ABSTRACT
DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
Subject(s)
DNA Repair Enzymes/genetics , DNA Repair , DNA/genetics , Huntingtin Protein/genetics , Huntington Disease/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Inhibitors of Activated STAT/genetics , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/genetics , Animals , Cell Differentiation , DNA/metabolism , DNA Damage , DNA Repair Enzymes/metabolism , Disease Models, Animal , Female , Humans , Huntingtin Protein/metabolism , Huntington Disease/metabolism , Huntington Disease/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/metabolism , Neurons/pathology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/pathology , Primary Cell Culture , Protein Inhibitors of Activated STAT/antagonists & inhibitors , Protein Inhibitors of Activated STAT/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Transcription, GeneticABSTRACT
Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1+ neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.
Subject(s)
Huntington Disease/pathology , Induced Pluripotent Stem Cells/pathology , Neurons/pathology , Wnt Signaling Pathway , Adult , Age of Onset , Cell Cycle/genetics , Cell Differentiation/genetics , Cells, Cultured , Epigenesis, Genetic , Humans , Huntington Disease/genetics , Mitosis , Neostriatum/pathology , Neural Stem Cells/metabolism , Transcription Factors/metabolism , Transcriptome/genetics , Up-Regulation/geneticsABSTRACT
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Subject(s)
Alleles , Huntingtin Protein/genetics , Huntington Disease/therapy , Mutation , Transcription, Genetic , Zinc Fingers , Animals , Cells, Cultured , Disease Models, Animal , Female , Humans , Huntington Disease/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Neuroprotection , Trinucleotide RepeatsABSTRACT
BACKGROUND: Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions. OBJECTIVE: Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation. METHODS: Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers. RESULTS: Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods. CONCLUSIONS: Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.
