Leslie Thompson - US grants

[unreadable] DESCRIPTION (provided by applicant): Accumulation of pathogenic proteins and peptides with expanded polyglutamine repeats is characteristic of Huntington's disease (HD) and related neurodegenerative diseases. Other than the fact that a polyQ expansion within the disease protein causes disease, the major cellular mechanisms of pathogenesis are not clear. Multiple cellular processes have been implicated, but proximal causative events have proven difficult to distinguish from distal correlative events. As polyQ disease proteins can be modified in ways that change their cellular function or fate, it is likely that Htt protein modifications contribute to pathology. We find that Htt can be SUMO modified and that this modification can affect its biochemical properties and pathogenic potential in a Drosophila model. These observations form the rationale for this proposal. I propose to investigate, in depth, the role of SUMOylation in HD pathogenesis and to investigate the biochemical mechanisms involved in the SUMO-1 modification and de-SUMOylation of mutant Htt using a multidisciplinary approach. Implicit in this research will be an effort to identify potential therapeutic targets and develop strategies for interventions that disrupt or prevent pathology in the biochemical pathways responsible for alteration of mutant Htt. Hypothesis 1: SUMO modification of Htt is involved in HD phenotypes Mutant Htt accumulates in the nuclei of neurons and induces the dysregulation of key cellular processes including transcription. The impact of SUMOylation of mutant Htt upon cellular functions will be tested using in vitro and in vivo systems. Hypothesis 2: Specific E3-SUMO ligases are responsible for SUMO modification of Htt. An overall reduction of SUMOylation suppresses pathogenesis in vivo, therefore the identification of the specific enzymes involved in the attachment of SUMO groups to the Htt protein or in the removal of SUMO from the Htt protein may provide novel therapeutic targets for treatment of HD. Hypothesis 3: SUMO modification of Htt is critical to disease pathogenesis in vivo. Here we will test the physiologic relevance of SUMO modification pathways in Drosophila. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This consortium aims to capitalize on an unprecedented "grand" opportunity to develop a novel and powerful model of Huntington's disease (HD), a fatal neurodegenerative condition with no current treatment. Skin cells from patients with HD can be reprogrammed to pluripotency and then differentiated into specific neuronal and glial cell types, permitting investigation of the effects of the genetic lesion in the susceptible human cell types. We hypothesize that the genetic changes that cause HD lead to specific alterations in neuronal function- perhaps even survival-that will give important clues as to the mechanism and progression of disease. Altered cellular phenotypes will also serve as the foundation for translational research and drug development. Stimulus funding will bring together a highly focused group that (i) has a strong track record of innovative HD research and of working together, (ii) is poised to engage in cutting-edge research with recently generated induced pluripotent stem (iPS) cells derived from HD patients and is committed to broad distribution of findings, protocols and iPS lines, (iii) can capitalize on this stimulus funding through further grant applications and collaborative studies, and (iv) is partnered with CHDI, an HD foundation with dedicated HD stem cell and translational/drug discovery programs. This infusion of funds will accelerate the coordinated analysis of iPS lines and leverage the complementary, synergistic skill sets that will move the field forward more rapidly than would be possible by any group alone. The proposed studies will provide an entirely novel genetically accurate model to test new drugs in the fight against this disease, a unique resource that will benefit the entire HD community. PUBLIC HEALTH RELEVANCE: We hypothesize that the genetic changes that cause Huntington's disease lead to specific alterations in neuronal function-perhaps even survival-that, in turn, give important clues as to the mechanism of disease and its progression and offer a potential basis for small molecule screening assays. Our studies will provide a more authentic way to study the consequences of the HD mutation in human target cells. They represent a unique and timely opportunity to enhance the investigation of disease mechanisms and will generate a validated resource freely available to the HD community to further accelerate HD research toward a successful treatment.

DESCRIPTION (provided by applicant): There are at present no disease-modifying treatments for Huntington's disease (HD) a highly complex neurodegenerative disorder caused by a polyglutamine repeat expansion within the gene encoding the Huntingtin (Htt) protein. Validated targets for therapeutic intervention in HD are currently limited to the HD gene transcript and its polypeptide product. The focus of this proposal is to explore a potential novel therapeutic target for HD, the histone demethylase SMCX/JARID1C. The impetus for this work comes from the study of expression of the gene encoding BDNF, a critical neurotrophic factor essential for survival of CNS neurons expressed in the cerebral cortex. The progressive depletion of BDNF and the decreased supply of BDNF to striatum through anterograde transport down corticostriatal axons is strongly implicated in neuropathology in HD. However, reductions in BDNF expression levels are only one aspect of the transcriptional dysregulation which characterizes the earliest phase of HD pathology. Other neuronal genes such as the dopamine receptor 2 (DRD2) and preproenkephalin (PPE) are reproducibly decreased in mRNA expression levels early in HD and the reduced expression of such transcripts other than BDNF may also be implicated in HD pathogenesis. We find that an epigenetic mark of active transcription, H3K4me3, is decreased at the BDNF promoter in cortex of HD mouse brain (R6/2). Furthermore, we find that a demethylase specific for H3K4me3, SMCX/JARID1C, is increased in levels in the cerebral cortex of R6/2 mice. We therefore hypothesize that increases in SMCX/JARID1C activity are a critical event in HD pathology and that effective therapeutic intervention in HD can be achieved through reductions in SMCX/JARID1C levels. This proposal is designed to gather data which test these hypotheses and provide a framework for the possible validation of SMCX/JARID1C as a target for therapeutic intervention in HD. Our specific aims are as follows: Aim 1) To perform a systematic genome wide analysis of the sites at which the H3K4me3 mark is altered in the pathological program modeling HD in the R6/2 mouse brain. Aim 2) Test the impact of acute knockdown of SMCX/JARID1C in mouse-derived primary neurons expressing mutant Htt. Aim 3) Test the impact of chronic reduction of SMCX/JARID1C levels on functional properties of mouse models of HD. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a devastating degenerative brain disease that inevitably leads to death. Current treatments do not change the course of the disease;therefore, a completely unmet medical need exists. The results of these studies will provide important preclinical data that will speak to the translational utility of targeting an enzyme that modifies DNA structure and transcription in HD systems.

assay development; Huntington Disease; induced pluripotent stem cell; Length; Phenotype;

DESCRIPTION (provided by applicant): The Huntington's disease (HD) IPS consortium, funded with ARRA support for two years, brings together leading groups in stem cell and HD research to establish whether newly created IPS cell lines show HD-related (i.e., GAG length-dependent) phenotypes. This consortium aims to capitalize on new technologies to use non-integrating approaches for reprogramming and promising phenotypes in current HD iPS lines to develop robust and validated assays for drug development for Huntington's disease (HD), a fatal neurodegenerative condition with no current treatment. Skin cells from patients with HD can be reprogrammed to pluripotency and then differentiated into specific neuronal and glial cell types, permitting investigation of the effects of the genetic lesion in the susceptible human cell types. We have worked closely together and established novel methods of generating standardized neural stem cell cultures (e.g., EZ spheres) from three HD IPS integrating lines with a wide range of GAG repeats (33, 60 and 180), which have shown a number of promising phenotypes. The current proposal will extend these studies by producing 15 additional lines using the latest non-integrating IPS technology and will apply novel differentiation and genetic tagging protocols to further optimize the system. This funding would continue the significant synergy of this very focused consortium that (i) has a strong track record of innovative HD research and of working together, (ii) is poised to continue in cutting-edge research with induced pluripotent stem (IPS) cells derived from HD patients and is committed to broad distribution of findings, protocols and IPS lines, and (iii) is partnered with a group of investigators with the gol to optimize neuron specific differentiation protocols. Continued funding will accelerate the coordinated analysis of IPS lines and leverage the complementary, synergistic skill sets that will move the field forward more rapidly than would be possible by any group alone. Our ultimate goal is to develop and validate methods and assays using >96 well format for GAG repeat length-dependent phenotypes that are amenable to high content/throughput screening methods. The proposed studies will provide an assessment of the power of iPS cell technology for modeling HD, and for drug discovery. The monogenic nature of HD and the existence of allelic series of IPSCs with a range of GAG repeat lengths confer tremendous power to model neurodegenerative disease. These cell lines will be an essential resource for academic groups and pharmaceutical companies for studying pathogenesis and for testing experimental therapeutics for HD.

assay development; Generations; induced pluripotent stem cell; Length; Phenotype;

DESCRIPTION (provided by applicant): Stem cell medicine promises to revolutionize the treatment of human diseases and injuries, and has captured the hopes of the scientific community and the public alike. Perhaps nowhere is the potential of stem cells to treat human disease and injury more promising than for neurologic disorders. For this promise to become a reality, not only must basic research in this rapidly evolving field advance, but these advances must be translated through pre-clinical and clinical development into clinical practice. This bench-to-bedside pathway represents an enormous multidisciplinary effort, and many hurdles, both scientific and technical, must be overcome to bring stem cell therapies safely and effectively to reality. Traveling a path from bench to bedside is a relatively new opportunity fo researchers and provides novel challenges for training graduate students. The advent of this new frontier also means that students need additional training in the clinical aspects of the disease they are studying to identify therapeutic targets with the greatest relevance to human disease, inform preclinical safety and efficacy testing, and define preclinical outcome measures that parallel clinical metrics. In addition, the complexities of the regulatory processes required for human clinical trials and the business of taking research products to patients require a working knowledge of bedside in translational applications of laboratory research. Finally, students need preparation for alternative careers outside academia that will advance the NIH goals of enhancing biomedical research in the context of health and human services as a whole. This proposal seeks to fill this training need in the translational application of stem cell biolog to neurological disorders, a need that is not met by traditional neurobiology, stem cell or clinical graduate programs. The goal of the proposed Training Program in Stem Cell Translational Medicine for Neurological Disorders is to train a new generation of scientists in the translational application of stem cell biology to neuroscience. This proposed training program will be the first training grant on campus that is specifically focused on three integrated areas: stem cells, neuroscience and translation to the clinic. To train the next generation of stem cell translational scientists we will provide a Regenerative Neuroscience Boot Camp, clinical experience to understand research in the context of clinical translation, industry Internships to learn commercial and practical aspects of moving discoveries to clinical trials, coursework focused on providing the specialized scientific background required, a Stem Cell Translational Medicine in Neuroscience Retreat to provide an annual education in the latest cutting-edge science, and Workshops to focus on specific technologies. Especially important will be real and unique engagement with clinical faculty, the inclusion of internships with successful pharmaceutical and biotech companies and support from the FDA who oversee all stem-cell based trials in the US. UCI is in a strong position to provide the highest quality training to fellows preparing for a carer in the new field of stem cell translational medicine in neurological disorders.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating inherited neurodegenerative disease that strikes in the prime of life with no available disease modifying treatment. The identification of bioavailable and brain penetrable drugs that can delay onset or slow progression of disease is therefore a crucial component to developing effective therapeutic interventions for HD. Methylene blue (MB), known commercially as rember, is a drug that successfully completed a Phase IIb clinical trial for the treatment of Alzheimer's disease (AD), showing a significant improvement in cognitive function after six months and slowing the progression of AD by 81% over the course of one year. MB has several desirable properties required for drug candidates that act in the central nervous system, including high solubility in aqueous media, the ability to cross the blood-brain barrier, the ability to act in the CNS, and low toxicity in rodent models and in humans. Because it is in human use and in multiple FDA clinical trials for neurological disorders, it also represents a candidate that can be rapidly moved to the clinic. We tested if MB could modulate formation of expanded polyglutamine repeat aggregation intermediates and provide therapeutic benefit in vivo. Our preliminary data demonstrates that MB may be a potent modulator of the mutant Htt aggregation process, is neuroprotective in cell-based and Drosophila models, increases production of BDNF and slows the time course of motor deficits in HD modeled R6/2 mice. To determine if MB may be appropriate for human clinical trials, a systematic assessment of MB in a long-term pre-clinical trial with multiple outcome measures and testing presymptomatic and symptomatic time of administration is required. Here we propose to use a full length BAC HD mouse model to investigate potential disease modifying effects of MB on behavior, neuropathology, molecular signatures and bioavailability. The proposed longitudinal study will lay the fundamental groundwork for understanding the temporal progression of aggregation species and relationship to disease, and future therapeutic application of MB for HD. The following specific aim is proposed: Aim: Efficacy of Methylene Blue treatment and modulation of aggregation in BACHD transgenic mice. Our aim is to evaluate the long-term benefit of MB treatment in the full length mouse model BACHD, determine bioavailability of MB and investigate the temporal relationship between aggregation intermediates, molecular signatures and neuropathology. Since data suggests that the timing of MB administration may be critical, treatment will begin during either a presymptomatic stage or when disease is evident. Our approach will utilize a battery of assays including motor function, behavior, assays of aggregation intermediates, brain pathology and gene expression analysis to elucidate MB effects.

Data Generation Component We propose three broad experimental aims based around the type of assay, perturbagens and technologies being applied that will overlap across the years. The first will use iPSCs from three disease states (non affected, SMA and ALS) in which we have shown specific phenotypes. We will use an iterative approach by first screening for a number of perturbagens of interest to the broad neuroscience community using cost effective assays including simple cell death models and a highly novel imaging analysis system. The second parallel effort will be to use the same iPSC lines but in this case test a set of known cell modifiers (Glutamate, ER stressor and SOD1 ASO) as perturbagens and perform massive parallel quantitative molecular phenotyping (QMP) to generate robust signatures and to define the responses of motor neuron cultures to these perturbagens. We will then perform QMP on neurons, astrocytes and oligodendrocytes from disease and control cells and in response to the same perturbations as above to elucidate signatures across broadly relevant neural cell types. This data will be compared to motor neuron cultures (where expected disease signature will be) with non motor neuron cultures (where no or a more restricted disease signature is expected) to resolve the question of cell type specificity. We will also generate new iPS lines from post mortem human patient tissues to allow clinical pathological signatures to be incorporated into the LINCS data, providing a unique resource to both the SMA and ALS scientific community and to researchers interested in larger questions relating to the CNS. The third is to bring in disease iPS lines from Huntington's and Parkinson's subjects (from the respective NIH consortia and in coordination with various foundations - see letters of support. Overall) and test the specificity of signatures seen in the motor neuron diseases with other neurodegenerative conditions (both disease and response to perturbagens). All of these studies will be done in close association with the data analysis component community section (being responsive to the needs of the community). Given the speed of discovery in iPSC and new molecule generation, we also aim to be flexible in our design to allow incorporation of breakthrough technologies or drugs should they arise.

DESCRIPTION (provided by applicant): There is a critical need to define the state and predict the behavior of human brain cells in health and disease. The number of different cell types in the CNS remains undefined, and despite a demographically ordained wave of neurodegenerative diseases, not a single disease-modifying therapy exists. Our knowledge of the CNS and the foundation for intervening rationally in disease would be dramatically advanced by generating quantitative molecular phenotypes essentially cell signatures of human neurons, astrocytes and oligodendrocytes from healthy people and from patients with motor neuron disease, Huntington's disease, and Parkinson's disease. The CNS is so unique that studying non-neuronal cells does not provide much assistance. Despite this desperate need, the inaccessibility of human brain cells meant studying them would have been impossible until the recent discovery of cellular reprogramming and induced pluripotent stem cell technology. Here we propose to form the NeuroLINCS consortium to accomplish these goals. We have handpicked the team to bring in critical expertise in iPSC technology, disease modeling, transcriptomics, epigenomics, metabolomics, whole genome sequencing, proteomics, high content, high throughput longitudinal single cell analysis, other cell-based assays, bioinformatics, statistics and computational biology. In addition, we are collaborating with Google to bring in special expertise in machine learning and the integration of signatures across platforms into highly predictive models of responses to perturbagens. Together, we expect to develop cell signatures of an array of human brain cell types under different conditions that should be broadly applicable to the LINCs community. We also anticipate generating innovative software tools and approaches that will make the signature generating process cheaper, faster, and more reliable. Besides the unique combination of expertise represented within NeuroLINCS, another distinguishing feature is the long track record that its members have of collaborating with each other. That collaborative spirit will be expressed in NeuroLINCS through its significant and multifaceted community outreach programs. These will involve specific and detailed plans to make the data and tools that NeuroLINCS generates available to the community, to interact with other LINCS sites, and to prepare for DCIC and the prospect of disseminating knowledge and resources at scale.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an inherited neurodegenerative disease that strikes in the prime of life and for which no disease-modifying treatments exist. The disease is caused by the expansion of a CAG repeat within the HD gene, leading to complex and extensive cellular dysfunction. The identification of validated cellular targets that impact the onset and progression of disease and a mechanistic understanding of these targets in HD systems are therefore critical to development of new and effective therapeutics. Mutant HTT (mHTT) and toxic fragments derived from the mutant protein are in a dynamic equilibrium poised to shift the protein homeostatic network from the appropriate balance of protein folding, misfolding, oligomerization and degradation to one in which that balance is disrupted. Upon disruption of this network, cellular proteins accumulate and degradation pathways become impaired. Our studies suggest that the E3 SUMO ligase, PIAS1, is a key modifier of this process and may act as an important regulatory switch in this dynamic equilibrium. In published findings, we identified PIAS1 as a novel modulator of both SUMO-1 and SUMO-2 modification and accumulation of mHTT protein in cultured cells. Further, reduction of the only PIAS in Drosophila delays expression of phenotypes caused by repeat expanded HTT, suggesting this enzyme may provide a selective therapeutic target. In addition to functioning as a SUMO E3 ligase, PIAS is implicated in regulating transcription of several pathways including proinflammatory cytokine signaling and the innate immune response, which is an emerging area of focus in HD. The communication and involvement between E3 SUMO ligases and protein clearance pathways are not well understood with respect to misfolded and accumulated proteins; therefore, understanding the behavior of the PIAS1 network in HD systems will contribute a crucial understanding as to its role in HD pathology. We hypothesize that PIAS1 is a key regulator of HTT SUMOylation and accumulation, that it can modulate HD pathogenesis and that it is a novel target for HD treatments. We propose to use a complementary set of cell based assays and in vivo studies to move forward our mechanistic understanding of PIAS1-mediated networks, and validate PIAS1 as a molecular target for HD drug development. Specifically, we will perform the following aims: Aim 1: Define the PIAS1 network in primary neurons and induced pluripotent stem cells. Aim 2: PIAS1 modulation in HD mouse models. Aim 3: Functional significance of PIAS1 domains in disease modifying pathways.

Data Analysis & Signature Generation Component Our goal is to generate cellular signatures of human neurons in response to perturbagens. Our studies will focus on human neurons, generated from induced pluripotent stem cells (iPSCs) (i-neurons) obtained from both healthy people and patients with neurodegenerative diseases. The cellular signature will be a composite picture of the molecular properties of a neuron that distinguish the state and determine the behavior of the cell. We will generate three classes of cellular signatures. The first will be static signatures based on quantitative molecular phenotyping involving OMIC analysis of the i-neurons. Analysis of the static signatures will highlight critical signaling pathways that distinguish a cellular response to a perturbagen. The second type of signature will be dynamic signatures generated with a novel high throughput, single cell longitudinal analysis system. Robotic Microscopy (RM). RM will be able to pinpoint critical times in the life of i-neurons as their physiology change in response to perturbagens. Analysis of dynamic signatures will guide selection of time points that will be investigated more in depth with methods that generate static signatures. In turn, elements of these static signatures will be perturbed genetically and analyzed by RM to elucidate the epistatic relationship of the components of a signature and to develop explicit multivariate predictive descriptions of cellular responses to perturbations. The third type of signature will emerge from an integration of the individual signatures using clustering methods and machine learning algorithms. The technology to analyze the data of the cellular signatures will be compatible with those produced at other sites in the LINCS network. A major innovation of our program is the implementation of novel data analysis platforms that will produce signatures that will have greater predictive value of a cell's biology than standard technologies. We will integrate Data Analysis and Data Generation, creating feedback loops to allow the cellular signatures that we generate to influence subsequent data generation. In turn, the use of machine learning algorithms in collaboration with Google will allow us to iteratively refine our signatures to make them more predictive in identifying cause and effect relationships from the cellular signatures.

Community Interactions & Outreach Component The NeuroLINCS Community project will plan to provide resources and tools for a broad user base of basic and clinical scientists. It has a structure to facilitate access to the various genetic and proteomic data sets, the signatures created, and the analysis tools. It is designed to be directed to researchers at the bench, clinicians developing biological disease readouts and those in computational roles. It will incorporate an assessment to demonstrate the utility of the generated resources, methodologies, and analytical tools to LINCS and non-LINCS scientific community. Importantly, it will develop and implement a plan to bring in external collaborators who may have data sets that bear on the development of cell signatures. There is an extensive plan to develop workshops, tutorials, and symposia in conjunction with the use of innovative online technologies for disseminating information to target the major LINCS goals. Finally it will develop bidirectional links with the neuroscience clinical and basic community through a series of collaborations with large National clinical data and tissue-based networks.

Administration Component The Administrative Core is responsible for setting the overall direction of the NeuroLINCS center and for ensuring that the resources and components of the Center are optimally utilized. The successful development and evolution of the NeuroLINCS center requires strong interactions between the leaders and co-leaders of each Component and of the center as a whole. Hence, the NeuroLINCS Administrative Component plays a vital role in facilitating these interactions. Moreover, the Administrative Component and its personnel provide the necessary administrative and fiscal oversight to ensure that the NeuroLINCS center is run efficiently. The NeuroLINCS center involves 5 principal sites with defined responsibilities of growing, differentiating and generating new induced pluripotent stem cell lines (iPSCs), performing data generation assays on human brain cells made from iPSCs in response to perturbagens, performing basic analyses and developing cell signatures through integrated data analysis .methods, and establish community interactions. An integrated and highly collaborative group of investigators with expertise in stem cell biology, IPS cells, quantitative molecular phenotyping (omics and single cell imaging) and bioinformatics will work closely together to generate significant and highly predictive cell signatures. The PIs of the NeuroLINCS center are Steven Finkbeiner (Gladstone), Ernest Frankel (MIT), Jeffrey Rothstein (JHU), Clive Svendsen (Cedars) and Leslie Thompson (UCI), who will serve as leaders and co-leaders of components. Each Component has identified co-investigators/collaborators/consultants appropriate for the planned scientific investigations. Component leaders and co-leaders will also be active participants in NeuroLINCS consortium working groups as they are developed to address specific issues. Results of the genetic, proteomic and other characterization conducted by consortium labs will provide important feedback for further enhancement of induction and differentiation protocols and related methodologies and it is anticipated that this collaborative and iterative approach will lead to the broadest success for the study. An Evaluation Program within the NeuroLINCS is in place to determine if the programs supported are meeting the needs of the research community, are efficiently managed, and demonstrably effective and annual objectives and milestones.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is a fatal neurodegenerative disease which strikes in the prime of life and progresses over 10-20 years, producing movement abnormalities, cognitive dysfunction, and psychiatric manifestations. HD is caused by a dominant expansion of a CAG trinucleotide repeat tract within the protein-coding region of the Huntingtin (Htt) gene and corresponding cortical dysfunction and striatal degeneration. As mutant HTT is the disease causing agent, one current strategy for disease intervention is to reduce the production of the HTT protein. In fact, inhibitory RNA strategies which knock down both mutant and wild-type Htt alleles have shown effectiveness in cell and mouse models at reducing HD-related phenotypes. However, the balance between efficacy in reducing Htt levels and side effects from lowering Htt must be considered given the important roles for normal HTT in neurodevelopment and other cellular functions. For this reason, alternative strategies have also focused on selectively reducing mHtt levels to diminish potential side effects. Recently, we and others have developed HD patient-derived induced pluripotent stem cells (iPSCs). These lines can be differentiated to mature neurons specified for striatal development and differentiated cells display HD- related phenotypes, thereby providing a system in which to examine the progression of symptoms in HD iPS cells and the degree to which these phenotypes are reversible by HTT reduction. Here we propose to use genome editing of existing HD iPSC lines to constitutively or inducibly express RNAi's that target either total HTT or preferentially target the mutant allele. This approach is distinct from isogenic lines where the expanded repeat is corrected to wild type range repeats through genome editing or homologous recombination in order to validate CAG-dependent phenotypes in the same genetic background. In the isogenic context, no disease phenotypes occur as the corrected iPS cell no longer expresses mHTT. In contrast, when considering RNAi or ASO approaches for treatment of HD, patients continuously express the expanded repeat mutation beginning in early development and the ability to modify the impact of that chronic mHTT expression is what is required of that RNAi or ASO. Questions that emerge are 1) whether CAG repeat dependent changes are modifiable in a mHTT background through reduction of HTT, 2) whether specific mHTT phenotypes can be ameliorated by total or mHTT reduction once disease phenotypes are manifest and 3) whether reducing HTT in general has consequences as even mHTT is likely to have functions that could be lost following knockdown strategies. The proposed aims are: Aim 1. To generate HD iPS Lines with constitutive or inducible silencing of Htt. Aim 2. To evaluate the consequence of lowering total versus mutant HTT in HD iPS cells.

? DESCRIPTION (provided by applicant): The primary cellular defense system against protein misfolding and impaired clearance is the functional network of chaperone proteins, such as TRiC, which are both responsible for the proper folding of normal proteins and the attempt to refold or initiate destruction of damaged and genetically abnormal proteins. Compelling evidence from model systems supports the view that the proteostasis network can be modulated to improve the outcome of the cellular challenges presented by the toxic protein species responsible for neurodegenerative disease. However, to date, no effective therapeutic intervention for any neurodegenerative disease has been developed based on any principle including modulation of the chaperone network. This proposal is focused on carrying out an integrated program of investigation whose goal is to create a strong framework in which basic science understanding of the structure of toxic species and their interaction with the proteostasis network is linked to translational approaches to reduce the accumulation of these species through reduced production and/or enhanced clearance. In so doing, the application addresses the critical societal goal of intercepting the oncoming epidemic of neurodegenerative disorders. We have chosen the paradigmatic neurodegenerative disease Huntington's disease and the TRiC chaperonin network as the focus of our program, based on strong data by our team that TRiC complex components provide clear beneficial effects on mutant HTT-induced phenotypes in model systems. In this Program Project we propose 3 integrated Projects and 2 supporting Cores to investigate mHTT-TRiC chaperonin interactions and determine how select TRiC related components contribute to or reduce mHTT-driven pathogenesis. The hypothesis that guides the proposal is that TRiC plays a critical role in regulating the accumulation of toxic form of the expanded repeat HTT protein and hence increasing the activity of TRiC and TRiC-derived proteins will abrogate and/or reverse mHTT-linked pathogenesis. We propose the following four overall Specific Aims: Aim 1: To characterize the interactions between TRiC reagents and aberrant forms of mHTT under in vitro and ex vivo conditions as well as in HD model cells and neurons. Aim 2: To investigate the impact of existing and novel TRiC reagents on the production and accumulation of mHTT species and cellular proteostasis. Aim 3: To systematically evaluate the impact of TRiC reagents on neuronal function and survival in cell culture. Aim 4: To systematically evaluate and compare the impact of TRiC reagent therapy in mouse models of HD through quantitative measures of neuronal structure, function and pathology. Our program will provide a framework for the extension of the study of TRiC based therapeutic strategies for other neurodegenerative disorders, including AD, PD, ALS or FTD. While our proposal will focus on TRiC-based therapeutics, our assays may point to the participation of chaperones that partner with TRiC in regulating mHTT effects and open the possibility of additional novel approaches to HD and related disorders.

Project Summary/Abstract Biochemical analysis and cell based phenotypic assays clearly establish the impact of modulation of the TRiC chaperonin system on mutant Huntingtin protein. To develop an effective TRiC based therapeutic strategy for blunting the pathological impact of mutant HTT, it is essential that studies on TRiC chaperonin interactions with mutant HTT be carried out in an animal model setting as close as possible to the human brain. The studies proposed in this project directly address this central issue. Aim 1: To perform an integrated evaluation of the impact of apiCCT1 delivery to the mouse striatum on mutant HTT biochemistry and quantitative measures of HD pathology. Preliminary data demonstrates that exogenous application of the apical domain of CCT1 in cells is sufficient to modulate aberrant accumulation of mHTT. To form an accurate and quantitative understanding of the impact of apiCCT1 delivery to CNS cells in vivo we will deliver apiCCT1 by several alternative modalities and 1) carry out assessments of the status and levels of aberrant forms of expanded repeat containing HTT including soluble, oligomeric and fibrillar forms, 2) assess the impact on HD pathological phenotypes in HD mouse models, including using reporters for the measure of transcriptional dysregulation, an early, characteristic HD phenotype, and 3) quantitatively assess the efficiency of uptake and subcellular localization of delivered apiCCT1. Aim 2: Evaluate the impact of the modulation of additional components of the TRiC system and TRiC inspired reagents on HD pathology and mutant HTT behavior in mouse brain. In projects 1 and 2 of this proposal, novel TRiC based or TRiC inspired reagents including novel forms of apiCCT1 optimized for therapeutic benefit, other components of the TRiC chaperonin system such as CCT3 and CCT5 and combinations of TRiC inspired reagents, will be developed. In this project, these novel TRiC inspired reagents will be tested in mouse brain for their impact on mutant HTT driven pathology and biochemistry using methodologies used for apiCCT1 in specific aim 1. Aim 3: Bring an optimized TRiC based therapeutic strategy to full scale behavioral testing and evaluation of neuronal trafficking in HD model mice to evaluate the potential clinical utility of this strategy. We will carry out full scale behavioral testing in fragment (R6/2) and full length (BACHD) HD model mice of optimized TRiC chaperonin based therapeutic intervention for HD. These studies will be designed to serve as initial preclinical studies to evaluate TRiC chaperonin therapy for human use. We will use neuronal trafficking assays based on studies already shown to demonstrate the striking impact of TRiC chaperonin activity in cell culture to assess the impact of TRiC chaperonin therapy on this significant parameter of neuronal health and function on full length HD model mice in vivo.  

CORE A: ADMINISTRATIVE CORE PROJECT SUMMARY/ABSTRACT This Program Project, ?From Structure to Therapy: The TRiC Chaperonin Network in Huntington's Disease? is focused on understanding the impact of modulating the TRiC chaperonin system on HD pathogenesis and developing effective TRiC based therapeutic strategies and reagents to alleviate the pathological impact of mutant huntingtin. The Administrative Core oversees the activities of the Program and provides infrastructure support for the investigators to facilitate achieving the proposed goals of the Program. The Core will provide support for oversight and governance, management and operations, as well as reporting and compliance needs of all three projects. The Administrative Core will also monitor and oversee access to the shared resource Repository core of the Program Project and establish a password protected interactive website for real-time data sharing between Program investigators. The Administrative Core will promote the multi- disciplinary and collaborative approach between the Projects by organizing bi-monthly conference calls and quarterly face-to-face meetings. The Core will also assemble an External Advisory Committee to review research progress and provide guidance for the Program Project and organize annual meetings with the Program Project investigators. The Administrative Core will coordinate the meetings and facilitate an environment to keep the program synergistic, productive and innovative. Finally, the Administrative Core will coordinate material transfer agreements, oversee animal use protocols, facilitate travel for lab members between Project sites and manage the financial budgets of the Cores and Projects.  

Huntington?s disease (HD), one of the first neurodegenerative diseases for which a genetic cause was determined, is an inherited neurodegenerative disorder that has no disease-modifying treatment. HD is caused by a CAG repeat expansion in the HTT gene encoding a polyglutamine (polyQ) tract within the amino terminal portion of Huntingtin (HTT). While the field has gained an understanding of the many cellular processes that are disrupted in HD, we do not yet understand the interplay between key proximal HD-associated events, such as the relationship between aberrant mutant HTT (mHTT) accumulation, RNA biology and epigenetic events in specific cell types in the brain. Similarly, we do not know how changes in these processes impact clinical manifestation of disease, where best to intervene therapeutically and what outcome measures may be the most informative in HD models. The overarching focus of the research proposed here is to fill vital gaps in our knowledge about how these factors impact onset and progression of HD and how that understanding might lead to new disease-altering therapies. The proposed research will leverage unique resources and methods developed in my lab and those of my collaborators and will utilize state-of-the-art technologies such as single-cell RNA-seq, mass spectrometry and cryo-electron tomography to dissect molecular mechanisms. Ultimately, treatments for this disease, including combination therapies, will likely require a much better fundamental understanding of how mHTT leads to HD pathology and death. Our recent data suggests unexpected relationships between protein posttranslational modification (PTM) pathways, aberrant mutant HTT accumulation and DNA damage responses in neurons, the latter now implicated as a critical modifier of HD age-of-onset. Using a systems biology approach we are learning how chronic expression and accumulation of mHTT impacts gene expression and now seek to develop a more comprehensive understanding of RNA biology and causal networks in specific cell types. Here I propose investigations aimed at addressing major gaps in our understanding of how the fundamental molecular and cellular events underlie how the mutant HD gene causes degeneration of specific cell populations in the brain to induce motor and cognitive decline and ultimately premature death of patients. My program benefits from the integrated use of patient iPSCs and HD mouse models and the extensive and productive collaborations we have established over many years. With the overall goal of understanding proximal and initiating events in the disease and developing therapies for HD, I propose two primary avenues of research relating to the integration of 1) protein homeostasis and 2) epigenetics and RNA biology in HD.

PROJECT SUMMARY For too long, studies of the Blood-Brain Barrier (BBB) have ignored the blood component of this interface, focusing almost exclusively on the cells of the Neurovascular Unit (NVU). The goal of the FOA to which we are responding aims to change this: ?The intent of this FOA is to stimulate the development of a new field of blood-based science by re-defining the neurovascular unit as a component of the blood-brain interface. This will facilitate development of human- based neurovascular-blood models to identify targets for diagnostics and regulation of the blood-brain interface?? The NVU is comprised of endothelial cells (EC), pericytes and astrocytes, and a complex basement membrane, which work together to severely limit the free movement of molecules from the blood into the brain parenchyma. In response to local signals during development BBB EC develop tight junctions and have very low rates of transcytosis. The side-effect of this is that access of potentially therapeutic drugs into the brain is also compromised. In this proposal we will build on our well-established human Vascularized Micro-Organ (VMO) platform to create a novel blood-brain interface model, the VMO-B. In this model a network of human microvessels anastomoses to microfluidic channels representing an artery and a vein and are induced to a BBB phenotype by Wnt signaling. The vessels are invested by pericytes and contacted by astrocyte foot-processes. Importantly, we will run a blood substitute ? VMOBlood ? through the vessels that will mimic the composition of blood, including protein and lipid content. We will then use the VMO-B to investigate the process of BBB breakdown in the pathogenesis of Huntington?s disease. We already have preliminary data suggesting that expression of mutant HTT protein in EC causes BBB deficits. We will investigate crosstalk between blood and the cells of the NVU, and how expression of mHTT in each cell type affects cell-cell communication and barrier function. In the R61 phase we will pursue three aims: Aim 1 Develop a stable MPS BBB model with perfused microvasculature; Aim 2 Incorporate flow of blood into BBB microfluidic model; and, Aim 3 Characterize key transporters at the blood-brain interface. In the R33 phase we will use this platform to examine the role of the blood-brain interface in the pathology of HD through an additional two aims: Aim 4 Test the hypothesis that expression of mHTT in EC disrupts transport across the BBB leading to changes in the neural micro-environment; and, Aim 5 Test the hypothesis that expression of mHTT disrupts multiple cell-to-cell interactions at the blood- brain interface. Completion of this project will not only shed light on the neuropathology of Huntington?s disease, but will also yield a platform ideally suited to drug development and investigating the role of the blood- brain interface in numerous neurological diseases including Alzheimer?s disease, Multiple Sclerosis, Parkinson?s disease, stroke, CADASIL, and traumatic brain injury.