Print HUNTINGTIN BINDS PARP ELEVATED PAR IN HD HUNTINGTIN BINDS POLY ADP RIBOSE DYSREGULATION OF POLY ADP RIBOSE SIGNALING IN HD and its potential as a therapeutic target Tamara Maiuri1, Carlos Barba1, Rachel J Harding2, and Ray Truant1 1Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada 2Structural Genomics Consortium, University of Toronto, Toronto, Canada Berman/Topper HD Career Development Fellowship @tam_maiuri raytruantlab.ca maiurit@mcmaster.ca Tamara Maiuri Figure 1: Huntingtin interacts with PARylated proteins A. Degree of overlap between huntingtin-interacting proteins puri�ed from STHdh cells and a compiled list of PARylated proteins from three independently generated databases (Gagne et al, 2008; Zhang et al, 2013; Jungmichel et al, 2013). B. RPE1 cells were treated with 400 uM H 2 O 2 for 10 min and proteins crosslinked with 1% PFA. Huntingtin was immunoprecipitated with EPR5526 and associ- ated proteins separated by SDS-PAGE and immunoblotted with the indicated antibodies (MABE1016: PAR detection reagent). BACKGROUND • DNA repair pathways affect HD age at onset (GeM-HD Consortium, 2015) • Huntingtin acts as a scaffold for DNA repair proteins (Maiuri et al, 2017) • Poly ADP ribose (PAR) is a post-translational modi�cation generated in response to DNA damage 268 Huntingtin interactors PARylated proteins 126 830 A B Figure 2: Huntingtin PAR- binding motifs A. Huntingtin se- quence analysis revealed �ve motifs matching the PAR- binding concensus. B. PBM-1, 2, and 3 map to the surface of the huntingtin struc- ture (Guo et al, 2018), while PBM-4 is not exposed. PBM-5 is exposed on the C-terminal domain. C. Dot blot PAR overlay assay with full length puri- �ed huntingtin (Harding et al, 2019) (top), hunting- tin fragment 78-426 (middle), and PBM peptides (bottom). Puri�ed proteins were dotted onto nitrocellulose then overlaid with 0.2 uM PAR (Trevigen). After washing, anti-PAR western was per- formed with PAR detection reagent MABE1016. A B C Figure 3: Huntingtin co-localizes with PAR RPE1 cells were treated with 10 uM PARG inhibitor for 15 min followed by 100 mM KBrO 3 for 30 min. Soluble proteins were ex- tracted with 0.2% Triton X-100 for 2 min on ice followed by �xation and staining with the indicated antibodies. Cells were imaged by super-resolution microscopy (SR-SIM). A. PAR co-localizes with SC35-positive nuclear speckles. B. Huntingtin co-localizes with PAR at speckles and redistributes to sites of PAR production upon oxidative stress. C. Co-localization of nuclear signals measured by Pearson correlation. n=1, 10 cells per condition. A B C Figure 4: Huntingtin interacts with Poly ADP ribose polymerase A. RPE1 cells were treated with 400 uM H 2 O 2 for 10 min and proteins crosslinked with 1% PFA. Huntingtin was immunoprecipitated with EPR5526 (Abcam) and associated proteins separated by SDS-PAGE and im- munoblotted with anti-PARP (BD Biosciences) and anti-huntingtin (MAB2166, Millipore). B. Puri�ed huntingtin (Harding et al, 2019) and PARP (Trevigen) were incubated in the presence of acti- vated DNA and NAD+. PARP was immunoprecipitated with anti-PARP antibody (BD Biosciences). Reactions were separated by SDS-PAGE and immunoblotted as in A. BACKGROUND • Levels of DNA damage are elevated in HD cells and tissues (Maiuri et al, 2017; Askeland et al, 2018; Castaldo et al, 2019) • Unrepaired DNA damage leads to prolonged activation of PARPs and overproduction of PAR • Unrelenting PAR production causes ATP depletion, mitochondrial failure and energy crisis • PAR acts as a mediator of cell death through parthanatos IS PARP INHIBITION A VIABLE THERAPEUTIC STRATEGY? Figure 5: Elevated PAR levels in HD patient �broblasts TruHD immortalized HD patient and control �broblasts (Hung et al, 2018) were pre-treated with 10 μM PARG inhibitor for 15 min followed by 100 mM KBrO 3 for 30 min. Soluble proteins were ex- tracted with 0.2% Triton X-100 for 2 min on ice, followed by �xation and staining with MABE1016 PAR detection reagent. Nuclei were identified as primary objects in CellProfiler (Carpenter et al, 2006) using Hoechst staining, then pixel intensity of the PAR staining within nuclei was calculated and the mean intensity recorded for each image. Ten images per well were captured, representing 750-1000 cells per experiment. The experiment was repeated 3 times for TruHD-Q43Q17 and TruHD-Q40Q50 cells, and four times for TruHD-Q21Q18 cells. P-values were calculated using Tukey’s test (****p<0.0001). MORE PRECLINICAL DATA NEEDED • PARP inhibition is beneficial in HD mouse model (Cardinale et al, 2015; Paldino et al, 2017) • High PAR levels in HD patient fibroblasts (Fig 5) • Are PAR levels high in CSF from HD patients? • Does PARP inhibition rescue phenotypes in clinically relevant models? REPURPOSING PARP INHIBITOR DRUGS • Veliparib, niraparib cross the blood brain barrier • Risk of genotoxicity, however PARP inhibitors are generally protective in postmitotic cells (Berger et al, 2018) References Askeland, G. et al., 2018. Increased nuclear DNA damage precedes mitochondrial dysfunction in peripheral blood mononuclear cells from Huntington’s disease patients. Scienti�c reports, 8(1), p.9817. Berger, N.A. et al., 2018. Opportunities for the repurposing of PARP inhibitors for the therapy of non-oncological diseases. British journal of pharmacology, 175(2), pp.192–222. Cardinale, A. et al., 2015. PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease. 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