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
Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKß, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKß on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKß phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKß to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKß knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKß in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKß knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKß is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKß is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKß may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.
Subject(s)
Huntington Disease , I-kappa B Kinase , Animals , Autophagy/genetics , Corpus Striatum/cytology , Corpus Striatum/pathology , Disease Models, Animal , Disease Progression , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/metabolism , Huntington Disease/pathology , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Male , Mice , Mice, Knockout , Microglia/cytology , Microglia/pathology , Phosphorylation/geneticsABSTRACT
BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder associated with aging, caused by an expanded polyglutamine (polyQ) repeat within the Huntingtin (HTT) protein. In HD, degeneration of the striatum and atrophy of the cortex are observed while cerebellum is less affected. OBJECTIVE: To test the hypothesis that HTT protein levels decline with age, which together with HTT mutation could influence disease progression. METHODS: Using whole brain cell lysates, a unique method of SDS-PAGE and western analysis was used to quantitate HTT protein, which resolves as a monomer and as a high molecular weight species that is modulated by the presence of transglutaminase 2. HTT levels were measured in striatum, cortex and cerebellum in congenic homozygous Q140 and HdhQ150 knock-in mice and WT littermate controls. RESULTS: Mutant HTT in both homozygous knock-in HD mouse models and WT HTT in control striatal and cortical tissues significantly declined in a progressive manner over time. Levels of mutant HTT in HD cerebellum remained high during aging. CONCLUSIONS: A general decline in mutant HTT levels in striatum and cortex is observed that may contribute to disease progression in homozygous knock-in HD mouse models through reduction of HTT function. In cerebellum, sustained levels of mutant HTT with aging may be protective to this tissue which is less overtly affected in HD.
