The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

Brain-Periphery Interactions in Huntington's Disease: Mediators and Lifestyle Interventions.

Prominent pathological features of Huntington's disease (HD) are aggregations of mutated Huntingtin protein (mHtt) in the brain and neurodegeneration, which causes characteristic motor (such as chorea and dystonia) and non-motor symptoms. However, the numerous systemic and peripheral deficits in HD have gained increasing attention recently, since those factors likely modulate disease progression, including brain pathology. While whole-body metabolic abnormalities and organ-specific pathologies in HD have been relatively well described, the potential mediators of compromised inter-organ communication in HD have been insufficiently characterized. Therefore, we applied an exploratory literature search to identify such mediators. Unsurprisingly, dysregulation of inflammatory factors, circulating mHtt, and many other messenger molecules (hormones, lipids, RNAs) were found that suggest impaired inter-organ communication, including of the gut-brain and muscle-brain axis. Based on these findings, we aimed to assess the risks and potentials of lifestyle interventions that are thought to improve communication across these axes: dietary strategies and exercise. We conclude that appropriate lifestyle interventions have great potential to reduce symptoms and potentially modify disease progression (possibly via improving inter-organ signaling) in HD. However, impaired systemic metabolism and peripheral symptoms warrant particular care in the design of dietary and exercise programs for people with HD.

TRIM37 is a primate-specific E3 ligase for Huntingtin and accounts for the striatal degeneration in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by preferential neuronal loss in the striatum. The mechanism underlying striatal selective neurodegeneration remains unclear, making it difficult to develop effective treatments for HD. In the brains of nonhuman primates, we examined the expression of Huntingtin (HTT), the gene responsible for HD. We found that HTT protein is highly expressed in striatal neurons due to its slow degradation in the striatum. We also identified tripartite motif-containing 37 (TRIM37) as a primate-specific protein that interacts with HTT and is selectively reduced in the primate striatum. TRIM37 promotes the ubiquitination and degradation of mutant HTT (mHTT) in vitro and modulates mHTT aggregation in mouse and monkey brains. Our findings suggest that nonhuman primates are crucial for understanding the mechanisms of human diseases such as HD and support TRIM37 as a potential therapeutic target for treating HD.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

HAP40 modulates mutant Huntingtin aggregation and toxicity in Huntington's disease mice.

Huntington's disease (HD) is a monogenic neurodegenerative disease, caused by the CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene. The HTT gene encodes a large protein known to interact with many proteins. Huntingtin-associated protein 40 (HAP40) is one that shows high binding affinity with HTT and functions to maintain HTT conformation in vitro. However, the potential role of HAP40 in HD pathogenesis remains unknown. In this study, we found that the expression level of HAP40 is in parallel with HTT but inversely correlates with mutant HTT aggregates in mouse brains. Depletion of endogenous HAP40 in the striatum of HD140Q knock-in (KI) mice leads to enhanced mutant HTT aggregation and neuronal loss. Consistently, overexpression of HAP40 in the striatum of HD140Q KI mice reduced mutant HTT aggregation and ameliorated the behavioral deficits. Mechanistically, HAP40 preferentially binds to mutant HTT and promotes Lysine 48-linked ubiquitination of mutant HTT. Our results revealed that HAP40 is an important regulator of HTT protein homeostasis in vivo and hinted at HAP40 as a therapeutic target in HD treatment.

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene.

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments.

PET Ligands for Imaging Mutant Huntingtin Aggregates: A Case Study in Non-For-Profit Scientific Management.

Positron emission tomography imaging of misfolded proteins with high-affinity and selective radioligands has played a vital role in expanding our knowledge of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The pathogenesis of Huntington's disease, a CAG trinucleotide repeat disorder, is similarly linked to the presence of protein fibrils formed from mutant huntingtin (mHTT) protein. Development of mHTT fibril-specific radioligands has been limited by the lack of structural knowledge around mHTT and a dearth of available hit compounds for medicinal chemistry refinement. Over the past decade, the CHDI Foundation, a non-for-profit scientific management organisation has orchestrated a large-scale screen of small molecules to identify high affinity ligands of mHTT, with lead compounds now reaching clinical maturity. Here we describe the mHTT radioligands developed to date and opportunities for further improvement of this radiotracer class.

A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers.

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-ß core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington's disease (HD), the httNT segment on the polyQ's N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer - either the tetramer or a larger multimer - plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein-protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

Impairment of the Glial Phagolysosomal System Drives Prion-Like Propagation in a Drosophila Model of Huntington's Disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.

Copper enhances aggregational toxicity of mutant huntingtin in a Drosophila model of Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder with clinical presentations of moderate to severe cognitive, motor, and psychiatric disturbances. HD is caused by the trinucleotide repeat expansion of CAG of the huntingtin (HTT) gene. The mutant HTT protein containing pathological polyglutamine (polyQ) extension is prone to misfolding and aggregation in the brain. It has previously been observed that copper and iron concentrations are increased in the striata of post-mortem human HD brains. Although it has been shown that the accumulation of mutant HTT protein can interact with copper, the underlying HD progressive phenotypes due to copper overload remains elusive. Here, in a Drosophila model of HD, we showed that copper induces dose-dependent aggregational toxicity and enhancement of Htt-induced neurodegeneration. Specifically, we found that copper increases mutant Htt aggregation, enhances the accumulation of Thioflavin S positive ß-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases ß-amyloid aggregates in the HD pathological model. These findings reveal a direct role of copper in potentiating mutant Htt protein-induced aggregational toxicity, and further indicate the potential impact of environmental copper exposure in the disease onset and progression of HD.

Progressive alterations in polysomal architecture and activation of ribosome stalling relief factors in a mouse model of Huntington's disease.

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.

Solid-state nuclear magnetic resonance in the structural study of polyglutamine aggregation.

The aggregation of proteins into amyloid-like fibrils is seen in many neurodegenerative diseases. Recent years have seen much progress in our understanding of these misfolded protein inclusions, thanks to advances in techniques such as solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, multiple repeat-expansion-related disorders have presented special challenges to structural elucidation. This review discusses the special role of ssNMR analysis in the study of protein aggregates associated with CAG repeat expansion disorders. In these diseases, the misfolding and aggregation affect mutant proteins with expanded polyglutamine segments. The most common disorder, Huntington's disease (HD), is connected to the mutation of the huntingtin protein. Since the discovery of the genetic causes for HD in the 1990s, steady progress in our understanding of the role of protein aggregation has depended on the integrative and interdisciplinary use of multiple types of structural techniques. The heterogeneous and dynamic features of polyQ protein fibrils, and in particular those formed by huntingtin N-terminal fragments, have made these aggregates into challenging targets for structural analysis. ssNMR has offered unique insights into many aspects of these amyloid-like aggregates. These include the atomic-level structure of the polyglutamine core, but also measurements of dynamics and solvent accessibility of the non-core flanking domains of these fibrils' fuzzy coats. The obtained structural insights shed new light on pathogenic mechanisms behind this and other protein misfolding diseases.

Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus.

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

A developmental component to Huntington's disease.

Huntington's disease is a dominantly inherited disorder characterized by the dysfunction and death of cortical and striatal neurons. Striatal degeneration in Huntington's disease is due, at least in part, to defective cortical signalling to the striatum. Although Huntington's disease generally manifests at the adult stage, mouse and neuroimaging studies of presymptomatic mutation carriers suggest that it may affect neurodevelopment. In support of this notion, the development of the cortex is altered in mice with Huntington's disease and the foetuses of human Huntington's disease gene carriers. We will discuss these studies and the contribution of abnormal brain development to the later appearance of the disease.

Alterations in Cerebrospinal Fluid Urea Occur in Late Manifest Huntington's Disease.

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by expanded cytosine-adenine-guanine (CAG) repeats in the Huntingtin gene, resulting in the production of mutant huntingtin proteins (mHTT). Previous research has identified urea as a key metabolite elevated in HD animal models and postmortem tissues of HD patients. However, the relationship between disease course and urea elevations, along with the molecular mechanisms responsible for these disturbances remain unknown. Objective: To better understand the molecular disturbances and timing of urea cycle metabolism across different stages in HD. Methods: We completed a global metabolomic profile of cerebrospinal fluid (CSF) from individuals who were at several stages of disease: pre-manifest (PRE), manifest (MAN), and late manifest (LATE) HD participants, and compared to controls. Results: Approximately 500 metabolites were significantly altered in PRE participants compared to controls, although no significant differences in CSF urea or urea metabolites were observed. CSF urea was significantly elevated in LATE participants only. There were no changes in the urea metabolites citrulline, ornithine, and arginine. Conclusions: Overall, our study confirms that CSF elevations occur late in the HD course, and these changes may reflect accumulating deficits in cellular energy metabolism.

The polyglutamine domain is the primary driver of seeding in huntingtin aggregation.

Huntington's Disease (HD) is a fatal, neurodegenerative disease caused by aggregation of the huntingtin protein (htt) with an expanded polyglutamine (polyQ) domain into amyloid fibrils. Htt aggregation is modified by flanking sequences surrounding the polyQ domain as well as the binding of htt to lipid membranes. Upon fibrillization, htt fibrils are able to template the aggregation of monomers into fibrils in a phenomenon known as seeding, and this process appears to play a critical role in cell-to-cell spread of HD. Here, exposure of C. elegans expressing a nonpathogenic N-terminal htt fragment (15-repeat glutamine residues) to preformed htt-exon1 fibrils induced inclusion formation and resulted in decreased viability in a dose dependent manner, demonstrating that seeding can induce toxic aggregation of nonpathogenic forms of htt. To better understand this seeding process, the impact of flanking sequences adjacent to the polyQ stretch, polyQ length, and the presence of model lipid membranes on htt seeding was investigated. Htt seeding readily occurred across polyQ lengths and was independent of flanking sequence, suggesting that the structured polyQ domain within fibrils is the key contributor to the seeding phenomenon. However, the addition of lipid vesicles modified seeding efficiency in a manner suggesting that seeding primarily occurs in bulk solution and not at the membrane interface. In addition, fibrils formed in the presence of lipid membranes displayed similar seeding efficiencies. Collectively, this suggests that the polyQ domain that forms the amyloid fibril core is the main driver of seeding in htt aggregation.

Imaging and Assay of the Dynamics of Cytotoxic Huntingtin (HTT) Protein Aggregates Regulated by lncRNAs.

Huntington's disease (HD) pathogenesis involves deregulation of coding and noncoding RNA transcripts of which the involvement of long noncoding RNAs (lncRNA) has been realized recently. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. In the present study, we formulate a methodology to visualize and quantify intracellular aggregates formed by mutant HTT protein. This method employs the use of both confocal laser scanning and super resolution (N-SIM) microscopy to accurately estimate aggregate numbers. Further, to determine the role of two lncRNAs Meg3 and Neat1 in the formation of aggregates of mutant HTT, we used commercially available siRNAs against Meg3 and Neat1 for transiently knocking them down in mouse Neuro2a and human SHSY5Y cells. Co-transfection of 83Q-DsRed and siRNA specific for Neat1 or Meg3 resulted in decreased intracellular aggregates of 83Q-DsRed in both the cell lines. We have established a quantitative method to estimate and directly or indirectly modulate the formation of mutant HTT aggregates.