Sheng Zhang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Huntington's disease (HD) is caused by an abnormal expansion of a polyglutamine (polyQ) tract within the Huntingtin (Htt) protein. Recent studies have demonstrated that normal functions of Htt protein play a critical role in determining the final disease outcome. However, functional studies on wildtype Htt have been hampered by the early lethality of murine Htt mutant models and by the unusual large size of Htt protein. Despite the identification of large number of Htt interacting proteins (HIPs), the normal cellular roles of Htt remain poorly defined, which is becoming a major obstacle in studying the pathogenesis of HD and developing rational therapies to treat this devastating disease. Characterizing a Htt homolog in a simple, genetically tractable system will complement the established mammalian models. Unlike in C. elegans or yeast, a single Htt homolog exists in Drosophila (dhtt), allowing us to characterize this Htt family protein in this well-studied genetic model system. In preliminary studies, we have established a null-mutant for dhtt, the first mutant allele for a Htt family gene in an invertebrate model organism. We found that contrary to the results from an earlier RNAi-based study, dhtt is dispensable for Drosophila development, but removing endogenous dhtt can significantly accelerate the neurodegenerative phenotypes associated with a Drosophila model of polyQ-expanded Htt toxicity, supporting that normal function of Htt is important for HD pathogenesis; Furthermore, dhtt is required for maintaining the mobility and long-term survival of adult animals, and its absence affects axonal terminal complexity in the adult brain. These studies allow us to use the powerful genetic system and abundant experimental tools in Drosophila to carry out more detailed characterization of the dhtt null mutant and perform systematic evaluation of potential functional interactions between dhtt and HIPs homologues. Outcome of this research will provide critical insights into the normal function of Htt and ultimately the mechanisms underlying HD. In this application, we propose the following Specific Aims: (1) Characterize dhtt-associated phenotypes by ultrastructural and gene expression analyses, and use established in vivo assays in Drosophila to directly test proposed cellular roles of Htt in axonal vesicle transport and endocytosis; (2) Use a genome-tagging approach to establish a versatile toolbox for in vivo analysis of dHtt protein, and perform deletion study to map the functional domains in dHtt protein; (3) Assess the physiological relevance of mammalian HIPs by testing genetic interactions between their Drosophila homologues and dhtt, and use the Tandem Affinity Purification (TAP)-based approach to directly isolate Drosophila HIPs.

PROJECT ABSTRACT The Hsp110 molecular chaperone is increasingly being recognized as a potent regulator of amyloidogenesis as well as a potential pathogenic modifier for multiple protein misfolding disorders including cystic fibrosis, Alzheimer?s disease and Huntington?s disease (HD). However, few systematic studies have been carried out on metazoan Hsp110 to examine its physiological roles, particularly in long-lived neurons that are more susceptible to protein misfolding and aggregate formation, largely due to lack of suitable metazoan model. In addition, Hsp110 has been determined at the biochemical level to function both as a chaperone ?holdase? and as a Hsp70 nucleotide exchange factor (NEF). However, how these two distinct activities contribute to its neuronal protective role is unknown. In this R21 application, we propose to generate Drosophila lines with designed mutations in the sole fly Hsp110 ortholog (dHsp110) that specifically abolish holdase or NEF activities, and use established Drosophila HD models to test the hypothesis that Hsp110 acts as an activator of Hsp70 cycling in concert with Hsp40, and/or as a stand-alone chaperone holdase to sequester amyloidogenic proteins and prevent aggregation and associated toxicity. Completion of this project will establish the feasibility of future pharmacological exploitation of Hsp110 in the nervous system to combat morbidity and mortality arising from proteopathies in the aging population.

Huntington's disease (HD) is caused by an abnormal expansion of the glutamine tract (polyQ) in Huntingtin (HTT). A clear understanding on how endogenous HTT is regulated in vivo is critical both for elucidating HD etiology and for identifying effective drug targets. HTT has numerous reported HTT associated partners (HAPs) and is functionally implicated in a growing list of cellular processes. However, little is known how HTT itself is regulated and whether such regulation is altered in HD. We previously characterized the HTT homolog (dHtt) in model organism Drosophila. Given the significant functional conservation of HTT from the fly to mammals, we hypothesized that the core regulators of HTT likely are among the numerous known HAPs and should also be conserved in Drosophila. In a proteomic study for such conserved central regulators of HTT in Drosophila, we isolated dHap40, the fly homolog of HAP40, as the strongest dHtt interactor. Importantly, converging evidence from studies in multiple species all support that in vivo HTT protein normally exists in a complex with HAP40, and HAP40 binding stabilizes the conformation of HTT. Further, in samples from HD patients, a ~10- fold increase of the levels of endogenous HAP40 were observed as compared to controls. However, despite these findings, by now there is no reported functional study of HAP40 in any physiological settings, and its effect on HTT's normal functions and mutant HTT toxicity remains unclear. Our preliminary studies support the significantly conserved physical and functional interactions between HTT and HAP40, implying a highly important regulatory relationship that constrains their co-evolution from flies to humans. Our findings not only establish Drosophila as a relevant genetic model to study the physiological roles of HAP40, but also lead to our hypothesis that HAP40 is a conserved central regulator of HTT and potentially a critical modulator of mutant HTT toxicity. Using established assays and HD models in Drosophila and cultured mammalian cells, we will systematically test this hypothesis. In Aim 1, we will carry out a comprehensive phenotypic analyses of dhap40 gene and test its genetic interactions with dhtt, so as to obtain a first systematic evaluation of HAP40 in a physiological setting and clarify its relationship with HTT at whole-animal level. In Aim 2, we will systematically test whether HAP40 is a central regulator of HTT's subcellular dynamics and its diverse cellular functions, so as to elucidate its relationship with HTT at molecular and cellular levels. In Aim 3, taking advantage of the well- established HD models in Drosophila and mammalian neurons, we will rigorously interrogate the role of HAP40 on mutant HTT toxicity. From these multidisciplinary studies, we will obtain a first comprehensive evaluation on the physiological functions of HAP40, its effect on endogenous HTT functions and on HD pathogenesis. The results potentially lay foundation on novel therapeutic avenues against HD via HAP40.