Potential function for the Huntingtin protein as a scaffold for selective autophagy

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51–like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.Hereditary Disease Foundation (U.S.)Cure Huntington’s Disease Initiative, Inc.Fox Family Foundatio

SUMO-2 and PIAS1 modulate insoluble mutant Huntingtin protein accumulation

A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression

SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

SUMMARY A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models.

Most neurodegenerative disorders are associated with accumulation of disease-relevant proteins. Among them, Huntington disease (HD) is of particular interest because of its monogenetic nature. HD is mainly caused by cytotoxicity of the defective protein encoded by the mutant Huntingtin gene (HTT). Thus, lowering mutant HTT protein (mHTT) levels would be a promising treatment strategy for HD. Here we report two kinases HIPK3 and MAPK11 as positive modulators of mHTT levels both in cells and in vivo. Both kinases regulate mHTT via their kinase activities, suggesting that inhibiting these kinases may have therapeutic values. Interestingly, their effects on HTT levels are mHTT-dependent, providing a feedback mechanism in which mHTT enhances its own level thus contributing to mHTT accumulation and disease progression. Importantly, knockout of MAPK11 significantly rescues disease-relevant behavioral phenotypes in a knockin HD mouse model. Collectively, our data reveal new therapeutic entry points for HD and target-discovery approaches for similar diseases

The ubiquitin conjugating enzyme Ube2W regulates solubility of the Huntington's disease protein, huntingtin

Huntington's disease (HD) is caused by a CAG repeat expansion that encodes a polyglutamine (polyQ) expansion in the HD disease protein, huntingtin (HTT). PolyQ expansion promotes misfolding and aggregation of mutant HTT (mHTT) within neurons. The cellular pathways, including ubiquitin-dependent processes, by which mHTT is regulated remain incompletely understood. Ube2W is the only ubiquitin conjugating enzyme (E2) known to ubiquitinate substrates at their amino (N)-termini, likely favoring substrates with disordered N-termini. By virtue of its N-terminal polyQ domain, HTT has an intrinsically disordered amino terminus. In studies employing immortalized cells, primary neurons and a knock-in (KI) mouse model of HD, we tested the effect of Ube2W deficiency on mHTT levels, aggregation and neurotoxicity. In cultured cells, deficiency of Ube2W activity markedly decreases mHTT aggregate formation and increases the level of soluble monomers, while reducing mHTT-induced cytotoxicity. Consistent with this result, the absence of Ube2W in HdhQ200 KI mice significantly increases levels of soluble monomeric mHTT while reducing insoluble oligomeric species. This study sheds light on the potential function of the non-canonical ubiquitin-conjugating enzyme, Ube2W, in this polyQ neurodegenerative disease

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

IKKβ slows Huntington’s disease progression in R6/1 mice

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 IKKβ, 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 IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ 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 IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset