxy- and deoxy-hemoglobin, water, and lipid, in addition to high-contrast wavelength-dependent maps of tissue scattering were recovered. Concurrent LSI acquisition yields average blood flow measurements. Together with inhaled gas challenges we measured baseline and dynamic changes in blood chromophore concentrations, blood flow, and brain tissue scattering in the AD triple transgenic and APP-/- mice.

DESCRIPTION (provided by applicant): Neurological disorders such as Alzheimer's and Huntington's disease, spinal cord injury, traumatic brain injury, multiple sclerosis, radiation injury, and brain developmental disorders are characterized by profound neuronal and glial injury, leading to significant impairments in cognitive and/or motor functions. Although the etiology of these disorders vary, the signature and defining pathological features for many of these conditions is the loss of neurons and synapses, loss of neuronal connectivity, tissue volume loss and expansion of the ventricular spaces, dysregulated neurogenesis, and rampant neuroinflammation, gliosis, and scarring. The investigators listed on this proposal are all members of the Institute for Memory Impairments and Neurological disorders at UC Irvine (UCI MIND), and are seeking funds to acquire a multichannel fluorescent structured illumination stereological imaging system to quantify tissue changes with unbiased methods. Neuropathological studies are severely limited by attempts to study three-dimensional interactions by observations made in two dimensions. Stereology, however, is a science that overcomes these limitations by furnishing three- dimensional interpretations of planar sections of biological tissues, as it is based on merging of the principles of geometry and statistics. Consequently, stereological studies of the nervous system provide a means to obtain more precise quantitative measurements of cell number and neural processes and tissue volume. In addition, for studies on neurodegenerative disorders, it is possible to determine the quantitative relationship between the accumulation of brain lesions such as amyloid plaques and cell number, for example. Standard brightfield stereology allows one to accurately quantify one immunolabel at a time in tisue sections. Double and/or triple labeling immunocytochemistry typically require fluorescent labeling and more specialized imaging techniques. An Apotome structured illumination system allows confocal-like 3D images to be acquired for both real-time and off-line analysis of multiple labels. Such a system would allow investigators to quantify two or three parameters simultaneously (e.g. total number of GFP-expressing transplanted cells, and a second or third fate marker to obtain total engraftment numbers and percentage of neural, astroglia and oligodendroglial fate in the same tissue sections).

DESCRIPTION (provided by applicant): This application seeks to renew the Alzheimer's Disease Research Center (ADRC) at the University of California, Irvine (UCI). Our overall objectives are to elucidate the factors that trigger the onset of AD and its conversion from the prodromal state of MCI, and to define the clinical and pathological features of AD. Our ultimate goal is to identify means to prevent, mitigate and eradicate this disorder. Towards this end, the UCI ADRC proposes four Cores and 3 Projects. The Administrative Core, directed by Dr. Carl Cotman and co- directed by Dr. Frank LaFerIa, will manage, guide and support the ADRC. The Clinical Core (Core 6), directed by Dr. Claudia Kawas, follows the standard cohort (controls, MCI and AD) and two unique human cohorts: (1) Down syndrome subjects, which represents the single largest cause of early-onset AD, and (2) individuals over 90 years, a particularly high incidence dementia group well-represented in the area for inclusion in an autopsy study. The Data Management and Statistics Core, directed by Dr. Dan Gillen, will manage ADRC data and support the Cores and ADRC investigators in standard and novel data analysis. The Neuropathology and Tissue Resources Core, co-directed by Dr. Ron Kim and David Cribbs, will collect postmortem and distribute brain tissues and body fluids from the clinical cohorts. The Core will also support investigator driven translational research with transgenic animal models, beta-amyloid peptides (AP) and conformation specific antibodies to A¿. The Education and Information Transfer Core, directed by Dr. Ruth Mulnard, will assist in subject recruiting and in education and information among ADRC investigators and the community. To support the overall goals of the ADRC and provide leadership in the field, the UCI ADRC proposes three Projects: 1) Neuroimaging of Hippocampal Subfields in Older Adults & MCI (Project Leader: Craig Stark, Ph.D.); (2) Astrocyte-related molecular mechanisms underlying altered neuronal plasticity in Down syndrome (Project Leader: Jorge Busciglio, Ph.D.) and (3) Neural stem cells to treat and model Alzheimer Disease (Project Leader and new investigator: Mathew Blurton-Jones, Ph.D.). These projects and Cores will interact extensively with other ADRC investigators to continue to build an exciting and productive research environment directed at elucidating the triggers, features and ultimately treatments for cognitive decline and development of AD.

DESCRIPTION (provided by applicant): Alzheimer disease (AD) impairs memory and causes cognitive and psychiatric deficits. Over 35 million people throughout the world are afflicted, including 5.4 million in the USA. All AD cases are marked by the accumulation of two lesions in the brain called plaques (consisting of a protein called A¿) and tangles (consisting of a protein called tau that becomes hyperphosphorylated. One of the most fundamental and unresolved questions in the field centers around elucidating the relationship between A¿ and tau and their role in cognitive decline. It is critical to determine if A¿ triggers pathology in human wild-type au, rather than the mutant used in previous models. Moreover, this proposal seeks to identify the interactions between these two critical proteins at the synapse using synaptoneurosomes, thereby enabling us to quantify A¿ and tau levels in the synapse and other key markers of synaptic function. Lastly, the broad goal of this application is to understand how these synaptic changes lead to cognitive deficits, and whether therapies that remove either A¿ or tau alone are sufficient to improve behavior. It is important to point out that research in the AD field can only progress if the tools evolve, hence there is an urgent need to develop and characterize new animal models until an effective treatment is discovered. Because the 3xTg-AD mice harbor mutant tau, addressing these issues necessitated the generation of innovative new transgenic models and viral vectors to manipulate gene expression in vivo. The first novel model we developed is a floxed human APP transgenic mouse that permits ablation of APP expression using Cre recombinase. We also created a novel human wild-type tau transgenic model that develops phosphotau pathology. After crossing these two lines, we can discover the impact A¿ has on the development of tau pathology during various stages (i.e., before, during, and after tau pathology is established). Results from this study will reveal the conditions under which A¿ induces wild-type tau pathology. Additionally, we will be uniquely positioned to determine if tau pathology continues to cause synaptic deficits even if A¿ is suppressed using Cre. Using viral gene delivery to the CNS, we will determine whether intracellular and extracellular A¿ plays a greater role in modulating tau pathology and cognitive decline. The development of the floxed human APP transgenic mice represents a significant advance for the field, as it enables abrogation of human APP gene expression using viral delivery of Cre recombinase during temporally-specified timepoints. Utilizing these novel models and innovative genetic approaches that add significantly to the research tool armatorium, we will unravel the distinctions between the cognitive loss due to A¿-dependent and -independent mechanisms, the pathological time point of A¿-induced tau dysfunction, and whether the presence of A¿ intracellularly or extracellularly facilitates tau pathology and cognitive decline. Because a better understanding of these pathways is critical not only for academic reasons but also for helping to identify novel drug targets, the translational impact of this work is substantial and significant.

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is the leading cause of age-related dementia, affecting over 5 million people in the United States alone. Unfortunately, current therapies are largely palliative and several promising drug candidates have failed in late-stage clinical trials. Hence, there is an urgent need to improve our understanding of the basic mechanisms that drive the development of AD and to develop new and effective therapies. Preclinical AD research has thus far relied heavily on transgenic mouse models but recent advances in stem cell biology have opened up an exciting new opportunity to model AD and test therapeutics with patient-derived human cells. Stem cell research is a major strength of the UCI ADRC as we were the first to show improved cognition in transgenic models of AD with neural stem cell transplantation and to use human embryonic stem cells to examine AD-associated mutations. The UCI ADRC therefore proposes the establishment of the first AD induced pluripotent stem (IPS) cell core. The goals of this core will be to generate, validate, and distribute a well-powered collection of AD, MCI, and control IPS cell lines to researchers within the UCI ADRC, within the whole ADC program, and worldwide. The significant advantage of having this core be part of the ADRC is that each cell line is linked to corresponding multi-dimensional clinical, biomarker, and pathological datasets. Hence, the IPS cell will provide scientists with a powerful, novel and innovative approach to understand the genetic and phenotypic basis of sporadic AD and identify and evaluate novel disease-modifying therapeutic interventions. To achieve these goals, the IPS cell core proposes the following 4 specific aims: AIM 1: Isolate, expand, and bank primary skin fibroblasts and peripheral blood mononuclear source cells from ADRC clinical cohort subjects. AIM 2: Generate integration-free IPS lines from fibroblasts of ADRC subjects. AIM 3: Validate, characterize, and expand ADRC iPS cell clones. AIM 4: Distribute ADRC iPS cell lines to investigators and facilitate access to corresponding clinical, biomarker, and pathological datasets.

Project 3: Role of Inflammation on Induction of Tau Pathology in the Brains of Transgenic IVIice Alzheimer disease (AD) impairs memory and causes cognitive and psychiatric deficits. The number of people with AD will quadruple to 115 million worldwide by 2050, with cumiulative costs of care in the absence of disease-modifying treatments exceeding $204rillion over the next 40 years alone in the USA. Should this expectation come to fruition, it will pose an unprecedented medical, social, and economic burden on our society. One of the most fundamental and unresolved questions in the field centers on elucidating the role that inflammation plays in disease progression, and in particular, how the cerebral buildup of p-amyloid (Ap) promotes inflammation and the development of hyperphosphorylated tau. Notably, our studies identified inflammation as an early and critical step that links Ap to tau pathology and cognitive decline. Supporting GWAS-derived evidence further reinforces the importance of inflammation, as single nucleotide polymorphisms in many immune-related genes significantly increase the probability of developing AD. Although inflammation is critical to disease progression, a detailed molecular analysis of specific mechanisms of the inflammatory response is greatly needed. Among numerous inflammatory pathways associated with AD, interleukin-ip (IL-ip) plays a critical pathogenic role. We hypothesize that AQ> alters intracellular protein clearance and trafficking, exacerbating IL-ip-mediated inflammation, eliciting tau pathology and synaptic and cognitive deficits. Our goal is to elucidate the impact of Ap on IL-ip signaling with emphasis on fiie relevance of protein clearance for IL-ip synthesis and protein trafficking for IL-1 receptor 1 (IL-1 Rl) levels. We developed several new and exciting transgenic models and viral approaches that add significantly to the field and enable us to dissect the molecular pathways by which Ap, IL-ip and tau interact and the mechanisms by which they adversely impact cognition during different stages of the disease process. Because a better understanding of these pathways is critical not only for academic reasons but also for helping to identify novel drug targets, the translational impact of this work is quite significant. RELEVANCE (See instructions): Inflammation plays both a protective and damaging role in Alzheimer disease (AD), so to identify a long lasting and effective treatment, it is important that we better understand its underlying processes. Our studies implicate a critical cytokine called interleukin-ip (IL-1P) as a factor that accelerates AD pathology. Here we propose to study the molecular mechanisms by which this cytokine alters basic cell biological functions and how these changes affect AD pathogenesis.

DESCRIPTION (provided by applicant): Understanding the factors that cause people to transition from normal aging to preclinical Alzheimer's disease (AD) to mild cognitive impairment, and subsequently convert to dementia is the central focus of the University of California Alzheimer's Disease Research Center (UCI ADRC). The UCI ADRC integrates basic and translational science and clinical domains to elucidate the heterogeneity in the clinical sub-types of AD, including early-onset, genetic, and late-onset forms. As such, we follow three valuable cohorts: (1) longitudinal cohort, which includes older adults with preclinical and early-stage AD, (2) Down syndrome cohort, representing the largest genetically at-risk population for AD, which enables us to describe the role of amyloid deposition in the early years of the life span and the particular vulnerability that this might impart for developing AD, and (3) the oldest-old, as part of the 90+ Autopsy Study, which show great disparity between AD pathology and cognitive loss. The UCI ADRC brings energetic and innovative multi-dimensional and multi- disciplinary approaches toward solving this insidious disease and also contributes to several national efforts.

Core A: Administrative Core Project Summary/Abstract The Administrative Core of the University of California, Irvine Alzheimer's Disease Research Center (UCI ADRC) provides innovative leadership, overall administrative support and oversight, and promotes the integration with the other cores, projects and pilot awards, thereby fostering the centeredness of our ADRC. The Administrative core establishes and implements policies to ensure the highest scientific and ethical studies, ensures compliance with HIPAA, NIH, and university policies and requirements related to human subjects, animal research, and stem cells. The Administrative Core oversees many critical areas to ensure the highest standards are achieved by providing administrative and financial supervision, development and promotion of scientific objectives, establishment and coordination of the External and Internal Advisory Committees, solicitation, review, and support for the pilot projects, interactions with the national ADC program, and recruitment, training and support of junior investigators and clinicians. A major function of the Administrative Core is to promote the ADRC with key leaders of the university administration and to procure resources. In short, the Administrative Core ensures that all operations of the UCI ADRC are functioning flawlessly and at the highest levels of productivity and standards.

PROJECT SUMMARY/ABSTRACT Alterations in the dichotomy between initiation and resolution of the immune response is a primary contributor to the reduced quality of life reported with aging. As we age, the immune system becomes dysregulated and is characterized by persistent inflammation. Changes in the immune system contribute to the increased susceptibility of the elderly to innumerous diseases including Alzheimer's disease (AD). A crucial aspect of this process is a failure to resolve inflammation, which normally involves the suppression of inflammatory cell influx, effective clearance of apoptotic cells, and promotion of inflammatory cell egress. Working under the hypothesize that the failure to properly resolve inflammatory pathways within the brain significantly contribute to the clinical manifestations of AD, our previous studies demonstrate that chronic inflammation mediated by interleukin-1? receptor 1 (IL-1R1), toll-like receptor-4 (TLR4), and tumor necrosis factor-? receptor (TNFR), represents a key mechanism by which ?-amyloid (A?) drives the development of tau pathology and cognitive decline in AD. The potent pro-inflammatory activities of these receptors are counter-regulated by target of Myb1 (Tom1) and its interaction partner toll-interacting protein (Tollip) via endocytosis and lysosomal degradation mechanisms to ensure proper resolution of immune responses. Remarkably, our preliminary data show that levels of Tom1 and Tollip are significantly reduced in human AD brains versus respective aged- matched controls. Further studies using AD model mice, show that reductions in Tom1 and Tollip occur vary early in the disease, prior to tau deposition and cognitive disruption. Therefore, reductions in Tom1 and Tollip may result in excessive expression of inflammatory receptors that in turn lead to exacerbated neuroinflammation, and represent an as yet identified link between A?, tau and cognitive disruption. Here we hypothesize that changes in the resolution of inflammatory receptors are an early A?-triggered event that leads to exacerbated neuroinflammation, which in turn evokes tau pathology, synaptic dysfunction and cognitive decline. To address properly our hypothesis, we will use cutting-edge in vitro and in vivo approaches, such as our novel APPKI-hA?wt that express wild-type human A? under the control of the endogenous mouse APP gene, to establish the connection between A? pathology and changes in Tom1-mediated endocytosis. Lastly, we will investigate the impact of persistent inflammatory receptor activation on tau pathology and cognition by using our innovative wild type human tau model (hTau). This research project will elucidate the underlying molecular mechanisms linking A? to Tom1 signaling, tau pathology and cognitive decline in AD. As we gain a better understanding of the age-related changes in the immune system, our overall goal is to craft therapies to proper activate the resolution of inflammation, with the broader purpose of sharply reducing the number of people suffering and dying from AD.

Project Abstract Alzheimer's disease (AD) is currently an incurable neurodegenerative disease that affects over 35 million people worldwide, including 5.4 million individuals in the USA with a new case diagnosed every minute. Over the past two decades, one of the most significant developments in the AD research field has been the generation of mouse models of AD. Although these have provided significant insight into the mechanism of AD, the findings have not yet translated into the development of any new disease-modifying therapies for the human condition. Moreover, there is concern about the discordance of treating AD in people versus mice, which may be due the incomplete modeling of the disease in mice. Along these lines, all of the currently available models are based on the rarer autosomal dominant form of the disease, whereas the majority of AD cases are sporadic, whose onset may still be influenced by genetics that display reduced penetrance compared to the autosomal dominant cases. Hence, there is an urgent need to develop the next generation of mouse models that have high face, construct, and translational validity, which means these models should be more closely aligned with sporadic AD (sAD). Over the past several years, the facility of GWAS has produced a rapid expansion in the list of risk factor genes that are associated with sAD. Here we propose to use a transdisciplinary/team approach to develop the next generation of mouse models that model sAD so that they can be used for preclinical therapeutic testing. Our strategy is to use our recently developed humanized wild- type APPKI mouse as the platform for introducing human tau, followed by other GWAS-identified risk polymorphisms that enhance the risk of developing sAD. We propose to develop the next generation of AD preclinical mouse models using the latest innovations in gene editing technology (CRISPR/Cas9 technology) to produce new mouse models that more accurately represent sAD. We will phenotype the mice using state-of- the-art quantitative methodology and make direct comparisons to the human condition and capitalize on novel reagents that have been developed at UCI, including unique conformation specific antibodies that identify multiple distinct forms of pathology. We will determine gene expression changes via RNA-seq and epigenetic disruptions, alterations in neuronal connectivity in hippocampal circuits via whole-cell patch clamping combined with laser scanning photostimulation, as well as LTP, behavior and cognition, and longitudinal functional imaging. We will also conduct biomarker development by performing plasma lipidomic and metabolomic analyses. We will distribute all data in an expeditious and accessible form for dissemination and will provide detailed protocols for characterization of the models for the field. Lastly, we have established an exciting partnership with The Jackson Laboratory to conduct second site validation of observed phenotypes and to re- derive, cryopreserve and distribute all new animal models so that they can be widely distributed to investigators in the field. Achieving these goals will be transformative for the AD research field.

The NexGen Alzheimer's Disease Models Center (NG-ADMC) will be administratively housed within the University of California, Irvine Institute for Memory Impairments and Neurological Disorders (UCI MIND). This Organized Research Unit at UCI administers the NIH funded ADRC, an Alzheimer's disease focused NIH Program Project Grant, an NIA Training Grant, and multiple other grants, requiring coordination of multiple investigators and multiple disciplines to achieve objectives that will transform the future. The Administrative/Coordination Core (ACC) of the NexGen Alzheimer's Disease Models Center will provide coordination of the entire Center and ensure fiscal accountability. Drs. LaFerla and Tenner have collaborated on multiple projects at UCI with complementing expertise in animal models, neuroinflammation, and therapeutic targetings. A Center Administrator with extensive administrative experience, as well as laboratory technical and management experience, will oversee daily transactions, organize routinely scheduled meetings, and contribute to fiscal planning and execution The Steering Committee will be convened at least every two months to review data and discuss any modification in procedures, direction, or priorities to facilitate the progress across the Center. The External Advisory Board will be convened every six months with the Steering Committee to affirm or modify Center activities toward achieving milestones of the multiple units. Importantly, a Project Manager who is knowledgeable about the various phenotype characterizations and experimental design will oversee the day-to-day placement of mice, including the efficient use of aged animals among the Center investigators. In addition, the Manager will maintain the catalog and inventory of the tissue repository, provide access to reagents/tissues under the guidance of the Co-PIs, and coordinate animal transfers to The Jackson Laboratory (JAX). The ACC will lead Center program evaluation activities, including annual reports of each core, and ensure communication among the units to accelerate progress toward achieving milestones. The ACC will communicate Center activities with the NIH Program Official and implement suggestions received from the NIH and the EAB.

PROJECT SUMMARY Increased attention is being paid to the need for a science of recruitment and retention in Alzheimer's disease clinical research. While barriers to successful and efficient recruitment are being elucidated, less work has focused on retaining longitudinal research participants. Poor retention stands to undue successful recruitment and can result in bias and scientific error. The purpose of this supplement application is to add the UC Irvine ADRC to a funded NACC Collaborative study to improve the retention of participants in longitudinal AD research studies. The objective of this study is to identify facilitators and barriers to longitudinal research participation and develop new guidelines to increase study retention. We will conduct a comprehensive survey of research participants age 45 years and older enrolled in our longitudinal cohort who have been noncompliant with their clinical visits using an AD research-specific survey; their study partners will also be surveyed. Findings will inform the development of guidelines that aim to improve retention in longitudinal AD studies.

Abstract/Summary A key goal of the UC Irvine MODEL-AD Disease Modeling Project (DMP) is to generate mice that express human TAU (hTAU) at physiological levels, with equivalent expression of the 3R and 4R hTAU isoforms. Identifying such mice requires screening of expression of hTAU protein isoforms in the CNS of multiple animals by quantitative western blot analysis. Once a suitable hTAU mouse model has been developed, additional quantitative analysis of hTAU expression will be required after the hTAU model has been combined with additional LOAD risk alleles ? e.g. APOE4; hAb-KI, Trem2R47H. Quantitative western blot analysis of hTAU expression in the adult brain of a relatively large number of mice can be performed efficiently using an instrument that combines accurate and sensitive quantification with relatively high throughput. This can be performed using a LI-COR near-infrared western blot imaging system. Hence, we request a Supplemental Award to purchase a LI-COR Odyssey CLx Infrared imager, with Image Studio Software, and a computer, for use in this project.

Project Summary/Abstract The goal of MODEL-AD is to generate improved mouse models of sporadic, late-onset Alzheimer's disease (AD). Ideally, such mice will develop facets of human AD pathology including neuritic plaques, neurofibrillary tangles, and widespread cortical and hippocampal neurodegeneration in advanced age. The C57BL/6J (B6J) mouse strain background has been selected as a uniform inbred strain to use for the production of mouse models by MODEL-AD. However, there is concern regarding the validity of using a single genetic background of mice to model a complex and polygenic human disease such as AD. Indeed, recent data from the UCI MODEL-AD DMP and that of our IU/JAX collaborators provide independent support which strongly suggests that the standard B6J mouse genetic background is not optimal for the formation of plaques or for neurodegeneration. We propose to test the hypothesis that genetic diversity affects pathology in mouse models of late-onset AD by using a unique genetic resource ? the Collaborative Cross (CC) strains. CC reference strains represent a multi-parental recombinant inbred panel derived from eight laboratory mouse inbred strains, which allow standardization of studies to investigate how genetic diversity impacts pathology. Importantly, the genetic diversity within CC lines is similar to that found in human populations. To address the concern of limiting MODEL-AD models to a single genetic background, we request a supplement to investigate the effect of introducing genetic diversity into the hAb-KI/Trem2/ApoE4 mouse on development of AD phenotypes. We anticipate that this study will identify genetic backgrounds with increased diversity that are more conducive to plaque formation and neurodegeneration compared to B6J and hence provide mice that more accurately develop AD pathology as they age. Mouse lines with optimum AD pathology will be sent to JAX for distribution to the international community.

Project Summary/Abstract A key goal of the UCI MODEL-AD Disease Model Development and Phenotyping Project (DMDPP) is to generate and phenotype new models of Alzheimer's disease (AD) that better recapitulate late onset AD (LOAD). Cognitive impairments characterize Alzheimer's disease, and are recapitulated in existing mouse models of the disease. Meaningful treatments for Alzheimer's disease will have to address these cognitive impairments, and thus it is imperative to characterize cognition and behavior in newly generated models of the disease. This will provide disease relevance and also to uncover any unexpected phenotypes that may arise as a consequence of disease modeling, and will complement the electrophysiology, gene expression, and pathological data that are collected from each of the mouse models. Additionally, this will allow for harmonization between the other MODEL-AD centers, which are conducting similar behavioral and cognitive testing of the models. To include behavioral and cognitive testing into our existing phenotyping protocol we are requesting funds to purchase a Noldus Ethovision XT Tracking System + chambers. !

Project Abstract Alzheimer's disease (AD) is currently an incurable neurodegenerative disease that affects over 35 million people worldwide, including 5.4 million individuals in the USA with a new case diagnosed every minute. Over the past two decades, one of the most significant developments in the AD research field has been the generation of mouse models of AD. Although these have provided significant insight into the mechanism of AD, the findings have not yet translated into the development of any new disease-modifying therapies for the human condition. Moreover, there is concern about the discordance of treating AD in people versus mice, which may be due the incomplete modeling of the disease in mice. Along these lines, all of the currently available models are based on the rarer autosomal dominant form of the disease, whereas the majority of AD cases are sporadic, whose onset may still be influenced by genetics that display reduced penetrance compared to the autosomal dominant cases. Hence, there is an urgent need to develop the next generation of mouse models that have high face, construct, and translational validity, which means these models should be more closely aligned with sporadic AD (sAD). Over the past several years, the facility of GWAS has produced a rapid expansion in the list of risk factor genes that are associated with sAD. Here we propose to use a transdisciplinary/team approach to develop the next generation of mouse models that model sAD so that they can be used for preclinical therapeutic testing. Our strategy is to use our recently developed humanized wild- type APPKI mouse as the platform for introducing human tau, followed by other GWAS-identified risk polymorphisms that enhance the risk of developing sAD. We propose to develop the next generation of AD preclinical mouse models using the latest innovations in gene editing technology (CRISPR/Cas9 technology) to produce new mouse models that more accurately represent sAD. We will phenotype the mice using state-of- the-art quantitative methodology and make direct comparisons to the human condition and capitalize on novel reagents that have been developed at UCI, including unique conformation specific antibodies that identify multiple distinct forms of pathology. We will determine gene expression changes via RNA-seq and epigenetic disruptions, alterations in neuronal connectivity in hippocampal circuits via whole-cell patch clamping combined with laser scanning photostimulation, as well as LTP, behavior and cognition, and longitudinal functional imaging. We will also conduct biomarker development by performing plasma lipidomic and metabolomic analyses. We will distribute all data in an expeditious and accessible form for dissemination and will provide detailed protocols for characterization of the models for the field. Lastly, we have established an exciting partnership with The Jackson Laboratory to conduct second site validation of observed phenotypes and to re- derive, cryopreserve and distribute all new animal models so that they can be widely distributed to investigators in the field. Achieving these goals will be transformative for the AD research field.

PROJECT SUMMARY/ABSTRACT The goal of MODEL-AD is to generate animal models of AD that recapitulate the sporadic form of the disease. We are accomplishing this by humanizing disease relevant genetic loci ? notably the Ab sequence in APP, the Tau gene, and appropriate AD-risk variants identified via GWAS. By design, we predict the pathologies will manifest in an age-dependent fashion. C57BL/6J mice have a mean lifespan of 26.3 months for males and 24.3 months for females. Reconciling corresponding mouse and human aging timelines, C57BL/6J mice would not be expected to spontaneously develop AD-relevant pathologies until 20+ months. However, the current award only proposes phenotyping of generated mouse models up to 18 months of age. To rectify this, we now request a 3-year supplement that will enable us to age and phenotype several models to 24 months of age, where the most disease relevant pathology is expected to be found. We will introduce a screening platform to identify which MODEL-AD generated mice have the greatest potential to drive sporadic AD pathologies. The two most promising GWAS-risk-allele MODEL-AD mice identified by this screen will then be bred onto the hAb-KI/hTau mouse background and then aged to 24 months (along with the relevant control strains), and then deep phenotyped and the data released to the community. Additionally, we are evaluating mouse strains with diverse genetic backgrounds that approximate the genetic diversity in the human population, to identify which are most permissive for AD-relevant phenotypes. Through an existing supplement, both UCI and the IU/JAX groups are currently evaluating the potential of 6 independent Collaborative Cross (CC) lines to promote AD-relevant pathologies. From this we will select the 2 most permissive CC lines and introduce the human Ab sequence via CRISPR. We will then age these newly generated CC-h Ab and control parental CC lines to 24 months and perform deep phenotyping. Collectively, this supplemental award will allow us to identify and prioritize GWAS- risk allele MODEL-AD mice and genetic backgrounds that are most permissive for the development of AD- relevant pathologies, generate them with human disease relevant sequences (i.e. human Ab and/or Tau), and then age and phenotype them, leading to the generation of models of sporadic AD.

Project Abstract Alzheimer's disease (AD) is currently an incurable neurodegenerative disease that affects over 35 million people worldwide, including 5.4 million individuals in the USA with a new case diagnosed every minute. Over the past two decades, one of the most significant developments in the AD research field has been the generation of mouse models of AD. Although these have provided significant insight into the mechanism of AD, the findings have not yet translated into the development of any new disease-modifying therapies for the human condition. Moreover, there is concern about the discordance of treating AD in people versus mice, which may be due the incomplete modeling of the disease in mice. Along these lines, all of the currently available models are based on the rarer autosomal dominant form of the disease, whereas the majority of AD cases are sporadic, whose onset may still be influenced by genetics that display reduced penetrance compared to the autosomal dominant cases. Hence, there is an urgent need to develop the next generation of mouse models that have high face, construct, and translational validity, which means these models should be more closely aligned with sporadic AD (sAD). Over the past several years, the facility of GWAS has produced a rapid expansion in the list of risk factor genes that are associated with sAD. Here we propose to use a transdisciplinary/team approach to develop the next generation of mouse models that model sAD so that they can be used for preclinical therapeutic testing. Our strategy is to use our recently developed humanized wild- type APPKI mouse as the platform for introducing human tau, followed by other GWAS-identified risk polymorphisms that enhance the risk of developing sAD. We propose to develop the next generation of AD preclinical mouse models using the latest innovations in gene editing technology (CRISPR/Cas9 technology) to produce new mouse models that more accurately represent sAD. We will phenotype the mice using state-of- the-art quantitative methodology and make direct comparisons to the human condition and capitalize on novel reagents that have been developed at UCI, including unique conformation specific antibodies that identify multiple distinct forms of pathology. We will determine gene expression changes via RNA-seq and epigenetic disruptions, alterations in neuronal connectivity in hippocampal circuits via whole-cell patch clamping combined with laser scanning photostimulation, as well as LTP, behavior and cognition, and longitudinal functional imaging. We will also conduct biomarker development by performing plasma lipidomic and metabolomic analyses. We will distribute all data in an expeditious and accessible form for dissemination and will provide detailed protocols for characterization of the models for the field. Lastly, we have established an exciting partnership with The Jackson Laboratory to conduct second site validation of observed phenotypes and to re- derive, cryopreserve and distribute all new animal models so that they can be widely distributed to investigators in the field. Achieving these goals will be transformative for the AD research field.

Project Summary/Abstract Alzheimer?s disease (AD) is characterized by the progressive appearance of amyloid plaques and neurofibrillary tangles, leading to neuroinflammation, neuronal loss and dementia. Microbial exposures modulate inflammation, and are now known to impact the onset and progression AD in both animal models and humans. However, the microbiomes of the UCI MODEL-AD animals are not currently being studied. Characterizing longitudinal microbiomes of MODEL-AD animals will have important impact in understanding disease progression as well ensuring the rigor and reproducibility of AD studies in animal cohorts that may have different microbiomes in different animal facilities or even different cages. Microbes and their metabolites may be part of AD prevention and treatment in the future. We therefore propose to pilot the generation of microbiome and metabolome data from fecal and cecal samples in young as well as older mice to characterize (a) longitudinal microbial community composition, (b) longitudinal metabolite composition, and (c) disease-associated microbes and metabolites. We will include cohorts of the early onset 5xFAD, late onset hAb-KI, the triple transgenic (3xTg-AD) mice that develop both plaque and tangle pathology, and several collaborative crosses along with WT controls. This will allow us to determine if there are microbes associated with each model, which timepoints and sample types are most associated with AD relevant physiology, and whether microbiome and metabolome markers will be useful in future studies. Microbiome data from UCI will be compared with microbiome data being generated at the other MODEL-AD sites.

PROJECT SUMMARY/ABSTRACT A major goal of the UC Irvine MODEL-AD project is to generate data and characterize models of late onset AD. Consistent with this goal, we propose to extend our miniscope imaging technology for functional phenotyping of AD mouse models, and leverage this new imaging approach to examine hippocampal circuit mechanisms that contribute to AD-related memory impairments. Dr. Xu's laboratory at UC Irvine recently has improved miniature microscope (?miniscope?) systems for live brain imaging. The head-mounted miniscope instrument enables our team to examine hundreds of brain cells in action at single cell resolution, as the animal explores freely in environments, and at multiple ages in the same animal. For the proposed study, we will examine spatial correlates of neural activity for single CA1 excitatory neurons in control and AD mice during open-field exploration, and track-based route-running behaviors. The imaging results will reveal maladaptive changes in place cell remapping, place field stability and size in AD mice, which will help to understand whether neurodegeneration in AD mice reduces spatially-specific mapping activity in hippocampal CA1. We will also use the object location memory (OLM) task which involves object-exploration based spatial learning. We will test whether and how control and AD neural activity differ in OLM encoding and retrieval, by imaging ensemble neural activation associated with object exploration across baseline exploration, OLM training and testing sessions. We predict that AD pathology evokes abnormal neuronal ensemble activities leading to impaired behavioral performance that relies on hippocampal circuits. If this approach is validated and proves its significant merit, this will be used to enhance functional phenotyping of mouse models for the MODEL-AD Consortium. This will help to advance our understanding of specific neural mechanisms underlying AD/ADRD etiology in humans.

Project Summary/Abstract The goal of the MODEL-AD consortia at UC Irvine is to generate improved mouse models of sporadic, late- onset Alzheimer?s disease (AD). Ideally, such mice will develop facets of human AD pathology including neuritic plaques, neurofibrillary tangles, and widespread cortical and hippocampal neurodegeneration in advanced age. In order to validate these models we are performing a host of tests including long-term potentitiation experiments, behavioral, transcriptomic and pathological profiling. As pointed out by the December 2018 external advisory board meeting synaptic loss (a key hallmark of AD and one that correlates best with cognitive decline) is currently not being investigated and was specifically requested as an endpoint to be determined. Traditionally, electron microscopy (EM) has been used to quantify synapse density/morphology but laborious sample preparation and stringent requirements in labelling of endogenous proteins precludes high-throughput analysis. On the other hand, fluorescence microscopy readily allows multiple protein species to be efficiently labelled and imaged in 3D. However, studying sub-synaptic structures is difficult because of the small size of synapses, which is near the diffraction-limited resolution of light microscopy. To determine synapse density/morphology in our AD models we request a supplement to perform superresolution imaging using Stochastic Optical Reconstruction Microscopy (STORM). This single molecule localization technique permits the position of proteins to be determined in 3D with nanometer precision and allows a large number of synapses in different brain regions to be imaged in a rapid manner facilitating systematic comparative analysis.

Project Summary/Abstract A key goal of the UCI Model-AD Disease Model Development and Phenotyping Project (DMDPP) is to characterize longitudinal gene expression levels of the new rodent models of sporadic AD being generated by the consortium during aging. This involves, at a minimum, the building of 60 RNA-seq libraries for every mouse model, plus early pilot RNA Seq to prioritize the models to be deep sequenced. We estimate close to 1500 libraries to be generated in the next 3 years. While the protocols for building these libraries are standardized for technicians to carry out as samples become available, subtle manual variations in these procedures can be a source of technical batch effects that can confound the interpretation of the resulting data. In order to minimize the amount of technical variation introduced by human involvement, we are requesting funds to purchase an Eppendorf epMotion 5075tC system in order to automate the reliable, reproducible building of RNA-seq libraries produced by UCI Model-AD. The resulting decrease in technical variation will increase the value of the resulting RNA-seq libraries for the characterization of the changes of gene expression during aging in the UCI model-AD rodent models. This reliability enhances the translational outcome when comparing with human data at multiple stages.

Project Summary/Abstract A key goal of the UCI MODEl-AD Disease Model Development and Phenotyping Project (DMDPP) is to generate and phenotype new models of Alzheimer's disease (AD) that better recapitulate late onset AD (LOAD). The LOAD models will be generated on a platform of humanized Ab and tau, two hallmark pathological proteins found in AD. Once humanized, we will then introduce AD-risk associated polymorphisms in genes to test their ability to drive characteristics of LOAD. The ultimate goal is the production and characterization of a mouse that can be used to explore the key drivers of AD pathology in the aging process, both genetic and environmental. Standard measures of A?40 and A?42 and total and phospho-tau levels, inflammatory markers will be performed for each genotype and age. In addition, neurofilament light, a newly investigated translatable biomarker, can be quantified in these animals. This involves, at a minimum, analysis of 1000 samples of plasma, hippocampus and cortex for every mouse model in the next 3 years. The Meso Quickplex SQ120 instrument provides several advances compared to pre-existing enzyme-linked immunosorbent assay (ELISA) system, as it enables simultaneous tests on a single sample and it is a high-performance electrochemilunescence immunoassay. Furthermore, the same system is being used in the other MODEL-AD centers to obtain these endpoints in their phenotyping protocols, and thus it is important for UCI to harmonize with the other consortium members.

Core A: Administrative Core Project Summary/Abstract The UCI ADRC Administrative Core provides administrative support and oversight, strong innovative leadership, and orchestrates the integration and centeredness with the cores of the Center (Clinical, Data Management and Statistical, Neuropathology, Outreach Recruitment and Engagement, Induced Pluripotent Stem Cell, Biomarker, Down Syndrome and 90+), and the Research Education Component (REC). The broad theme of the UCI ADRC is ?to identify, quantify, and validate factors that influence the risk of AD across the lifespan.? Beyond attending to the administrative details of the Center, the Administrative Core focuses on five broad activities that have a significant impact on our overall vitality and productivity: promote centeredness, engage and recruit new faculty, facilitate AD research, ensure that our researchers have state-of-the-art facilities, and actively engage in philanthropy. The UCI ADRC Administrative Core supports the broad goals of our ADRC as we perform basic and targeted clinical studies into the mechanisms of dementia and builds on our rich history in neurobiology research into age-related cognitive decline and AD. Maintaining a highly functioning and efficient Center enables the Administrative Core to foster innovation in AD research, facilitate collaborations (including core-to-core interactions and within the ADRC network), and support the education of the next generation of scientists, clinicians, and members of our community. The Administrative Core facilitates the Center's overall goals of collaboration, innovation, and education, and promotes the National Alzheimer's Project Act (NAPA) research implementation milestones and goals. The Administrative Core capitalizes on opportunities to grow our Center and played a major role in recruiting ten new faculty to the ADRC over the past four years, many of whom are leaders in their subdiscipline and have become leaders of one of the Cores in the Center. The leaders of the Administrative Core are deeply committed and devote a significant amount of time to ensure that our Center is successful and cutting-edge. The active engagement of the Administrative Core leadership has helped to transform our Center over the past several years by promoting innovation and new ideas, such as the establishment of our novel Special Populations Cores (Down Syndrome Core and 90+ Core), our new participant recruitment registry (Consent-to-Contact, C2C), and our induced pluripotent stem cell (iPSC) Core.

Project Summary/Abstract A key goal of the UCI Model-AD Disease Model Development and Phenotyping Project (DMDPP) is to characterize the gene expression levels of the new rodent models of sporadic AD being generated by the consortium during aging. This involves the building of numerous bulk and single-cell RNA-seq libraries for Model- AD mice. All of these libraries are currently sequenced on an Illumina NextSeq 500 purchased in 2013. As this instrument is nearing its end-of-life support by Illumina, we are requesting funds to upgrade the Illumina NextSeq 2000 system in order to increase the reliability and throughput of sequencing the bulk and single-cell RNA-seq libraries produced by UCI Model-AD. The upgrade will ensure the timely sequencing of the RNA-seq libraries for the characterization of the changes of gene expression during aging in the UCI model-AD rodent models.

Project Summary/Abstract Neurodegenerative disease mechanisms encompassing Alzheimer?s disease (AD) are complex and have remained largely unknown to date despite the identification of risk factors and genetic mutations associated with the disease. One of the major goals of the UC Irvine MODEL-AD project is to characterize rigorous, reproducible and translatable animal model studies of Alzheimer?s disease (AD), the most common form of neurodegenerative dementia. In this respect, the deep characterization of mouse models of AD using high throughput gene-expression studies at bulk (RNA-seq) and single-cell resolutions (single-nuclei RNA-seq; snRNA-seq) have the potential to unravel novel biology and define drivers of the disease, which is of great therapeutic value. However, current bulk and single-nuclei transcriptomics approaches lack the ability to encode spatial, brain region-specific information, which are crucial to decipher gene-expression changes in the local environment and address long standing questions on how expression of neuronal and glial genes changes during progression of AD-related pathology. The current project represents a major advance in the field by taking a comprehensive approach to data-driven discovery to identify spatial organization of resident neural cells and how they change during the progression of AD. Finally, the project will identify shared transcriptomic signatures between human and mouse model systems in a spatially-resolved manner.

PROJECT SUMMARY During homeostasis and proper acute immune responses, interleukin-18 (IL-18) acts as a facilitator of higher cerebral functions and as an alarmin molecule to signal incoming danger to cells. Our studies led us to discover endogenous counter-regulatory components in the IL-18 pathway that are impaired in Alzheimer's disease (AD), which results in excessive and prolonged signaling. More importantly, we found compelling evidence that this process plays a major role in the dysregulation of tau and accumulation of its pathological species. Investigating the underlying mechanisms by which the IL-18 pathway is affected by and drives tau pathology in AD could, therefore, yield novel means to mitigate this insidious disease. Here we propose three aims to investigate this important issue. In aim 1, we hypothesize that ?-amyloid (A?) disrupts the endocytic protein Tollip (toll interacting protein), which is the leading regulator driving the homologous desensitization of the IL-18 receptor (IL-18R). We will genetically up- or downregulate Tollip in neuronal cell cultures and in an AD mouse model. The outcome of this aim may show that the buildup of pathological forms of tau induced by chronic IL-18 signaling in neurons is, at least in part, caused by the upregulation of IL-18R due to the impairment of its trafficking and degradation mediated by Tollip. For the second aim, we will test the hypothesis that the deficiency in the production of IL- 18's endogenous decoy receptor, IL-18 binding protein (IL-18BP), also plays a role in the chronic activation of this pathway in AD. We will use an adeno associated virus to upregulate IL-18BP levels in AD mice and determine its therapeutic efficacy on AD-like pathology. Finally, our last aim utilizes biochemical and proteomic methods to map the neuronal intracellular networks affected in response to IL-18. We will apply pharmacological and genetic tools to link potential candidates altered by IL-18, such as protein kinases and phosphatases, to the hyperphosphorylation of tau and subsequent synaptic loss and cognitive decline. Establishing these intracellular cascades could allow us to identify novel strategies to inhibit the pathological effects of IL-18, while preserving its relevant physiological functions. Overall, our studies are significant for two reasons: first, they will provide greater insights into the IL-18 signaling cascade and second, they will uncover the critical steps specifically involved in the IL-18 pathway in AD, and may result in the identification of new therapeutic candidates to potentially translate our discoveries into the clinic.