The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

Huntington disease (HD) is a neurological disorder caused by polyglutamine expansions in mutated Huntingtin (mHtt) proteins, rendering them prone to form inclusion bodies (IB). We report that in yeast, such IB formation is a factor-dependent process subjected to age-related decline. A genome-wide, high-content imaging approach, identified the E3 ubiquitin ligase, Ltn1 of the ribosome quality control complex (RQC) as a key factor required for IB formation, ubiquitination, and detoxification of model mHtt. The failure of ltn1∆ cells to manage mHtt was traced to another RQC component, Tae2, and inappropriate control of heat shock transcription factor, Hsf1, activity. Moreover, super-resolution microscopy revealed that mHtt toxicity in RQC-deficient cells was accompanied by multiple mHtt aggregates altering actin cytoskeletal structures and retarding endocytosis. The data demonstrates that spatial sequestration of mHtt into IBs is policed by the RQC-Hsf1 regulatory system and that such compartmentalization, rather than ubiquitination, is key to mHtt detoxification.

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

Studies have suggested that amyloid precursor protein (APP) regulates synaptic homeostasis, but the evidence has not been consistent. In particular, signaling pathways controlling APP transport to the synapse in axons and dendrites remain to be identified. Having previously shown that Huntingtin (HTT), the scaffolding protein involved in Huntington's disease, regulates neuritic transport of APP, we used a microfluidic corticocortical neuronal network-on-a-chip to examine APP transport and localization to the pre- and post-synaptic compartments. We found that HTT, upon phosphorylation by the Ser/Thr kinase Akt, regulates APP transport in axons but not dendrites. Expression of an unphosphorylatable HTT decreased axonal anterograde transport of APP, reduced presynaptic APP levels, and increased synaptic density. Ablating in vivo HTT phosphorylation in APPPS1 mice, which overexpress APP, reduced presynaptic APP levels, restored synapse number and improved learning and memory. The Akt-HTT pathway and axonal transport of APP thus regulate APP presynaptic levels and synapse homeostasis.

Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand our understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.

Huntington's disease (HD) is initially characterized by an inability to suppress unwanted movements, a deficit attributable to impaired synaptic activation of striatal indirect pathway spiny projection neurons (iSPNs). To better understand the mechanisms underlying this deficit, striatal neurons in ex vivo brain slices from mouse genetic models of HD were studied using electrophysiological, optical and biochemical approaches. Distal dendrites of iSPNs from symptomatic HD mice were hypoexcitable, a change that was attributable to increased association of dendritic Kv4 potassium channels with auxiliary KChIP subunits. This association was negatively modulated by TrkB receptor signaling. Dendritic excitability of HD iSPNs was rescued by knocking-down expression of Kv4 channels, by disrupting KChIP binding, by restoring TrkB receptor signaling or by lowering mutant-Htt (mHtt) levels with a zinc finger protein. Collectively, these studies demonstrate that mHtt induces reversible alterations in the dendritic excitability of iSPNs that could contribute to the motor symptoms of HD.

Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

Loss of cellular homeostasis has been implicated in the etiology of several neurodegenerative diseases (NDs). However, the molecular mechanisms that underlie this loss remain poorly understood on a systems level in each case. Here, using a novel computational approach to integrate dimensional RNA-seq and in vivo neuron survival data, we map the temporal dynamics of homeostatic and pathogenic responses in four striatal cell types of Huntington's disease (HD) model mice. This map shows that most pathogenic responses are mitigated and most homeostatic responses are decreased over time, suggesting that neuronal death in HD is primarily driven by the loss of homeostatic responses. Moreover, different cell types may lose similar homeostatic processes, for example, endosome biogenesis and mitochondrial quality control in Drd1-expressing neurons and astrocytes. HD relevance is validated by human stem cell, genome-wide association study, and post-mortem brain data. These findings provide a new paradigm and framework for therapeutic discovery in HD and other NDs.

Neurotransmitters are released at synapses by synaptic vesicles (SVs), which originate from SV precursors (SVPs) that have traveled along the axon. Because each synapse maintains a pool of SVs, only a small fraction of which are released, it has been thought that axonal transport of SVPs does not affect synaptic function. Here, studying the corticostriatal network both in microfluidic devices and in mice, we find that phosphorylation of the Huntingtin protein (HTT) increases axonal transport of SVPs and synaptic glutamate release by recruiting the kinesin motor KIF1A. In mice, constitutive HTT phosphorylation causes SV over-accumulation at synapses, increases the probability of SV release, and impairs motor skill learning on the rotating rod. Silencing KIF1A in these mice restored SV transport and motor skill learning to wild-type levels. Axonal SVP transport within the corticostriatal network thus influences synaptic plasticity and motor skill learning.

Dynamic changes in protein S-palmitoylation are critical for regulating protein localization and signaling. Only two enzymes - the acyl-protein thioesterases APT1 and APT2 - are known to catalyze palmitate removal from cytosolic cysteine residues. It is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. Using a dual pulse-chase strategy comparing palmitate and protein half-lives, we found knockdown or inhibition of APT1 and APT2 blocked depalmitoylation of Huntingtin, but did not affect palmitate turnover on postsynaptic density protein 95 (PSD95) or N-Ras. We used activity profiling to identify novel serine hydrolase targets of the APT1/2 inhibitor Palmostatin B, and discovered that a family of uncharacterized ABHD17 proteins can accelerate palmitate turnover on PSD95 and N-Ras. ABHD17 catalytic activity is required for N-Ras depalmitoylation and re-localization to internal cellular membranes. Our findings indicate that the family of depalmitoylation enzymes may be substantially broader than previously believed.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Catching primal functional changes in early, 'very far from disease onset' (VFDO) stages of Huntington's disease is likely to be the key to a successful therapy. Focusing on VFDO stages, we assessed neuronal microcircuits in premanifest Hdh150 knock-in mice. Employing in vivo two-photon Ca2+ imaging, we revealed an early pattern of circuit dysregulation in the visual cortex - one of the first regions affected in premanifest Huntington's disease - characterized by an increase in activity, an enhanced synchronicity and hyperactive neurons. These findings are accompanied by aberrations in animal behavior. We furthermore show that the antidiabetic drug metformin diminishes aberrant Huntingtin protein load and fully restores both early network activity patterns and behavioral aberrations. This network-centered approach reveals a critical window of vulnerability far before clinical manifestation and establishes metformin as a promising candidate for a chronic therapy starting early in premanifest Huntington's disease pathogenesis long before the onset of clinical symptoms.

Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in HttQ111 mice, of Hdac2 or Hdac3 in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with Hdac2 knockout decreasing nuclear huntingtin pathology. Hdac2 knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the HttQ111 allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded Htt allele and Hdac2 with implications for targeting transcriptional dysregulation in HD.

Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntington's disease (HD), we profiled the gene expression alterations of Drosophila expressing human mutant Huntingtin (mHTT) in either glia or neurons and compared these changes to what is observed in HD human and HD mice striata. A large portion of conserved genes are concordantly dysregulated across the three species; we tested these genes in a high-throughput behavioral assay and found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. To our surprise, reducing dNRXN3 function in glia was sufficient to improve the phenotype of flies expressing mHTT in neurons, suggesting that mHTT's toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD.

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

Previously, we reported that ATXN1 oligomers are the primary drivers of toxicity in Spinocerebellar ataxia type 1 (SCA1; Lasagna-Reeves et al., 2015). Here we report that polyQ ATXN1 oligomers can propagate locally in vivo in mice predisposed to SCA1 following intracerebral oligomeric tissue inoculation. Our data also show that targeting these oligomers with passive immunotherapy leads to some improvement in motor coordination in SCA1 mice and to a modest increase in their life span. These findings provide evidence that oligomer propagation is regionally limited in SCA1 and that immunotherapy targeting extracellular oligomers can mildly modify disease phenotypes.