Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease HD is a progressive neurodegenerative disorder caused by a single gene mutation, the CAG repeat expansion in Huntingtin (Htt). The best-validated therapeutic target in HD remains Htt itself, and RNAi approaches are under development to lower mutant Htt in HD patients. However, these approaches have daunting obstacles of delivery large molecules to the CNS. Small molecule therapeutics remains an important alternative. We have identified near 40 PTMs (phosphorylation and acetylation) on endogenous Htt from human and mouse brain using quantitative proteomics. We find that the PTMs are arranged in clusters, and we have initial evidence for crosstalk among certain sites in these clusters.. Furthermore, we find that alteration of the residues for several of the PTMs abrogates mutant Htt neuronal toxicity, showing that PTMs are modulators of mutant Htt toxicity. These data indicate that identification of the enzymes catalyzing these PTMs will have potential for yielding defined molecular targets for disease-modifying therapy for HD. We now propose the next series of steps in these studies, in order to identify additional PTMs, and to identify enzymes that catalyze modifications, as potential defined molecular targets for rational therapeutics for HD. We will take advantage now of our newly developed HD iPSC-derived immortalized striatal precursor cell lines. In Aim 1, we will identify novel PTMs on Htt using our novel striatal precursor cell lines (SPNs) derived from HD iPS cells. These can be differentiated into neurons with a medium-spiny neuron phenotype. We will generate PTM-specific antibodies to high-priority PTMs, in order to evaluate localization in cellular compartments using immunofluorescence, and cell fractionation followed by Western blot, in SPNs and human brain. In Aim 2 we will identify kinases and other modifying enzymes. In Aim 3 we will confirm the role of PTMs and kinases in HD cellular pathogenesis and their validation as therapeutic targets. We will perform CRISPR/Cas9 PTM alterations for highly-ranked functional PTMs, as well as CRISPR/Cas9 knock-out of most relevant kinases in striatal precursor neuron model in collaboration with Gene Edit BioLab and Xiao and Shihua Li, who are expert at gene editing in relation to HD. Taken together, these studies will further elucidate the basic biology of mutant Htt. The identification of modifying enzymes has the potential to provide defined molecular targets for preclinical therapeutic studies.