Identifying Huntingtin Interactions in DNA Damage Repair

This project, funded by the HDSA Berman/Topper HD Career Development Fellowship, expands on the finding that reactive oxygen species (ROS) stress promotes the interaction of huntingtin with DNA repair proteins (Maiuri, et al. Hum Mol Genet. 2017 Jan 15;26(2):395-406.) Using immunoprecipitation and mass spectrometry, we have now identified a list of proteins that interact with huntingtin and overlap significantly with databases of proteins modified by poly ADP ribose (PAR). This finding has led to two hypotheses currently under investigation: that huntingtin binds PAR, and that PAR signaling is dysregulated in HD. The findings of this project are reported in real time through the Truant lab blog and open notebook entries deposited to the Truant Lab Zenodo Community.

Spinocerebellar Ataxia Type 1: Role of Ataxin-1 in DNA Damage Repair

Looking at the role of another CAG expansion disease protein, ataxin-1 and it’s role in DNA damage repair in Spinocerebellar Ataxia 1 (SCA1). This project revisits previous work from the lab as it is apparent ataxin-1 and huntingtin both have roles in DNA damage repair (Ross, C.A. and Truant, R. DNA repair: A unifying mechanism in neurodegeneration. Jan 2017.) and HD GWAS pathways have been found to be relevant for age of onset in SCA1. As exomic sequencing is more common in the clinic, more spontaneous age-onset ataxias are being identified as mutations in DNA repair factors.

Spinocerebellar Ataxia Type 7: Cellular Bioenergetic Defects

Understanding how cellular bioenergetics are disturbed in neurodegenerative disorders and how this can lead to dysfunction and disease.

Targeting N17 Phosphorylation as an HD Therapeutic

This project began with a focus on using high content screening and unbiased
computer analysis to identify compounds that affect the phosphorylation state of
the huntingtin protein. Increasing phosphorylation of huntingtin in the context of
HD is a known protective modification, so the goal of this venture was to identify
potential protective compounds or novel pathways for drug targeting. Out of this
initial goal, several compounds and pathways of interest were identified and the
follow-up validation utilized a number of classic biochemical techniques as well as
biophotonic imaging. During the course of this follow-up, the nature of the
compounds of interest from the various screens have led this project to touch on a
number of areas including: the role of huntingtin in DNA damage, novel pathways of
huntingtin phosphorylation, and the role of huntingtin in oxidative stress.

Regulation of Huntingtin Localization by HMGB1

Regulation of huntingtin localization to and from the nucleus by a small protein called high mobility group box 1 (HMGB1). Elucidating how and where the interaction between huntingtin and HMGB1 takes place and how this relationship changes in the presence of oxidative stress so that this knowledge can become applicable in the context of mutant huntingtin and HD.

Patient-Derived Immortalized TruHD Cells

Characterizing novel cell lines for modelling Huntington’s Disease to uncover disease phenotypes at the cellular level. Most cell and mouse models are generated with irrelevantly long CAG repeats that may overlook the importance of researching why some patients with the same CAG mutation length develop disease much sooner than other patients. These cell lines are based on patient fibroblasts to address subtle aspects of the disease relevant to humans and not more obvious phenotypes from mouse models that use extreme CAG repeat expansions. We are working on identifying novel phenotypes between cells from different HD patients, not just between HD and control patients, to gain insight on early disease mechanisms that are relevant to studying the huntingtin protein’s role in cellular stress response, DNA damage and cell death pathways.

FLIM-FRET Methodology

Creating fluorescent protein coated microbeads to calibrate FLIM-FRET microscope to collect data on the lifetime of specific fluorescent tags. Using standardized data per a specific cellular condition, we can monitor in vitro cell models to gain information on protein protein interactions in HD and SCA1 by observing their modified fluorescent lifetime.