Scott Zeitlin - US grants

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a dominant neurodegenerative disease that is caused by the expansion of a stretch of CAG triplet repeats encoding polyglutamine (polyQ) within huntingtin (htt), the protein product of the HD gene. HD is considered to be the consequence of a deleterious gain-of-function caused by the expanded polyQ stretch that is unrelated to htt's normal function. Recent work suggests that although gain-of-function may play an important role in HD pathogenesis, a corresponding loss of normal htt function also contributes to the disease process. Our long-term objective is to use genetic and biochemical approaches to understand the role of htt's normal function in HD pathogenesis, and to discover new potential therapeutic strategies for the treatment of HD based on restoring normal htt function in HD. To accomplish this objective, we propose three specific aims that are designed to help us understand how the expanded polyQ stretch can affect normal htt function, and how a version of htt that lacks its normal short stretch of polyQ (?Q-htt) is able to rescue HD phenotypes in a mouse model for HD. In Aim 1, we will test the hypothesis that ?Q-htt is able to rescue HD mouse model phenotypes by enhancing autophagic clearance of mutant htt. We will use both cell culture and mouse models to focus on two potential mechanisms that may be responsible for ?Q-htt's effects. First, ?Q-htt may mediate the enhanced recognition of mutant htt aggregates by p62/SQSTM1, a polyubiquitin binding protein that can target such aggregates for autophagic degradation. Second, ?Q-htt may affect autophagic degradation of mutant htt aggregates indirectly by enhancing retrograde transport of autophagosomes to lysosomes. To test these mechanisms, we will characterize brains and primary neurons derived from mice expressing ?Q-htt with or without 140Q-htt expression, and conditional knockout mice lacking neuronal htt expression, for p62/SQSTM1 function. In addition, the efficiency of retrograde transport will be characterized in primary neuronal cultures by measuring organelle and dynein complex movement. Recently, we have also observed that increasing the length of the mouse htt polyQ stretch from 7Q to the normal human average length of 20Q can accelerate interactions of normal and mutant htt. To test the hypothesis that an interaction between normal and mutant htt can affect HD pathogenesis, we will compare in Aim 2, behavioral and neuropathological phenotypes in mice expressing 7Q/140Q htt, and in mice expressing 20Q/140Q htt. Finally, in Aim 3, we will test the hypothesis that ?Q-htt may interact with a novel set of binding partners and/or is resistant to 140Q-htt's potential to influence the interaction of htt with its binding partners, by using mice expressing epitope-tagged htt alleles to detect differences in the repertoire of normal and ?Q-htt interacting proteins in the presence and absence of mutant htt expression. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a hereditary neurodegenerative disease affecting ~1 in 10,000 people that is caused by a mutation in the protein huntingtin (htt). There is currently no cure for this disorder, and once symptoms are detected, the disease progresses over 10-20 years and ends inevitably in death. We propose experiments that will help us to understand how the mutation affects htt normal function so that we can discover new therapeutic strategies based on restoring htt function in people affected with HD.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a dominant hereditary disease that is caused by the expansion of a CAG triplet repeat encoding a stretch of polyglutamine (polyQ) within Huntingtin (Htt), the protein product of the HD gene. The expansion of the Htt polyQ stretch confers a deleterious gain-of-function while simultaneous loss of normal Htt function can also contribute to pathogenesis. In HD and other adult onset neurodegenerative diseases, evidence is accumulating suggesting that dysfunction during development can contribute to later pathogenesis. Alternatively, compensatory mechanisms acting during development can delay the onset of neurodegeneration and motor/behavioral symptoms. Our overall objective is to use novel repressible/inducible knockin mouse models for HD to explore the role of developmental expression and compensation in the progression of Huntington's disease, and to determine if there are critical times during development or at different ages in the adult, when normal htt expression must be maintained. To this end, we are developing knockin mouse models that express either normal (7Q) or mutant (140Q) versions of the mouse HD gene (Hdh) that have lactose operator (LacO) sequences inserted within their promoters. When these HdhLacO alleles are expressed together with a transgene encoding a version of the bacterial lactose repressor (LacIR) that functions in mice (HdhLacO-3xFLAG-7Q/-; -actin LacIR-tg and HdhLacO-140Q/+; -actin LacIR-tg mice, respectively), wild-type or mutant htt expression can be turned-on or turned-off by administering or withdrawing isopropyl - D thiogalactoside (IPTG, a lactose analog) in the mouse's drinking water. In Aim 1, we propose to study the consequences of developmentally-restricted mutant htt expression on adult behavioral and neuropathological phenotypes by turning-on the expression of mutant htt at conception, and turning-off mutant htt expression at 1 month of age in HdhLacO-140Q/+; -actin LacIR-tg mice. Similarly, we will turn-on mutant htt expression at one month of age to study the role of compensation in HD pathogenesis. In Aim 2, we propose to determine if there are critical periods during the life of the mouse when normal htt expression must be maintained by turning-off normal htt expression at different ages in HdhLacO-3xFLAG-7Q/-; -actin LacIR-tg mice. Moreover, to model the therapeutic efficacy and safety of reducing overall Htt expression (Aim 3), we will combine both LacO-modified Hdh alleles in one mouse model (HdhLacO-140Q/LacO-3xFLAG-7Q; -actin LacIR-tg mice), and suppress both normal and mutant htt expression at 6 months or 1 year of age.

? DESCRIPTION (provided by applicant): In Huntington's disease (HD), flanking protein domains modulate the toxicity of mutant Huntingtin's (HTT''s) expanded polyQ stretch. In mammals, a proline-rich region (PRR) is located at the C-terminal end of the polyQ stretch, and it has co-evolved with the polyQ stretch, increasing in size as the normal HTT polyQ stretch has lengthened during evolution. In yeast model systems, deletion of the HTT PRR in the context of an expanded polyQ stretch interferes with aggresome formation and increases toxicity of an N-terminal fragment of mutant HTT. To determine the role of the HTT PRR in modulating mutant HTT pathogenesis in mouse models for HD, we have generated three knock-in alleles of the mouse HD gene (Hdh) that express normal or mutant huntingtin (htt ) with deletions of the PRR - Hdh?P, Hdh140Q?P, and Hdh3xFlag140Q?P. We have found, in contrast to the results of the yeast studies, that deletion of the mutant htt PRR ameliorates several phenotypes exhibited by the CAG140 knock-in mouse model for HD, resulting in a significant delay in aggregate formation, alterations in aggregate conformation, normalization of striatal Darpp-32 expression, and the rescue of activity deficits. Using these new mouse models, we propose three complementary aims to determine the mechanisms by which deletion of the htt PRR affects HD mouse model pathogenesis. In Aim 1, we will test the hypothesis that deletion of the PRR modulates mutant htt's toxicity by altering the association of htt-interacting proteins. We propose using anti-FLAG immunoaffinity purification to enrich for proteins associating with 3xFlag140Q?P- tt and 3xFlag140Q-htt in the striatum and cortex, and characterize their identity using mass spectrometry. In addition, we will characterize the protein composition of htt aggregates purified from the Hdh140Q/+ and Hdh140Q?P/+ brain to determine if differential sequestration of cellular proteins by 140Q-htt and 140Q?P-htt aggregates contributes to pathogenesis. In Aim 2, we will test the hypothesis that deletion of the mutant htt PRR alleviates mutant htt's perturbation of gene expression by performing RNA-seq analysis of wild-type, Hdh140Q/+, and Hdh140Q?P/+ striatal gene expression to identify those genes whose expression is altered in the Hdh140Q/+ brain but restored by the mutant htt PRR deletion in the Hdh140Q?P/+ brain. In Aim 3, we will genetically test the role of the htt PRR on aggresome formation (in cis or in trans), and the potential difference between the murine and human PRR in HD pathogenesis by characterizing behavior and neuropathology in Hdh140Q?P/?P, Hdh140Q?P/+, Hdh140Q?P/7QhuPRR and Hdh140Q?P/20QhuPRR mice as they age. In addition, we will characterize microtubule-based transport and autophagy (two pathways involved in aggresome formation and protein degradation) in early postnatal (P5) primary cortical and striatal neuronal cultures generated from wild-type, Hdh?P/?P, Hdh140Q/140Q and Hdh140Q?P/140Q?P mice.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is caused by the expansion of a CAG triplet repeat encoding a stretch of polyglutamine (polyQ) within Huntingtin (HTT), the protein product of the HD gene. The HD mutation confers a deleterious gain-of-function and potential loss-of- function on mutant HTT that affects a variety of cellular pathways. Gene-silencing is a promising therapeutic strategy for HD which can circumvent the challenge of finding treatments targeting all the cellular pathways that are affected by mutant HTT. To determine the optimal time for reducing mutant HTT expression for achieving maximal therapeutic benefit, and to evaluate the consequences if selective targeting of the mutant HTT allele cannot be achieved, we propose using novel HD knock-in mouse models (HdhLacO-140Q and HdhLacO- 20Qhu mice) in which Lac operators have been inserted into the mouse HD locus (Hdh). After crossing these mice with a strain of transgenic mice ubiquitously expressing the Lac repressor (ß-actin-LacIR-tg), we can globally de-repress or repress mouse mutant huntingtin (Htt) expression, or both mutant and normal Htt expression at different ages by administering or withdrawing Isopropyl-ß-D-1-thiogalactopyranoside (IPTG) in their drinking water. In Aim 1, we will characterize the effect of repressing either mutant Htt or or both mutant and wild-type Htt expression at weaning, 3-, 6-, and 9-months of age in HdhLacO-140Q/+; ß-actin-LacIR tg, HdhLacO-140Q/LacO-20Qhu; ß-actin-LacIR tg, and control mice by characterizing their behavior, neuropathology, and htt expression levels at 2- to 24- months of age. In addition, to examine the effect of de-repressing mutant htt expression in an aged mouse (modeling discontinuation of a gene therapy in an older patient), IPTG will be administered to 12-month old HdhLacO-140Q/+; ß-actin-LacIR tg mice. Their phenotypes will be characterized at 12- to 24-months of age and compared to controls. In Aim 2, in order to identify potential biomarkers for evaluating the efficacy of a gene-silencing therapy, we propose to characterize by RNA-seq the proximal gene expression changes that occur in the cortex and striatum following repression of mutant htt or both mutant and wild-type htt expression at 3-, 6-, and 9- months of age. Validation of candidate genes will be performed first with brain tissue and cultured primary neurons. Validated genes will then be further examined using blood samples obtained from mice prior to and following mutant htt repression. Together, the results of these analyses should contribute to the design of future gene-silencing therapies for HD, and to our understanding of HD pathogenesis.