CHARACTERIZING THE HTT-HAP40-PARP1 INTERFACE, THE FUNCTIONAL CONSEQUENCES OF CDK5 MODIFICATION OF HUNTINGTIN, AND GENERATING AN ISOGENIC HD RPE1 CELL LIBRARY

Abstract

Huntington’s disease (HD) is an autosomal dominant late-onset neurodegenerative disease caused by a CAG expansion in the HTT gene. This expansion results in an expansion of the polyglutamine tract in huntingtin (HTT), thereby producing mutant huntingtin (mHTT). Production of mHTT disrupts numerous cell pathways, including several DNA damage repair (DDR) pathways. One such pathway is the poly-ADP ribose (PAR) response, where PAR polymerases (PARPs) synthesize and attach PAR chains to numerous substrates upon detection of DNA damage to orchestrate timely DDR. Evidence suggests HTT and PARP1, the most prominent PARP, directly interact, but there is no information regarding the structure of this interaction. Also, HTT is modified by numerous kinases during DDR, but the exact functional consequences of several of these modifications are unclear. Furthermore, studying the precise effects of the mHTT polyglutamine expansion has proven difficult due to the challenges of defining a structure for full-length HTT/mHTT and the lack of a biologically accurate isogenic HD model. This project aimed to overcome these limitations with the following aims: generating predictive models of the HTT-HAP40-PARP1 interface and validating them using biochemical methodologies, defining a new functional consequence of cyclin-dependent kinase 5 (CDK5) phosphorylating HTT on serines 1181 and 1201, and generating a library of isogenic HD cells using the immortalized retinal pigment epithelial cell 1 (RPE1) cell line. Although the findings of this project are limited, it describes powerful methodologies that can be used to investigate aspects of HTT biology and HD pathology that otherwise may have been deemed futile.