Andrea J. Tenner - US grants

The long term objective of this research program is to define the mechanisms by which the interaction between humoral and cellular elements of the human immune system result in complementary and synergistic responses to invading pathogens. We have chosen to investigate the interaction of C1q, a protein found in blood and additionally secreted by inflammatory cells, with the phagocytic cells of the immune system. These cells ingest and subsequently destroy infectious agents and additionally clear immune complexes and other deleterious substances from the circulation and from tissues. C1q is the recognition subunit of the classical complement pathway and as such interacts with antibody-antigen complexes and various other structures such as bacterial and viruses in the absence of antibody. Thus it has an uncommon potential for participating in a cellular-humoral immune network. The recent demonstration that C1q enhances Fc-mediated and C3b- mediated phagocytosis provides supportive evidence for our current postulate that C1q is an activation ligand for phagocytosis. Furthermore, C1q enhances the generation of toxic oxygen radicals critical in the destruction of invading microorganisms. This proposal specifically outlines the biochemical and immunological characterization of the C1q receptor on human phagocytic cells. The C1q receptor will be isolated, and monoclonal antibodies to the receptor will be generated and used to definitively establish he relationship between the isolated C1q receptor and the C1q-mediated enhancement of phagocytic cell function. Amino acid sequence determination of C1qR will be used to direct synthesis of specific peptides to use as antigens to unique domains of the receptor molecule, as probes of the structure and function of this receptor and as the basis for the synthesis of oligonucleotide probes for use in cloning the receptor gene. These studies will provide the basis for determining the molecular events involved in the C1q enhancement of phagocytic cell function, which compared with that of other ligand-induced effects, should further the general understanding of this critical element in host defense. Ultimately, this research should aid in the development of beneficial therapeutic or prophylactic procedures to augment appropriate protective responses.

DESCRIPTION (provided by applicant): The complement component C1q is a member of the defense collagen family of proteins that have been shown to enhance phagocytosis of pathogens, cellular debris and apoptotic cells. Phagocytosis, while resulting in the killing and clearance of infectious agents, is also a first step in antigen processing and presentation necessary for the induction of an adaptive response. It is now being recognized that pattern recognition molecules of the innate immune system have significant influence in determining the nature of the subsequent adaptive response. Just as TLRs influence both effector function and gene expression profiles in response to pathogens, it is becoming increasingly evident that other recognition molecules, including specific complement components, not only play a role in the effector phase of the immune response, but also act as "biosensors" contributing to the programming of a subsequent immune response. As such, this first response assesses the level of "danger" of an intrusion or injury and initiates an appropriate program of protection (when the challenge is a pathogen) and/or suppression of detrimental responses (to avoid autoimmune responses and/or a tissue-damaging level of inflammation). Our preliminary results demonstrate that under specific conditions, C1q, while facilitating phagocytosis, can suppress specific proinflammatory cytokine production. We propose here to test the hypothesis that specific gene expression patterns are activated by ligation of the cell by C1q and other defense collagens, and that those patterns are modified by both the differentiation state of the phagocyte and the other simultaneous signals received by the cell. We propose to characterize the signaling pathways and parameters that lead to the specific cytokine gene expression. Therapeutic targeting of these "biosensors", exemplified by C1q/MBL and the cell surface molecules/complexes that transfer the signals to the cell, should facilitate more effective protective responses to pathogens (including biodefense strategies), the development of more efficient and protective vaccine strategies, and suppression of autoimmunity.

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is a neurodegenerative disorder associated with the loss of cognitive function and the presence of characteristic neuropathological changes that include synaptic and neuronal loss, neurofibrillary tangles and extracellular senile plaques composed of beta-amyloid (Abeta) protein deposits. The association of complement proteins, as well as acute phase proteins and reactive glia, with senile plaques in AD brain suggests that inflammatory processes may play a role in this disease, and that complement activation may contribute to the initiation of these inflammatory events. However, the actual in vivo contribution of complement activation to pathology and dementia in AD has not yet been determined. Our recently completed study provides compelling evidence for a detrimental role for C1q (the initiation component of the classical complement pathway) in the progression of pathology in murine models of Alzheimer's Disease. Animals overexpressing the amyloid precursor protein but lacking the ability to activate the classical complement pathway showed less inflammation and less loss of critical neuronal structures than those with a functioning classical complement pathway. Since sites of interaction between fibrillar Abeta and C1q critical for this activation are known, the development of potential therapies that would block the amyloid interaction with C1q that leads to complement activation should be feasible. The research program described here focuses on the generation of animal models of AD that more closely and critically mimic the human disease. The models will be genetically manipulated to either eliminate or enhance specific features of the complement system, and the age-related consequences on brain pathology and behavior compared. These novel models will test the hypothesis that complement activation promotes the progression of pathology and cognitive dysfunction in AD at a stage when fibrillar Abeta is present, and will be valuable for testing candidate therapies for AD in an in vivo context that more closely mimics the human condition.

DESCRIPTION (Adapted from the application): AD is a common dementia or loss of cognitive abilities, which is linked to degeneration of brain tissue. The cause of this neurodegeneration is under intense investigation, as a critical step toward designing therapies for this debilitating and costly disease. In a variety of test systems, fibrillar beta-amyloid displays neurotoxic properties via its direct interaction with neurons but also via its interaction(s) with microglia and its ability to activate the complement system. Multiple studies have demonstrated that reactive microglia, astrocytes and proteins of the complement system are associated with the senile plaques in AD brain, consistent with the hypothesis that inflammation initiated by the activation of the complement system may contribute to the generation of pathology that leads to the cognitive loss seen in this disease. The complement (C) system is a powerful effector mechanism of the immune system. Tissue damage can result however, from chronic or unregulated activation of this system. Nevertheless, it is also becoming increasingly evident that some complement components provide protective functions in areas of injury. Thus, in this research program novel mouse models will be utilized to test the hypothesis that complement plays a role in the pathogenesis of AD. Potential protective effects of specific complement components in this disorder will be assessed and specific hypotheses of the protein-protein interactions that regulate these functions will be tested. Organotypic culture systems will be used to assess the ability of specific complement components to modify amyloid-induced microglial activation and its effect on neurodegeneration. Finally, the investigators will utilize Down Syndrome tissue to further assess the correlation between complement activation, inflammation and dementia. These studies should provide solid data on the significance of complement activation and inflammatory events in AD and other forms of dementia, events that could be targeted to slow the progression of the disease. Since complement has been implicated in a number of other neurodegenerative diseases, it is likely that the findings will be relevant to other diseases as well.

DESCRIPTION (adapted from investigator's abstract): The innate immune system is composed of "hard-wired" elements of immunity, such as natural killer cells, phagocytes, defensins, and complement, that mount an initial protective response to kill, clear and/or limit deleterious invaders. A relatively newly defined family of proteins, designated as defense collagens, is an example of the constitutive recognition components of the innate system. Each of these defense collagens recognizes selective motifs displayed on pathogenic material, and mediates a protective response. Interaction of several soluble defense collagens with a novel transmembrane protein, C1qRP, has been shown to facilitate the rapid ingestion of suboptimally opsonized particles, a potentially critical mechanism in host defense particularly at early stages of infection/disease when little or no adaptive response is yet present. In addition, this receptor, which is selectively expressed on monocytes, macrophages, neutrophils, endothelial cells and platelets, may contribute to the rapid clearance of apoptotic and/or damaged cells, a critical process during tissue remodeling. This would be particularly advantageous, as ligation of this receptor by monocytes does not trigger proinflammatory cytokine production. This proposal will focus, first, on determining the physiologic role of this receptor by generating, characterizing, and testing specific hypotheses in a murine model in which this gene has been ablated, and secondly, on the intracellular signaling mechanisms involved in triggering enhanced phagocytosis and in regulating proinflammatory cytokine induction. The ability of C1qRP to regulate the phagocytic capacity of myeloid cells could be extremely valuable as a prophylactic treatment for individuals at risk for infection, such as individuals with genetic immunodeficiencies or pathogen-induced immunosuppression, patients undergoing cancer chemotherapy, or patients undergoing high risk surgery. However, this surface molecule also has the potential for modulation of processes contributing to damaging inflammation (such as vasculitis, nephritis, neurodegeneration), and understand physiologic clearance of apoptotic cells and/or cellular debris which would be potentially relevant to both inflammation and certain types of autoimmunity.

Project 4: Neuroprotection and neuroinflammation induced by the complement proteins Clq and C5a The complement system is a component of the innate immune system whose function is to recognize deviations from the norm (such as an infection or tissue injury) and to initiate a response that will protect and initiate repair of the injured area. If not properly regulated however, tissue damage results. Previous observations suggest a detrimental effect of the activation of the complement cascade at a late stage of Alzheimer's disease when amyloid plaques containing the fibrillar, and thus complement activating, form of the amyloid peptide, AB, accumulate. Clq, the recognition component of one pathway of complement activation, binds to fibrillar amyloid and activates the pathway. One downstream product of complement activation is C5a which is known to enhance inflammation by binding to specific receptors. In this proposal, a C5a receptor antagonist which has proven effective in limiting complement mediated inflammation in other models, is being tested as a potential targeted therapy in AD mouse models. This point of inhibition would leave the remainder of the complement cascade intact, thereby permitting potentially beneficial effects of complement, such as enhanced clearance of abnormal (amyloid) deposits (by C3b), apoptotic cells and/or cellular debris (by Clq and C3b). However, it is also becoming increasingly evident that there are both activating and modulating/decoy receptors for C5a expressed in brain, and thus we propose to determine whether the balance of expression of these receptors dictates the degree of inflammation in the AD brain. In addition, Clq, which is known to be synthesized and secreted as a response to injury, has recently been shown to down regulate proinflammatory cytokines in peripheral macrophages and to provide survival signals to neurons in vitro. Using several in vitro approaches, the basis for these neuroprotective events will be defined. Results of these proposed studies should provide solid data on the significance of the contribution of complement-induced inflammatory events in AD and likely other neurodegenerative disease in the aging individual. Therapeutic inhibitors of detrimental processes identified here as well as reagents or treatments that promote the neuroprotective functions induced can subsequently be designed to slow the progression of this pervasive disease of the elderly.

DESCRIPTION (provided by applicant): The annual meeting of Society for Leukocyte Biology, a leading scientific society addressing immune activation by infection or injury and the organization and control of that response, has a tradition of highlighting the most innovative and timely work on the mechanisms underlying host-pathogen interactions, dysregulation as a result of trauma or injury and leukocyte functions in the pathogenesis of disease. The 2011 meeting "Infection, Inflammation and Immunity" will continue this tradition by bringing together scientific researchers of all levels in an environment geared to maximize interactions and the exchange of ideas. Objective 1. To provide a forum for approximately 300-350 participants from academic, governmental, and industrial laboratories worldwide to share the latest most significant advances and new discoveries in inflammation and immunity, with an added focus on the impact of the immune system in the nervous system. The program will feature presentations by invited leaders in the field, as well as talks and poster presentations by junior investigators and trainees chosen from among the submitted abstracts from laboratories worldwide. These areas include the molecular pathways involved in inflammation and infection, the accelerating field of neuroinflammation, emerging importance of lipids in activation, signaling, and effector functions, epigenetic regulation of inflammation and Immunity, the paradigm shifting discoveries of monocyte subsets and the growing awareness of the extent of plasticity in the innate immune response afforded by malleable macrophage, the extent of interaction and cross regulation between the innate and adaptive immune response and the influence of the vasculature on the immune system (and vs versa). Objective 2. To present a program that captures significant newly emerging areas in which the immune systems contributes significant influence over proper function, and thus provides potential targets for therapies. A pre-meeting Satellite Symposium on "The Immune System in Psychiatric Disorders" (separate organization and funding) will provide attendees with insight into this rapidly emerging area, in addition to a complementary meeting plenary session on Neuroinflammation. Objective 3. To provide an opportunity for young investigators and trainees to interact closely with well- established researchers in their fields both formally and informally and to present their research in both poster and podium sessions. Objective 4. To provide an opportunity for women, members of under-represented minority groups, and persons with disabilities to participate in a meeting that showcases their work and facilitates one on one interactions with other scientists in their fields, as well as providing a forum to address obstacles and explore solutions that will optimize their success. PUBLIC HEALTH RELEVANCE: The scientific advances in the complementary and interactive fields of inflammation, innate immunity, infectious disease and inflammatory disorders continues to accelerate, with increased emphasis and the development of technology. The annual conference of the Society for Leukocyte Biology with a guest session sponsored by The Inflammation Research Association and a satellite Symposium provided by the Psychoimmunolgy Research Society in 2011 will provide a much needed opportunity for a highly productive exchange of scientific information on the latest advances and best work in these fields, which have enormous relevance to a broad range of clinical disorders currently prevalent in the US and worldwide populations. Moreover, the conference will strongly promote effective interactions among scientists from different fields, between established and developing junior investigators, and with women and minority scientists with the hope of developing productive collaborations and/or accelerating productivity and success.

Description (as provided by the applicant): The annual meeting of Society for Leukocyte Biology, a leading scientific society addressing immune activation by infection or injury and the organization and control of that response, has a tradition of highlighting the most innovative and timely work on the mechanisms underlying host-pathogen interactions, dysregulation as a result of trauma or injury and leukocyte functions in the pathogenesis of disease. The 2012 meeting Inflammation in Innate and Adaptive Immune Mechanisms will continue this tradition by bringing together scientific researchers of all levels in an environment geared to maximize interactions and the exchange of ideas. Objective 1. To provide funding to expand the number of individuals that can attend the annual meeting of the Society of Leukocyte Biology, by providing travel awards for trainees and increase the capacity for additional attendees. The meeting is a forum for approximately 300-350 participants from academic, governmental, and industrial laboratories worldwide that will include presentation and discussion of emerging concepts in inflammatory response as they impact on innate and adaptive immunity. These will include (1) identification of new pattern recognition receptors, (2) the role of the epithelium in initiation of inflammatory response and the connection to adaptive immunity, (3) new features of inflammasome function in propagation of inflammatory response, (4) the emerging importance of tumor infiltrating myeloid cells in the link between inflammation and cancer, and (5) the contribution of metabolic controls in lymphoid differentiation and function. The program will feature presentations by invited leaders in the field, as well as poster presentations by junior investigators and trainees and oral presentations selected from among the abstracts submitted by laboratories worldwide. Objective 2. To provide additional scientific programmatic content covering new developments in the regulation, activation and function of the diverse cell populations of the innate and adaptive immune system. This will be accomplished through sessions focused on macrophages and dendritic cells, ?/¿ T cells and NK cells, B cells, and Th17 and Treg cells. In addition, aspects of leukocyte functionality will also be included in the program and will capture new developments in cytokine signaling, post-transcriptional regulation of inflammatory gene expression, host pathogen interactions, and the connection of immunity and neuroscience. A pre-meeting satellite symposium focused on the impact of alcohol on aspects of signaling in inflammation and immunity will be organized by the Alcohol and Immunology Research Interest Group and will occur the day before the opening of the SLB conference. Our meeting will broaden exposure of basic and clinical immunology to the attendees of the satellite meeting. Objective 3. To provide an opportunity for young investigators and trainees to interact closely with well- established researchers in their fields both formally and informally and to present their research in both poster and podium/oral sessions. Objective 4. To provide an opportunity for women, members of under-represented minority groups, and persons with disabilities to participate in a meeting that showcases their work and facilitates one on one interactions with other scientists in their fields, as well as providing a forum to address obstacles and explore solutions that will optimize their success. Public Health Relevance: The scientific advances in the complementary and interactive fields of inflammation, innate immunity, infectious disease and inflammatory disorders continue to accelerate with novel findings and implications for human health. The annual conference of the Society for Leukocyte Biology in 2012 will provide a much needed opportunity for a highly productive exchange of scientific information on the latest advances and best work in these fields, which have enormous relevance to a broad range of clinical disorders currently prevalent in the US and worldwide populations. Moreover, the conference will strongly promote effective interactions among scientists from different fields, between established and developing junior investigators, and with women and minority scientists with the hope of developing productive collaborations and/or accelerating research productivity and success.

DESCRIPTION (provided by applicant): Cognitive decline and Alzheimer's disease (AD) will increase dramatically in the coming decades as the number of elderly rises from 1 out of 5 over 65 to a projected 1 out of 3 by 2050. The loss of cognitive function will impact the quality of lif, the available elderly workforce in the nation and our economic viability. We therefore urgently need to discover new prevention and treatment strategies. Biomedical research and the training of a new generation of scientists devoted to studying the mechanisms associated with aging and age-related disorders hold the greatest promise for identifying strategies that allow individuals to age successfully. Our training emphasizes preparation and instruction in the application of molecular and quantitative approaches to the elucidation of the cellular and molecular mechanisms of age-related neurodegeneration, brain plasticity, and learning and memory. Overall, our training program has 5 primary features and strengths: 1. A team of highly innovative researchers studying cutting edge questions in the field. We have 26 faculty from 11 Departments dedicated to training in areas including basic mechanism of brain dysfunction, brain plasticity and learning and memory, inflammation and inflammatory cascades, and stem cells and other therapeutics to delay and treat age-related neurological decline; 2. An excellent collaborative training environment and an informative and thought provoking set of core courses, seminars, symposia and workshops; 3. A mini-clinical internship for trainees to experience interacting with individuals with mild cognitive impairment (MCI) and AD and instruction and experience on brain clinical pathological case studies; 4. An emphasis and training on the translation of basic research findings to humans, to reduce the incidence and progression of age-related cognitive decline and AD; and 5. Finally, individual guidance and counseling is included to optimize the potential of a diverse trainee pool and to help them realize their specific career goals. Our Program has a solid track record over its 30-year history of producing quality and highly successful scientists who enter academia or apply their training and knowledge in industry to address a challenging and serious health problem for the nation and our growing senior population.

Project 4: Neuroprotection and neuroinflammation induced by the complement proteins and receptors In order to increase the successful identification of novel therapeutic targets for degenerative disorders commonly seen in the elderiy, the interrelations between new discoveries in genetics (GWAS identified risk association genes), cell biology (autophagy), immunology (inflammasomes) and neurobiology must be delineated using a combination of experimental approaches. One rapidly emerging area is the link between the immune system and cellular homeostasis in neurobiological systems, consistent with a role for the immune system components in normal and pathological brain function. The complement system is a powerful effector mechanism of the innate immune system that contributes to protection from infection and resolution of injury. Complement component deposition has been associated with Alzheimer's disease (AD), macular degeneration, Parkinson's disease and other age-related neurodegenerative disorders. While the activation of the complement cascade may be detrimental in these diseases, C l q , the initiator molecule of the classical complement pathway, has anti-inflammatory effects in the absence of the other components of the complement system. Unexpectedly, it has become apparent that in the brain C l q synthesis is induced in response to a variety of insults and injury initially in the absence of synthesis of other complement components. In addition, our lab made the novel observation that in vitro C l q improves neuronal survival under nutrient stressed conditions and suppresses amyloid beta-induced neuronal death suggesting that C l q may have a neuroprotective role. This novel neuroprotection triggered by C l q was associated with induction of expression of neuronal genes which if suppressed abrogated the C l q neuroprotective effects. Here we will collaborate with other members of the Program Project to identify the mechanisms of this C l q neuroprotection/survival, determine if this coordinates with a GWAS identified risk polymorphism in CRI, and create new animal models to elucidate the significance of this pathway in in vivo models of neurodegenerative diseases in aging populations. The molecular delineation of this novel pathway should uncover a number of potential therapeutic targets which have not been explored which can then be translated into innovative strategies (perhaps in combination with other therapeutic targets) in the clinic for the enhancement of neuron survival to improve the quality of life for individuals suffering from neurodegenerative or neurodevelopmental disorders. RELEVANCE (See instructions): This project will identify key signaling pathways and molecular interactions that are required for a novel neuroprotection mechanism discovered in our laboratory and determine if this pathway improves neuronal survival in an animal model of Alzheimer's Disease (AD). In a complementary approach the biological basis of the AD risk association of a polymorphism in an immune regulatory protein, CRI, will be determined. These results will provide the first step in design of novel pharmacologic interventions for degenerative diseases in humans.

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 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.

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 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: By 2050 it is estimated that there will be 13.8 million individuals in the US with Alzheimer's disease (AD), at a cost of over $1.2 trillion per yr, if no disease-modifying therapy is developed. The relative contributions of the AD defining pathological markers, amyloid and hyperphosphorylated tau, to cognitive dysfunction remains controversial, but studies in both AD patients and transgenic mouse models of AD, have shown that amyloid is necessary but not sufficient for the development of cognitive loss which is the key clinical target of AD. There is a growing consensus that it is the response of glial cells in the brain to amyloid that is relevant to neuronal damage and thus cognitive impairment, and that the capacity to phagocytose and clear amyloid and perhaps other deleterious material in the brain may have a substantial influence on the initiation and progression of the disease. The complement cascade, a powerful effector mechanism of the immune system that is directly activated by fibrillar A? (fA?), can both enhance clearance and induce inflammation. In addition, complement activation dependent excessive synapse pruning occurs in models of AD and other neurological disorders. Our data demonstrate that pharmacologic inhibition or genetic deletion of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppresses neurite and cognitive loss in mouse models of Alzheimer's disease/amyloidosis. The effects of blocking this C5a receptor interaction on astrocyte activation, inflammatory and clearance related gene expression, neuronal integrity and synaptic density are proposed in two aims here with the ultimate goal of determining mechanistic steps between activation of C5aR1 and loss of neuronal function, as well as the potential for C5aR1 antagonists as therapeutic candidates for clinical trials in humans to prevent cognitive impairment. In a third aim, we proposed to use a newly generated mouse with a floxed C5aR1, to temporally and cell specifically ablate the receptor and assess gene expression, neuronal integrity and function in an AD mouse model, to more closely mimic the adult inhibition of this receptor that would occur with receptor antagonist treatment of individuals with or at risk for AD. Selective modulation of complement activation products or their receptors may be an optimal strategy for retaining the neuroprotective and phagocytic functions of complement components, as well as systemic protection from pathogens lysis, while dampening induced inflammatory damage. Importantly, blockage of this ligand-receptor system has not shown adverse effects in humans, suggesting that C5aR1-targeted therapeutics for AD may be safely administered.

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 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 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 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 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 SUMMARY/ABSTRACT At present 5.3 million US citizens are affected by Alzheimer's disease and countless others are impacted by age-related cognitive decline. The cost of care in the US is currently more than $220 billion annually, and with the increase in cases will grow to an unsupportable $1.2 trillion annually by 2050.  The loss of cognitive function will impact the quality of life, the available elderly workforce in the nation and our economic viability. We urgently need to discover new prevention and treatment strategies. Biomedical research and the training of a new generation of scientists devoted to studying the mechanisms associated with aging and age-related disorders hold the greatest promise for identifying strategies that allow individuals to age successfully. Our training program focuses on preparation and instruction in the application of molecular and quantitative approaches to the elucidation of the cellular and molecular mechanisms of age-related neurodegeneration, brain plasticity, and learning and memory. We emphasize training on mechanisms but also the discovery and translation of effective therapeutics including lifestyles such as physical activity. Overall, our training program has five primary features and strengths: 1. A team of innovative and scholarly preceptors who have a strong record of accomplishment for training young scholars and an excellent collaborative environment fostering team science 2. A core set of courses on Brain Aging along with seminars and symposia (eg. ReMIND), training on brain pathology through Clinical-pathological case presentations and a mini-clinical internship 3. A unique environment that allows for students from many departments and programs across the campus to have a customized program of study, an opportunity that a single department based program cannot provide 4. Specific training to help trainees reach their individual career goals that will include Training in Communication skills such as our recent training for students on delivering ?elevator pitches? and brief lay descriptions of research 5. An Individual Development Plan (IDP) to prepare them for their own independent careers in the neurobiology of aging, and continual monitoring to ensure they develop broad understanding of the bench to clinic translation of research to improve the lives of the elderly and ensure sustainably healthcare for the nation. Overall, our Training program in Brain Aging is designed to develop a uniquely trained cadre of investigators who over the years will develop successful careers.  

By 2050 it is estimated that there will be 13.8 million individuals in the US with Alzheimer's disease (AD), at a cost of over $1.2 trillion per yr, if no disease-modifying therapy is developed. The relative contributions of the AD defining pathological markers, amyloid and hyperphosphorylated tau, to cognitive dysfunction remains controversial, but studies in both AD patients and transgenic mouse models of AD, have shown that amyloid is necessary but not sufficient for the development of cognitive loss which is the key clinical target of AD. There is a growing consensus that it is the response of glial cells in the brain to amyloid that is relevant to neuronal damage and thus cognitive impairment. The capacity of these cells to phagocytose and clear amyloid and perhaps other deleterious material in the brain may have a substantial influence on the initiation and progression of the disease. The complement cascade, a powerful effector mechanism of the immune system that is directly activated by fibrillar A? (fA?), can both enhance clearance and induce inflammation. In addition, complement activation dependent excessive synapse pruning occurs in models of AD and other neurological disorders, and is postulated to contribute to the loss of cognition. Our data demonstrate that pharmacologic inhibition or genetic deletion of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppresses cognitive loss in mouse models of Alzheimer's disease/amyloidosis. This study proposes to investigate the effect of a C5a receptor antagonist on inflammation- and clearance-related gene expression (using single cell RNA-seq to evaluate multiple cell types affected), protein expression and synaptic density (via super resolution microscropy) in order to determine if C5aR1 plays a direct or indirect role in neuronal damage and whether C5aR1 antagonists may be promising therapeutic candidates for future clinical trials in humans to prevent cognitive impairment.

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.

Project Summary. Alzheimer?s disease (AD) is the most prevalent neurodegenerative disease of the elderly. The complement cascade, a powerful effector mechanism of the innate immune system that can be directly activated by fibrillar A?, is implicated as a player in this inflammatory scenario. In brain, expression of most complement components increases during aging and further increases in AD patients and animal models of AD, consistent with a role for complement immune activation in progression of the disease. Complement activation fragment C5a has been a major focus, as inhibition of its proinflammatory receptor, C5aR1, leads to less activation of microglia and astrocytes, preservation of neuronal complexity and reduction of cognitive loss in AD models. These critical observations strongly suggest C5a binding to its receptor C5aR1 initiates cellular activation leading to changes in protein expression in microglia and astrocytes, which result in pathological phenotypes and disease progression. The primary goal of this proposal is to systemically understand alterations in cell-specific protein expression that result from signaling via C5a-C5aR1 in the context of Alzheimer?s disease. Specifically, this will extend our knowledge of induction of specific RNAs to production of the proteins, and the contribution of each cell type to those functional proteins, that ultimately accelerate pathogenesis and neuronal dysfunction in Alzheimer?s disease 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/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. Alzheimer?s disease (AD) is the most prevalent neurodegenerative disease of the elderly. The complement cascade, a powerful effector mechanism of the innate immune system that can be directly activated by fibrillar A?, is implicated as a player in this inflammatory scenario. In brain, expression of most complement components increases during aging and further increases in AD patients and animal models of AD, consistent with a role for complement immune activation in progression of the disease. Complement activation fragment C5a has been a major focus, as inhibition of its proinflammatory receptor, C5aR1, leads to less activation of microglia and astrocytes, preservation of neuronal complexity and reduction of cognitive loss in AD models. These critical observations strongly suggest C5a binding to its receptor C5aR1 initiates cellular activation leading to changes in protein expression in microglia and astrocytes, which result in pathological phenotypes and disease progression. The primary goal of this proposal is to systemically understand alterations in cell-specific protein expression that result from signaling via C5a-C5aR1 in the context of Alzheimer?s disease. Specifically, this will extend our knowledge of induction of specific RNAs to production of the proteins, and the contribution of each cell type to those functional proteins, that ultimately accelerate pathogenesis and neuronal dysfunction in Alzheimer?s disease models.