A closer look at the hits generates 2 new hypotheses

Blog post by Dr. Tamara Maiuri

Forgive me readers for I have been busy, it has been 3 months since my last blog post. During this time, we published a very exciting story identifying a lead compound and how we think it works in HD. Also during this time, I have gone through the list of huntingtin interactors identified in my HDSA-funded project to find drug targets that are relevant to the process of DNA repair. We now have some interesting results!

As explained in my last post, the original aim to identify huntingtin interactors from human cells was not technically feasible and mouse cells were used instead. Several hundred protein-protein interactions were detected, then the list was further refined by only considering high and medium confidence hits. We now have a list of 314 proteins that interact with huntingtin reproducibly across biological replicates. See the end of this post for the lists of ROS-dependent interactors (Tables 1-3), proteins that interacted with huntingtin only in untreated cells (Table 4), and proteins found to be modified by poly ADP-ribose (PARylated proteins; Table 5). These tables have also been deposited to Zenodo.

Indeed, the most notable finding of the ROS-dependent interactome analysis was the high degree of overlap with datasets of “PARylated” proteins. PAR is of interest to us because it is generated in response to DNA damage, and acts to recruit DNA repair proteins to damage sites.

This result led us to pursue two hypotheses with the aim of identifying drug targets relevant to DNA repair:

Huntingtin is a PAR-binding protein: Huntingtin may use PAR binding to interact with PARylated proteins. If so, this is likely to be the mechanism by which huntingtin interacts with chromatin and assembles DNA repair factors in its role as a scaffold, and this may be dysregulated in HD.

PAR signaling is dysregulated in HD: Regardless of whether huntingtin physically binds PAR, it is still possible that we have uncovered a previously unexplored aspect of HD pathology: there is strong genetic evidence that DNA repair genes influence disease [1-3], huntingtin participates in the process of DNA repair [4], and elevated levels of damaged DNA are common to HD tissues and models [4-8].

In response to DNA damage, PAR chains are the first scaffold generated for DNA repair factor assembly: a sticky web bound by proteins involved in DNA repair. So it’s possible that in HD, excess DNA damage accumulates due to sub-optimal huntingtin function in the repair process, leading to Poly ADP-Ribose Polymerase (PARP) hyperactivation and elevated PAR levels.

High PAR levels could not only interfere with huntingtin function if it binds PAR, but also cause an “energy crisis” in high-energy-consuming neurons [9-11]. In fact, energy deficits have been frequently observed across HD models and in HD patients [12]. Similarly, PARP hyperactivation is linked to cerebellar ataxia [13], and PARP inhibition has been reported to improve phenotypes in a HD mouse model, although by a different mechanism [14,15].

In the coming blog posts, I will share preliminary data that suggests huntingtin can bind PAR in a test tube, and that PAR is at least partially responsible for huntingtin recruitment to chromatin. We have also detected increased amounts of PAR and of chromatin-bound huntingtin in cells from an HD patient. Stay tuned!

References:

1. Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Identification of Genetic Factors that Modify Clinical Onset of Huntington’s Disease. Cell. 2015;162: 516–526.
2. Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79: 983–990.
3. Moss DJH, Pardiñas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol. 2017;16: 701–711.
4. Maiuri T, Mocle AJ, Hung CL, Xia J, van Roon-Mom WMC, Truant R. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2017;26: 395–406.
5. Bogdanov MB, Andreassen OA, Dedeoglu A, Ferrante RJ, Beal MF. Increased oxidative damage to DNA in a transgenic mouse model of Huntington’s disease. J Neurochem. 2001;79: 1246–1249.
6. Kovtun IV, Liu Y, Bjoras M, Klungland A, Wilson SH, McMurray CT. OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature. 2007;447: 447–452.
7. Enokido Y, Tamura T, Ito H, Arumughan A, Komuro A, Shiwaku H, et al. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol. 2010;189: 425–443.
8. Askeland G, Dosoudilova Z, Rodinova M, Klempir J, Liskova I, Kuśnierczyk A, et al. Increased nuclear DNA damage precedes mitochondrial dysfunction in peripheral blood mononuclear cells from Huntington’s disease patients. Sci Rep. 2018;8: 9817.
9. Morales J, Li L, Fattah FJ, Dong Y, Bey EA, Patel M, et al. Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for targeting in cancer and other diseases. Crit Rev Eukaryot Gene Expr. 2014;24: 15–28.
10. Andrabi SA, Umanah GKE, Chang C, Stevens DA, Karuppagounder SS, Gagné J-P, et al. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis. Proc Natl Acad Sci U S A. 2014;111: 10209–10214.
11. Fouquerel E, Goellner EM, Yu Z, Gagné J-P, Barbi de Moura M, Feinstein T, et al. ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion. Cell Rep. 2014;8: 1819–1831.
12. Dickey AS, La Spada AR. Therapy development in Huntington disease: From current strategies to emerging opportunities. Am J Med Genet A. 2017; doi:10.1002/ajmg.a.38494
13. Hoch NC, Hanzlikova H, Rulten SL, Tétreault M, Komulainen E, Ju L, et al. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2017;541: 87–91.
14. Cardinale A, Paldino E, Giampà C, Bernardi G, Fusco FR. PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease. PLoS One. 2015;10: e0134482.
15. Paldino E, Cardinale A, D’Angelo V, Sauve I, Giampà C, Fusco FR. Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington’s Disease. Front Neuroanat. 2017;11: 61.