Xiangdong Yang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating neurodegenerative disease caused by a polyglutamine expansion mutation in the Huntingtin gene (Ht). The mutant Huntingtin (mHt) exerts dominant toxicity to cause selective neuronal dysfunction and degeneration. Currently, there are very few valid drug targets for HD. One method to identify new drug targets is through genetic modifier studies. Genetic = modifier screenings in Yeast, Drosophila, C. elegans and mammalian cell culture have produced a number of genes that can be manipulated to suppress mutant Huntingtin toxicity. However, the efficacy of these genes to suppress mutant Huntingtin toxicity in the mammalian brain is unclear. This proposal seeks to establish a mouse genetic system that can readily and systematically test genetic suppressors for HD in vivo. Using a relatively novel mouse genetic technology, called Bacterial Artificial Chromosome (BAC) mediated transgenics, we have succeeded in creating novel transgenic mouse models of HI) expressing full-length mutant Huntingtin under the endogenous regulation. Some of these full-length transgenic founders have already demonstrated early and severe motor deficits. The specific aims of this proposal are to: 1. Characterize the fulfull-lengthC models of HD and select lines suitable for genetic modifier studies. 2. Apply the newly developed rapid BAC modification system to generate mice over expressing two modifier genes and to test their efficacy in suppressing mHt toxicity in the full-length transgenic models. If proven effective, our BAC transgenic System can readily be scaled up to systematically study a large number of putative HD genetic modifiers in vivo. Identification of effective genetic modifiers in mice may provide novel insights into disease pathogenesis and new targets to develop therapeutics.

DESCRIPTION (provided by principal investigator): Huntington's disease (HD) is one of the most common dominantly inherited neurodegenerative disorders affecting 30,000 patients in the US with another 150,000 at risk. HD is characterized by motor, cognitive and psychiatric symptoms that often progress and result in the patient's death in about 10-20 years after disease onset. HD is caused by a polylgutamine repeat expansion in mutant Huntington, resulting in the relentless progression of neurodegeneration primarily targeting the striatal neurons but also affecting the cortical neurons. Currently, there is no treatment or cure for HD. Since mutant huntingtin (mhtt) is widely expressed in the brain and in the body but neuro degeneration in HD primarily targets the striatal and cortical neurons, a critical yet unanswered question in HD is how does the widely distributed mhtt cause such selective patterns of neuro degeneration? To address this question, we first will ask where mhtt expression is critical for disease pathogenesis. Using a novel series of HD mouse models that can express mhtt or its toxic fragments in different types of cells in the brain, we discovered that mhtt fragments can induce not only the intrinsic toxicities to the neurons in which it is expressed but also toxic interactions between different types of neurons. Furthermore, we found that switching off mhtt expression in the cortical neurons results in a significant but partial rescue of both the behavioral deficits and striatal toxicities in HD mice. These exciting findings underscore the need to study whether and how the cortical and striatal neurons may act together to elicit HD. We designed the following aims to address this critical question: Aim 1. Is mhtt expression in the striatum necessary for HD pathogenesis? Aim 2. Can switching off full length mhtt expression in both the cortex and striatum synergically reduce key disease phenotypes in HD mice? Aim 3. Can switching of full length mhtt only in the cortical pyramidal neurons and striatal medium spiny neurons be sufficient to induce key aspects of HD phenotypes in vivo? The completion of our study may reveal whether mhtt exert synergistic toxic effects from within two types of neurons (i.e. the striatal and cortical neurons) to elicit the major disease phenotypes in HD. Our study may support the molecular dissection of HD pathogenic mechanisms in these two cell types, and may further inform us on the optimal strategies of local delivery of mhtt reducing therapeutics (i.e. RNA interference, antisense oligonucleotides or intrabodies).

[unreadable] DESCRIPTION (provided by applicant): A common theme in the pathogenesis of the neurodegenerative diseases is that a widely expressed mutant protein can cause selective degeneration of a subset of neurons in the brain. In most of the cases, the molecular and cellular mechanisms underlying such selective neurodegeneration remain unclear. Using Huntington's disease (HD) as a model, we will investigate whether selective degeneration of the striatal and cortical neurons in HD requires mutant Hungtingtin (mHt) toxicities originated from outside these neurons (non-cell-autonomous toxicities). In our preliminary studies, we have developed two conditional mouse models in which mHt expression can either be activated or inactivated in specific neuronal populations. We propose to use these mouse models to achieve the following three Specific Aims: Specific Aim 1. Phenotypic analyses of the cortical activation mouse model and the whole brain activation mouse model to determine whether non-cell autonomous toxicities are involved in dysfunction and degeneration of the vulnerable cortical and striatal neurons. Specific Aim 2. Phenotypic analyses of the striatal activation mouse model to determine if the non-cellautonomous toxicities are involved in dysfunction and degeneration of the striatal and cortical neurons. Specific Aim 3. Generation of a full length mHt cortical-inactivation mouse model to study whether non-cell autonomous toxicities are required in disease pathogenesis in a full length mHt mouse model. In summary, we propose to use a novel series of conditional mouse models of HD to study the role of neuronal circuitry in HD pathogenesis. Our studies may reveal that mHt toxicities originated from outside the most vulnerable neurons may be required for their dysfunction and degeneration. Identification of such toxicities will have important implications for developing therapeutics for HD as well as for other neurodegenerative diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A common theme in the pathogenesis of the neurodegenerative diseases is that a widely expressed mutant protein can cause selective degeneration of a subset of neurons in the brain. In most of the cases, the molecular and cellular mechanisms underlying such selective neurodegeneration remain unclear. Using Huntington's disease (HD) as a model, we will investigate whether selective degeneration of the striatal and cortical neurons in HD requires mutant Hungtingtin (mHt) toxicities originated from outside these neurons (non-cell-autonomous toxicities). In our preliminary studies, we have developed two conditional mouse models in which mHt expression can either be activated or inactivated in specific neuronal populations. We propose to use these mouse models to achieve the following three Specific Aims: Specific Aim 1. Phenotypic analyses of the cortical activation mouse model and the whole brain activation mouse model to determine whether non-cell autonomous toxicities are involved in dysfunction and degeneration of the vulnerable cortical and striatal neurons. Specific Aim 2. Phenotypic analyses of the striatal activation mouse model to determine if the non-cellautonomous toxicities are involved in dysfunction and degeneration of the striatal and cortical neurons. Specific Aim 3. Generation of a full length mHt cortical-inactivation mouse model to study whether non-cell autonomous toxicities are required in disease pathogenesis in a full length mHt mouse model. In summary, we propose to use a novel series of conditional mouse models of HD to study the role of neuronal circuitry in HD pathogenesis. Our studies may reveal that mHt toxicities originated from outside the most vulnerable neurons may be required for their dysfunction and degeneration. Identification of such toxicities will have important implications for developing therapeutics for HD as well as for other neurodegenerative diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders characterized by three domains of behavioral deficits: impaired social interaction, delayed language development, and motor stereotypy. Although etiologies underlying the heterogeneous clinical manifestations of autism remain unclear, one hypothesis is that ASDs are the result of neurodevelopmental deficits targeting the specific neural circuits/networks that mediate the behaviors affected in ASDs. Our preliminary studies of a mouse model that carries a CNS-specific deficiency of the striatonigral medium spiny neuron (MSN)-enriched transcription factor, Zfp521 (Zfp521cko) show postnatal developmental onset of motor stereotypic behaviors resembling the motor stereotypies associated with ASDs. The motor stereotypies exhibited by the Zfp521cko mice, which include facial/head grooming with the forelimbs, body licking, rapid flapping of one hindlimb, and rapid head twitches, are highly frequent, debilitating, cause self-injurious lesions, and impair an ethologically normal behavior, nest building. Based on these compelling preliminary studies, we hypothesize that Zfp521 orchestrates a critical transcriptional program within the striatonigral MSNs which is crucial for their normal maturation, and that disruption of this molecular program can result in motor stereotypies. To this end, our proposed studies will focus on the following Aims: (1) Genetic rescue of the motor stereotypies and striatal circuitry dysfunction by selective transgenic expression of Zfp521 in striatonigral MSNs or striatopallidal MSNs;and (2) Developmental expression profiling of purified striatonigral MSNs in Zfp521cko, D1-BAC-Zfp521 transgenic mice and wildtype mice using the FACS-array technology. The latter study may help to identify the critical downstream transcriptional targets of Zfp521 in the striatonigral MSNs prior to and after the onset of motor stereotypies. Our proposed study is highly significant since it may provide novel circuitry and molecular insights into the neurodevelopmental basis of motor stereotypy, a critical domain affected in ASDs. Furthermore, the molecular insights gained in the study may be a basis to further study the genetic etiology of motor stereotypy in ASDs and to develop novel therapeutics to ameliorate such behavioral deficits in ASDs and other neuropsychiatric disorders. PUBLIC HEALTH RELEVANCE: Motor stereotypy is a core domain of behavioral impairment in Autism Spectrum Disorders (ASDs). Our study will investigate novel basal ganglia circuitry and molecular mechanisms underlying the neurodevelopmental onset of motor stereotypy using a mouse genetic model. Our study will establish whether selective striatonigral neuron dysfunction may be the cause of motor stereotypy and will identify basal ganglia circuitry specific candidate genes and molecular pathways which may underlie the motor stereotypy in our model. Our study may provide novel molecular and circuitry level neurobiological insights into the pathogenesis and treatment of motor stereotypy in ASDs and related neuropsychiatric disorders.

DESCRIPTION (provided by applicant): The first 17 amino acids of huntingtin (NT17 domain) has been implicated as a critical regulator of mutant huntingtin (mhtt) mediated disease pathogenesis in Huntington's disease (HD). In the current study, we created a novel BAC mouse model of HD expressing full-length mhtt lacking this domain (BACHD-dNT17), and showed that these mice exhibit accelerated nuclear mhtt accumulation (hence supporting its role as a cytoplasmic retention signal in vivo), and exacerbate HD-like disease pathogenesis including movement disorder and striatal neurodegeneration. Based on these exciting findings, we hypothesize that the NT17 domain and its cytoplasmic interactors are critical in preventing mhtt nuclear accumulation and HD-like disease pathogenesis in vivo. To test this hypothesis, we proposed the following two Aims: Aim One. Using independent transgenic mouse lines to show the NT17 domain can prevent nuclear mhtt accumulation and HD-like disease pathogenesis in a polyQ-length dependent manner in vivo. Aim Two. Validating novel NT17 interacting proteins as cytoplasmic acceptors that mediate NT17- dependent htt cytoplasmic retention in cell models, and one such interactor, Tcp1, as modifier of HD pathogenesis in vivo. Our study may provide novel therapeutic insights based on the NT17 domain and its interacting proteins to ameliorate nuclear mhtt toxicity and prevent the onset of movement disorder and neurodegeneration in HD.

? DESCRIPTION (provided by applicant): Cajal revolutionized the study of the brain through the use of the Golgi stain to label cells sparsely and stochastically in a fashion that revealed a neuron's morphology in its entirety. Although genetic tools for sparse and stochastic labeling and manipulation of single neurons in Drosophila have been used extensively over the past 15 years, they have only recently become available for mammalian systems, but the latter tools are limited to only a few systems for which cell-type specific reagents (e.g. enhancers) are available or otherwise involve cumbersome manipulations. Thus, there is an important need in the field to develop robust reagents for analysis of neurons at the level of single cells. Indeed, analysis of neurons at the single identified cellular level provides critical information on the control of neuronal morphology, connectivity, physiology and plasticity. This application is in response to BRAIN Initiative RFA-MH-14-216. We provide proof-of-concept preliminary data for a general method to label and manipulate single neurons in the mouse central nervous system. Using this method (called MORF), we created a novel dopamine D1 receptor BAC transgenic mouse that can sparsely and stochastically label a subset of D1-expressing striatal direct pathway medium spiny neurons as well as hippocampal pyramidal neurons. The labeled neurons reveal detailed morphology including dendritic arbors and synapses. We propose to further validate and expand this technique to be of general use for both single-neuron genetic labeling as well as genetic manipulation for multiple neuronal cell types in the mammalian brain. In addition, we propose to modify MORF to facilitate epitope tagging of synaptic proteins from their endogenous loci to image synapses of single identified cell types. We will develop and streamline imaging and computational tools to acquire and register brain-wide single neuron morphological information in a standard brain atlas for rapid dissemination of data to the research community. In summary, our proposed plan will develop a novel genetically-directed single neuron labeling tool that is conceptually different and drastically simpler than those currently available, along with streamlined imaging and mapping methods to facilitate the use of rich single neuron information provided by the models. The novel tools and mouse resources developed here should be immediately useful and impactful for neuroscience and brain disease-related fields.

DESCRIPTION (provided by applicant): (HD) is the most common autosomal dominant neurodegenerative disorder (NDD), and is characterized by involuntary movement, cognitive and psychiatric symptoms, and relentless disease progression. HD is caused by a CAG repeat expansion in the mutant Huntingtin (mHTT) gene; however the molecular mechanisms underlying HD pathogenesis remain poorly understood. Currently, there is an urgent need to validate novel therapeutic targets to prevent or modify HD pathogenesis. Growing evidence suggests that a hallmark of age-dependent NDDs, including HD, is early neuronal dysfunction and loss of CNS synapses, followed by progressive neurodegeneration. Recent research supports an unexpected role of the complement system in synaptic pruning during development and disease. During development, and in a mouse model of glaucoma, complement proteins C1q and C3 localize to synapses and mediate synapse elimination. Our recent studies support a model in which weaker or less active synapses in the developing brain are tagged by complement and then eliminated by microglia, the primary phagocytic immune cells in CNS that express phagocytic complement receptors (i.e. CR3/Cd11b). We propose that similar complement and microglia-dependent mechanisms contribute to synapse loss, which in turn is crucial to pathogenesis of behavioral deficits and selective neurodegeneration in HD. Complement upregulation and microglial activation have been observed in human HD brain tissue and several HD mouse models, and our substantial new data showed complement factors (C1q and C3) are upregulated and tagged the synapses in the vulnerable brain regions (i.e. striatum) with known synapse loss in a full-length mHTT HD mouse model (BACHD). Recent study from Yang lab showed that behavioral deficits, selective brain atrophy and striatal synaptic toxicities in BACHD mice dependent on interaction between cortico-striatal neuronal interactions. Based on these strong preliminary data, we hypothesize that the complement system contributes to early stages of HD pathogenesis through complement- and microglia-mediated synaptic loss as a result of reduced cortico-striatal neuronal activity in HD mice. To test this hypothesis, we will study an innovative conditional HD mouse model developed by the Yang lab to determine whether complement targets cortico-striatal circuits for elimination, and whether optogenetic restoration of neuronal activities or genetic reduction/therapeutic antibody-based inhibition of the complement cascade (C3 and C1q) could ameliorate synapse loss, neurodegeneration, and behavioral impairments in HD mice. Our study could validate complement cascade (e.g. C3) and its interactions with microglia as a critical mechanism in HD pathogenesis, and provide support on the inhibition of this pathway in HD therapy.

Project Summary/Abstract Alzheimer's disease (AD) is the most common neurodegenerative disorder (NDD), and the leading cause of dementia in late adult life. Pathologically it is characterized by amyloid plaques, neurofibrillary tangles, and neuroinflammation (e.g. microglia and astrocyte activation). Studies in AD field so far have been focused on proteins that are misfolded and aggregated in the AD brains (e.g. amyloid beta peptide and Tau). However, the role of neuroinflammation in AD pathogenesis remains unclear and underexplored. Recent genome-wide analyses of AD risk genes have lead to the discovery of over 20 genes that modulate AD risk. One exciting insight gained from these studies is that multiple AD risk genes function in the innate immune system, which are known to mediate neuroinflammation in AD and related NDDs. Among the newly discovered AD risk genes, the R47H variant of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is conferring by far the highest risk (i.e. 2-4 fold higher risk compared to the controls). TREM2 is solely expressed in microglia and peripheral myeloid cells. TREM2 appears to modulate several important function of microglia/myeloid cells, including phagocytosis of debris, suppressing proinflammatory cytokine release, increasing neurotrophic factor synthesis, and supporting microglia survival. However, it remains unclear how TREM2 function or TREM2-R47H dysfunction may modulate AD risk in intact animal models of AD. In this proposal, we developed novel human genomic transgenic models expressing either the wildtype TREM2 variant (BAC-TREM2) or the AD-associated R47H variant (BAC-TREM2-R47H). We designed a series of in vivo genetic experiments, by crossing these models with two existing AD transgenic mouse models, to test our hypothesis that overexpressing TREM2 (hence boosting TREM2 signaling) may promote the beneficial function of microglia and ameliorate AD pathogenesis. Furthermore, we will be able to test whether the TREM2-R47H variant exerts partial loss-of-function or dominant toxicities to modulate AD. In addition, we will apply an integrative genomic approach to compare the transcriptome networks from our mouse models to those derived from AD patients. Finally, we will conduct in vitro signaling assays using primary microglia derived from our models to study how TREM2 and its AD-associated variant may alter signaling. Our study may help to validate the possible neuroprotective effects of microglial TREM2 overexpression in ameliorating AD pathogenesis, and elucidate mechanisms through which TREM2 and its R47H variant may modify AD risk.

ABSTRACT The Technical Advancement Core (TA-Core) has the overall goal of providing critical and appropriate expertise to use new technologies within CSORDA Projects and Pilots, as well as to advance and optimize these technologies for addiction-related research. The Core will also provide consultation (through Dr. Art Arnold) regarding analysis of potential sex differences observed in the animal experiments. The primary areas of technological contributions to the Center are in transcriptomics and bioinformatics (William Yang and Giovanni Coppola) and in MRI imaging (Brigitte Kieffer), which is reflected by the majority of resource allocation to these areas. The Core will additionally provide expertise and technical development for miniaturized microscopes (Peyman Golshani), wireless optoprobe (Wendy Walwyn) and silicon microprobe multi-recording devices (Sotiris Masmanidis). The Core Leaders, (Drs. Yang and Golshani) are both productive researchers, highly collaborative and experienced in leadership of shared resources and will have oversight of the administrative aspects and reporting of the core The Transcriptomics and Bioinformatics component will provide expertise for Projects I and IV as well as Pilot I and aid in characterization of mice for the AB-Core. The TA-Core leverages existing UCLA and NIH- supported infrastructure at UCLA specifically developed to support neuroscience investigators with genetics, genomics, sequencing, and bioinformatics experiments, therefore providing CSORDA faculty with a depth of technical expertise. The MRI imaging component will contribute to aims in Projects II and IV. This is an emerging technology to determine high-resolution brain region connectivity with the potential to guide future CSORDA research to areas connected with the circuitry investigated in current CSORDA Projects. The Miniaturized Microscope component will be utilized in Project I and Pilot IV for calcium imaging and striatum circuit analysis following opioid treatments. Development of the technology for addiction and CSORDA-related research will focus on combining the miniaturized microscopes with drug delivery systems for future experiments allowing direct analysis of neuronal networks by local drugs perfusion. The Silicon Microprobe Multi-Recording Devices component will be utilized in Pilot III to assess modulation of striatal network response to natural rewards during opioid withdrawal. Development of the technology will employ optogenetics for cell-specific analysis and activity modulation of D1 and D2 Medium Spiny Neurons. The Wireless Optoprobe component will be utilized in Projects I, III and IV. The core will improve the battery, modify the design to include bilateral probes and alter the communication system to enhance range. The TA-Core will be integral to current and future technical approaches utilized by CSORDA.

ABSTRACT A major force driving relapse in drug addiction, including opiate addiction, is the almost intolerable aversive effects of drug withdrawal. The biological bases of learned aversion to the drug withdrawal state have important mechanistic overlap with those for aversion to non-drug-related noxious stimuli (e.g. bitter taste, footshock, social defeat, traumatic stress) and involve multiple brain regions. Among them, the interconnected basal ganglia (BG) nuclei, including nucleus accumbens (NAc), ventral pallidum (VP) and dopaminergic (DA) neurons in the ventral tegmental areas (VTA) appear to be particularly crucial. In this proposal, we will focus our study on the NAc, not only because prior studies have implicated this brain region as a critical substrate for both rewarding and aversive effects of opiates, but also because recent studies suggest a particular neuronal cell type in the NAc, the D2 medium spiny neuron (D2-MSN), is essential to establishment of avoidance behaviors elicited by noxious environmental stimuli as well as the intense aversive effects of opiate withdrawal. Since manipulating the activities of D2-MSNs using the latest chemogenetic or optogenetic tools have demonstrated pivotal roles of these neurons in aversive behaviors, we reason the critical next step is to dissect the roles of D2-MSNs in non-drug-related and opiate withdrawal-related aversion through genetic analyses of endogenous genes that are highly selectively expressed in D2-MSNs. To this end, we hypothesize three such genes - two receptors (Gpr6 and Adora2a) and one peptide precursor (pre-pro-enkephalin (Penk)) - play important roles in opiate withdrawal aversion. We propose that signaling through these two Gs-linked receptors, by opposing the signaling imparted by the Gi/o-coupled dopamine D2 receptor, is crucial for D2- MSN mediation of both non-drug and opiate withdrawal-related aversion. To test this, we will investigate the behavioral and signaling effects of single or double deletion of these receptors. Conversely, our recent data demonstrate that pro-enkephalin-derived peptides in D2-MSNs play a crucial role in mediating an endogenous opioid hedonic tone, hence we will test the idea that removal of Penk in D2-MSNs will exacerbate the aversive effects of opiate withdrawal. Finally, we have obtained exciting preliminary data, using RNA-seq and network analyses, showing aberrant changes in NAc molecular networks upon the establishment of opiate dependence. We will apply such powerful integrative systems biology approaches to investigate whether genetic perturbation of key D2-MSN genes or opioid network genes alters the molecular signatures of opiate dependence.

PROJECT SUMMARY Integrating molecular, morphological, and connectomic properties is critical for unbiased classification of neuronal cell types in the mammalian brain. Here we propose a novel approach to classify neuronal cell types by brainwide comprehensive profiling of the dendritic morphology of genetically-defined neurons in the mouse brain. We have developed an innovative mouse genetic tool, called Mosaicism with Repeat Frameshift (or MORF), which enables sparsely and stochastically labeling of genetically-defined neurons in mice. MORF reporter mice can label in exquisite detail single neurons from dendrite and spines to axons and axonal terminals at a labeling frequency of 1-5% of a given neuronal population. We propose to cross our new MORF lines with Cre mouse lines for striatal medium spiny neurons (MSNs) of direct- and indirect pathways, and for cortical pyramidal neurons of distinct cortical layers (i.e. L2/3/4, L5 and L6). Each MORF/Cre mouse will allow us to image the detailed dendritic morphology for thousands of genetically-defined striatal and cortical neurons (i.e. dendritome). We have also developed and streamlined imaging and computational tools to acquire and register brainwide single neuron morphological data onto a standard reference mouse brain atlas. We will digitally reconstruct hundreds of thousands of MORF-labeled neurons using our novel program called G-Cut. Reconstructed neurons will subsequently used for morphology based clustering to define new morphological subtypes, which in turn can be analyzed for the expression of novel molecular markers neuronal cell types (e.g. from single cell RNA-sequencing). Finally, we will disseminate the data to the Brain Cell Data Center (BCDC) for data integration with those from other BRAIN Initiative Cell Census Network (BICCN) and for data access by the broader neuroscience research community. In addition to dendritome data generation and analyses, we will further advance our MORF method by generating new MORF reporter mouse lines with logarithmic fold decrease in the Cre- dependent labeling frequencies, which will permit imaging of the complete, brainwide morphology of genetically-defined single neurons that include both dendritic and axonal arborization. Such tool should greatly facilitate the neuronal morphology based cell type classification. Finally, we will develop integrated computer hardware and software for domain-specific computing for automated image processing and neuronal reconstruction, a major bottleneck in analyzing large bioimage datasets. Altogether we will provide rich dendritome information to enable unbiased, morphology-based neuronal cell type classification, and novel mouse genetic tools and computer software and hardware to advance the field of large-scale neuronal morphological imaging and analyses for the comprehensive study of the mammalian brain.

PROJECT SUMMARY Huntington's disease (HD) is one of the most common autosomal dominant neurodegenerative disorders affecting 30,000 patients in the US. HD patients typically experience motor, cognitive and psychiatric symptoms, which is associated with the loss of the striatal and cortical neurons. The disease inexorably progresses after the onset and patients typically succumb to the disease about 10-20 years after disease onset. HD is caused by a CAG repeat expansion, that encodes a polyglutamine repeat, in mutant Huntingtin (mHTT). Currently, there is no treatment to prevent the onset or slow the progression of HD. A recent genome wide association study (GWAS) identified several loci associated with modification of HD age of disease onset. The most significant loci identified were in Chr. 15 and encompasses a DNA repair enzyme called FAN1. Interestingly, FAN1 and two other genes identified in the HD modifier GWAS (MLH1 and MSH3) are all implicated in the instability of mHTT CAG repeat in somatic tissues. However, it is unclear whether FAN1's roles in CAG repeat instability is related to its function as a modifier of HD pathology including behavioral impairment (locomotor activity, sleep disturbance), striatal and cortical neuronal electrophysiological and pathological changes, and transcriptional dysregulation. In this proposal, we will address these critical questions using two HD mouse models, the Q140 murine Htt knockin model and a novel human genomic BAC transgenic mouse model of HD with >120 pure CAG repeats. The latter model is the first human mHTT model that shows CAG repeat instability that is correlated with behavioral deficits, and robust striatum-selective transcriptional dysregulation. We will cross these two HD mouse models with novel Fan1/FAN1 genetic models to address the following key questions: 1. Does reducing endogenous murine Fan1 levels accelerate the pathogenesis in the two HD mouse models (Aim 1)? 2. Do mice with murine Fan1-R510H knockin alleles (equivalent to the human patient-derived, deleterious FAN1 variant R507H) show disease-exacerbating effects, similar to the Fan1 knockdown mice (Aim 2)? and 3. Whether genetic mouse models with elevated expression of human FAN1 (BAC-FAN1) can ameliorate the behavioral, electrophysiological, pathological and molecular (i.e. transcriptomic) phenotypes in the two HD mouse models (Aim 3)? The findings from our study will be crucial to unravel where in the brain and what molecular mechanisms underly how Fan1 mutants modify HD pathogenesis in vivo. Furthermore, the mouse resources we have developed and the phenotyping platforms that will use in this study will be invaluable to future investigations of other HD modifiers and for developing therapeutics to target the HD human genetic modifiers to prevent, slow or stop progression of HD.

PROJECT SUMMARY A major challenge in studying the mammalian brain is to characterize the integrative properties of individual neurons, such as molecular profiles, complete morphology (dendrites, axons, synapses), connectivity, and activity; furthermore, this must be done at a scale that is commensurate with the goal of understanding all the neurons and their circuitry in the brain. While current single-cell transcriptomic and epigenomic profiling techniques are highly quantitative, scalable and informative, the technologies to study other neuronal cell-type defining properties(e.g. single-neuron brain-wide morphology and synaptic connectivity) are low throughput, labor intensive, poorly scalable and often yield partial data. Emerging neuronal cell type classification studies in invertebrates (e.g. Drosophila) and in rodents suggest that the neuronal morphological data such as axonal projection patterns are correlated, but may also be independent to the cell classes defined by single-cell gene expression. Thus, a complete and unbiased survey of mammalian neuronal cell census should include orthogonal data types consisting of both molecular profiles and brainwide morphology of single neurons. Finally, for emerging new cell types defined by unique transcriptomic profiles, the causal links between the cell-type-defining ?neuronal identity? genes and other cell-type-specific features, such as morphology, synaptic connectivity and activity, remain elusive and cannot be readily characterized in a scalable manner. In this proposal (in response to RFA MH-21-140), we will address these challenges by building upon a novel neurotechnology called Mosaicism with Repeat Frameshift, or MORF. MORF mice can confer cell- type specific, sparse and brightly labeling of neurons and glia to illuminate their complete morphologies in the mouse brain. The innovative aspect of the MORF mice is the use of an out-of-frame mononucleotide repeat as a stochastic translational switch; and its random frameshift leads to the expression of an extremely bright membrane-bound immunoreporter protein in 1-5% of genetically-defined neurons. In this proposal, we will generate four next-generation MORF mouse models that will allow: (1). precise and sparse labeling of neuronal cell types based on two genetic drivers (i.e. two molecular markers that define the neuronal cell type); (2). Cre-dependent labeling of endogenous presynaptic proteins in sparsely labeled GABAergic and cortical glutamatergic neurons; (3). selective expression of genome-editing tools in genetically and sparsely labeled neurons to support perturbation and multiplex subcellular labeling; and (4). development of an innovative and integrative multiscale imaging and registration pipeline to provide proof-of-concept data that analyzes brainwide morphology and connectivity of genetically-defined single neurons. Together, our grant may help to develop generalizable, scalable and democratizable tools to advance the study of neuronal morphology, synapses and connectivity, and genetic perturbation. These tools will facilitate the construction of mammalian brain cell census and advance the study of brain development, function and disease at the resolution of single neurons.