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

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

Finding balance in Huntington's disease: PIAS1 and huntingtin in protein homeostasis

The disruption of protein quality control networks that ensure proper folding and degradation of cellular proteins is likely central to pathology in Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and other “protein misfolding” diseases (La Spada and Taylor, 2010; Wilkinson et al., 2010). A detailed understanding of the proteostasis network components and their contributions to pathology are therefore crucial to developing improved therapeutic interventions. HD is caused by the abnormal expansion of a CAG repeat within the HD gene resulting in an expanded stretch of polyglutamines in the Huntingtin (HTT) protein (Group, 1993a). A key pathological feature is the accumulation of mutant HTT protein (mHTT) (Cisbani and Cicchetti, 2012; Zhao et al., 2016). Post-translational modifications of HTT, including SUMOylation and phosphorylation (Ehrnhoefer et al., 2011; Pennuto et al., 2009), appear to contribute to mechanisms underlying mHTT function, clearance, and accumulation (O'Rourke et al., 2013; Ochaba et al., 2016; Zhao et al., 2016).The work presented here represents an innovative conceptual shift to address questions fundamental to HD and other diseases where protein homeostasis is affected. This approach may therefore provide insight into a broad spectrum of protein misfolding disorders.. My dissertation utilized gain-of and loss-of-function approaches to query the importance of HTT function, modulation of PIAS1-regulatory networks, and SUMO-interaction motifs in the context of cell culture and in vivo mouse model systems to assess the impact on critical disease regulatory networks and HD pathogenesis. The precise mechanisms involved in the aforementioned have not yet been elucidated, nor has the PIAS and SUMO pathways been tested in vivo for its relevance to the following: the accumulation of neurodegenerative disease-causing proteins, neuroinflammation, protein clearance networks, or overall impact on disease phenotypes. My dissertation suggests that PIAS1 may link protein homeostasis and neuroinflammation in HD through a combination of modulating accumulation of toxic HMW species of HTT and compensating for dysfunctional inflammatory signaling cascades between neurons and microglia, potentially allowing improved flux through protein clearance pathways, of which HTT itself is likely involved in regulating

Finding balance in Huntington's disease: PIAS1 and huntingtin in protein homeostasis

The disruption of protein quality control networks that ensure proper folding and degradation of cellular proteins is likely central to pathology in Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and other “protein misfolding” diseases (La Spada and Taylor, 2010; Wilkinson et al., 2010). A detailed understanding of the proteostasis network components and their contributions to pathology are therefore crucial to developing improved therapeutic interventions. HD is caused by the abnormal expansion of a CAG repeat within the HD gene resulting in an expanded stretch of polyglutamines in the Huntingtin (HTT) protein (Group, 1993a). A key pathological feature is the accumulation of mutant HTT protein (mHTT) (Cisbani and Cicchetti, 2012; Zhao et al., 2016). Post-translational modifications of HTT, including SUMOylation and phosphorylation (Ehrnhoefer et al., 2011; Pennuto et al., 2009), appear to contribute to mechanisms underlying mHTT function, clearance, and accumulation (O'Rourke et al., 2013; Ochaba et al., 2016; Zhao et al., 2016).The work presented here represents an innovative conceptual shift to address questions fundamental to HD and other diseases where protein homeostasis is affected. This approach may therefore provide insight into a broad spectrum of protein misfolding disorders.. My dissertation utilized gain-of and loss-of-function approaches to query the importance of HTT function, modulation of PIAS1-regulatory networks, and SUMO-interaction motifs in the context of cell culture and in vivo mouse model systems to assess the impact on critical disease regulatory networks and HD pathogenesis. The precise mechanisms involved in the aforementioned have not yet been elucidated, nor has the PIAS and SUMO pathways been tested in vivo for its relevance to the following: the accumulation of neurodegenerative disease-causing proteins, neuroinflammation, protein clearance networks, or overall impact on disease phenotypes. My dissertation suggests that PIAS1 may link protein homeostasis and neuroinflammation in HD through a combination of modulating accumulation of toxic HMW species of HTT and compensating for dysfunctional inflammatory signaling cascades between neurons and microglia, potentially allowing improved flux through protein clearance pathways, of which HTT itself is likely involved in regulating

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

Microglial depletion prevents extracellular matrix changes and striatal volume reduction in a model of Huntington's disease.

Huntington's disease is associated with a reactive microglial response and consequent inflammation. To address the role of these cells in disease pathogenesis, we depleted microglia from R6/2 mice, a rapidly progressing model of Huntington's disease marked by behavioural impairment, mutant huntingtin (mHTT) accumulation, and early death, through colony-stimulating factor 1 receptor inhibition (CSF1Ri) with pexidartinib (PLX3397) for the duration of disease. Although we observed an interferon gene signature in addition to downregulated neuritogenic and synaptic gene pathways with disease, overt inflammation was not evident by microglial morphology or cytokine transcript levels in R6/2 mice. Nonetheless, CSF1Ri-induced microglial elimination reduced or prevented disease-related grip strength and object recognition deficits, mHTT accumulation, astrogliosis, and striatal volume loss, the latter of which was not associated with reductions in cell number but with the extracellular accumulation of chondroitin sulphate proteoglycans (CSPGs)-a primary component of glial scars. A concurrent loss of proteoglycan-containing perineuronal nets was also evident in R6/2 mice, and microglial elimination not only prevented this but also strikingly increased perineuronal nets in the brains of naïve littermates, suggesting a new role for microglia as homeostatic regulators of perineuronal net formation and integrity

PIAS1 Regulates Mutant Huntingtin Accumulation and Huntington’s Disease-Associated Phenotypes In Vivo

The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation