The evolutionary history of the polyQ tract in huntingtin sheds light on its functional pro-neural activities

Huntington’s disease is caused by a pathologically long (>35) CAG repeat located in the first exon of the Huntingtin gene (HTT). While pathologically expanded CAG repeats are the focus of extensive investigations, non-pathogenic CAG tracts in protein-coding genes are less well characterized. Here, we investigated the function and evolution of the physiological CAG tract in the HTT gene. We show that the poly-glutamine (polyQ) tract encoded by CAGs in the huntingtin protein (HTT) is under purifying selection and subjected to stronger selective pressures than CAG-encoded polyQ tracts in other proteins. For natural selection to operate, the polyQ must perform a function. By combining genome-edited mouse embryonic stem cells and cell assays, we show that small variations in HTT polyQ lengths significantly correlate with cells’ neurogenic potential and with changes in the gene transcription network governing neuronal function. We conclude that during evolution natural selection promotes the conservation and purity of the CAG-encoded polyQ tract and that small increases in its physiological length influence neural functions of HTT. We propose that these changes in HTT polyQ length contribute to evolutionary fitness including potentially to the development of a more complex nervous system.

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR–dysregulated transcriptional activity.

A novel imaging ligand as a biomarker for mutant huntingtin-lowering in Huntington's disease

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (PolyQ) tract. While several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, no method is currently available to visualize mHTT levels in the living brain. Here we demonstrate the development of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region- and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum have a functional restorative effect that can be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

Stress granules, RNA-binding proteins and polyglutamine diseases: too much aggregation?

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context.

Topological Insights into Mutant Huntingtin Exon 1 and PolyQ Aggregates by Cryo-Electron Tomography

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

Topological Insights into Mutant Huntingtin Exon 1 and PolyQ Aggregates by Cryo-Electron Tomography

ABSTRACTHuntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we observed some filaments in both types of aggregates under ∼2 nm in width, thinner than previously reported, while other regions form large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy, slab-shaped morphology in both aggregates, supportive of the “polyQ core” model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling and their identification in cells without fusion tags.

Molecular Characterisation of Canine Osteosarcoma in High Risk Breeds

Dogs develop osteosarcoma (OSA) and the disease process closely resembles that of human OSA. OSA has a poor prognosis in both species and disease-free intervals and cure rates have not improved in recent years. Gene expression in canine OSAs was compared with non-tumor tissue utilising RNA sequencing, validated by qRT-PCR and immunohistochemistry (n = 16). Polymorphic polyglutamine (polyQ) tracts in the androgen receptor (AR/NR3C4) and nuclear receptor coactivator 3 (NCOA3) genes were investigated in control and OSA patients using polymerase chain reaction (PCR), Sanger sequencing and fragment analysis (n = 1019 Rottweilers, 379 Irish Wolfhounds). Our analysis identified 1281 significantly differentially expressed genes (>2 fold change, p < 0.05), specifically 839 lower and 442 elevated gene expression in osteosarcoma (n = 3) samples relative to non-malignant (n = 4) bone. Enriched pathways and gene ontologies were identified, which provide insight into the molecular pathways implicated in canine OSA. Expression of a subset of these genes (SLC2A1, DKK3, MMP3, POSTN, RBP4, ASPN) was validated by qRTPCR and immunohistochemistry (MMP3, DKK3, SLC2A1) respectively. While little variation was found in the NCOA3 polyQ tract, greater variation was present in both polyQ tracts in the AR, but no significant associations in length were made with OSA. The data provides novel insights into the molecular mechanisms of OSA in high risk breeds. This knowledge may inform development of new prevention strategies and treatments for OSA in dogs and supports utilising spontaneous OSA in dogs to improve understanding of the disease in people.

CRISPR/Cas9-based silencing of the ATXN1 gene in Spinocerebellar ataxia type 1 (SCA1) fibroblasts

Spinocerebellar Ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. Actually, there are no treatments available to delay the onset, stop or slow down the progression of this pathology. Many approaches developed over the years involve the use of siRNAs and antisense oligonucleotides (ASOs). Here we develop and validate a CRISPR/Cas9 therapeutic strategy in fibroblasts isolated from SCA1 patients. We started from the screening of 10 different sgRNAs able to recognize regions upstream and downstream the CAG repeats, in exon 8 of ATXN1 gene. The two most promising sgRNAs, G3 and G8, whose efficiency was evaluated with an in vitro system, significantly downregulated the ATXN 1 protein expression. This downregulation was due to the introduction of indels mutations into the ATXN1 gene. Notably, with an RNA-seq analysis, we demonstrated minimal off-target effects of our sgRNAs. These preliminary results support CRISPR/Cas9 as a promising approach for treated polyQ-expanded diseases.