11 research outputs found
PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.
Correction for Morozko et al., PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington’s disease
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PIAS1 modulates the transcriptional landscape and DNA damage repair in Huntington’s disease
Disruption of protein homeostasis, leading to accumulation of insoluble high molecular weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated proteins, and transcriptional dysregulation are key features in Huntington’s disease (HD). Genetic modifiers contributing to HD age of onset have recently been identified and have critical roles in DNA damage repair (DDR) pathways. The mechanisms involved in DDR rely strongly on signaling cascades and post-translational modifications such as SUMO to maintain genomic integrity. Further, the Huntingtin (HTT) protein itself scaffolds DDR proteins. We previously showed that striatal reduction of the E3 SUMO ligase PIAS1 was neuroprotective and modulated disease associated pathologies including accumulation of mutant HTT in a mouse model of HD. However, the exact mechanistic contributions of PIAS1 towards HD pathogenesis have not yet been fully elucidated. To further evaluate PIAS1 function in the context of HD, knock-down was investigated in human patient medium spiny neurons differentiated from induced pluripotent stems cells and two disease mouse models. My findings suggest that PIAS1 functions as a key regulator of post-translational modification and protein homeostasis in HD neurons and mediates the functional activity of the transcription-coupled DNA damage repair complex in the striatum. Reduction of PIAS1 facilitated DNA repair, normalized aberrant transcriptional profiles related to synaptic function, and may stabilize the CAG-repeat within HTT. The results of this research provide the first mechanistic link between SUMOylation and DNA damage repair in the central nervous system. Specifically, they provide insight into how DNA damage repair pathways and post-translational modifications might contribute towards HD, and overall for targeting pathway mediators to restore homeostatic balance, with broad implications for HD and other neurodegenerative diseases
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PIAS1 modulates the transcriptional landscape and DNA damage repair in Huntington’s disease
Disruption of protein homeostasis, leading to accumulation of insoluble high molecular weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated proteins, and transcriptional dysregulation are key features in Huntington’s disease (HD). Genetic modifiers contributing to HD age of onset have recently been identified and have critical roles in DNA damage repair (DDR) pathways. The mechanisms involved in DDR rely strongly on signaling cascades and post-translational modifications such as SUMO to maintain genomic integrity. Further, the Huntingtin (HTT) protein itself scaffolds DDR proteins. We previously showed that striatal reduction of the E3 SUMO ligase PIAS1 was neuroprotective and modulated disease associated pathologies including accumulation of mutant HTT in a mouse model of HD. However, the exact mechanistic contributions of PIAS1 towards HD pathogenesis have not yet been fully elucidated. To further evaluate PIAS1 function in the context of HD, knock-down was investigated in human patient medium spiny neurons differentiated from induced pluripotent stems cells and two disease mouse models. My findings suggest that PIAS1 functions as a key regulator of post-translational modification and protein homeostasis in HD neurons and mediates the functional activity of the transcription-coupled DNA damage repair complex in the striatum. Reduction of PIAS1 facilitated DNA repair, normalized aberrant transcriptional profiles related to synaptic function, and may stabilize the CAG-repeat within HTT. The results of this research provide the first mechanistic link between SUMOylation and DNA damage repair in the central nervous system. Specifically, they provide insight into how DNA damage repair pathways and post-translational modifications might contribute towards HD, and overall for targeting pathway mediators to restore homeostatic balance, with broad implications for HD and other neurodegenerative diseases
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Fractionation for Resolution of Soluble and Insoluble Huntingtin Species.
The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution. For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts
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Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.
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
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Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice.
BackgroundBiochemical 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.ObjectiveProvide 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.MethodsEstablished 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.ResultsDetection 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.ConclusionsConformational 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
ILDR1 null mice, a model of human deafness DFNB42, show structural aberrations of tricellular tight junctions and degeneration of auditory hair cells
In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs
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Mutant Huntingtin Disrupts the Nuclear Pore Complex
Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets
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PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.
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