Characterisation of novel cell models of Huntington's disease; insights into pathogenesis

Huntington’s disease (HD) is an adult-onset, autosomal dominant neurodegenerative disease characterised by progressive movement disorder, psychiatric and cognitive symptoms. The causative genetic mutation is an abnormally expanded CAG triplet repeat near the N-terminus of exon 1 of the huntingtin gene (HTT), and longer CAG repeat lengths are associated with earlier age of disease onset. The aim of this thesis was to create a HTT allelic series cell model of HD in which the effects of increasing CAG repeat length, against a stable genetic background, could be observed within human neurons. Initially, recombinant adeno-associated virus (rAAV) was used to knock-in different CAG expansions into an immortalised human neural stem cell line (ReNcellVM), but due to low rates of homologous recombination, this was not successful. Instead, work was undertaken to optimise and characterise a transgenic HTT exon 1 allelic series in the ReNcellVM line; there is much evidence to suggest that HTT exon 1 is the pathogenic species in HD and generates phenotypes over a faster timescale than full-length HTT models. In ReNcellVM neurons the expression of pathogenic HTT exon 1 led to the formation of mutant HTT aggregates in a proportion of cells, in a manner that was related to CAG-repeat length and levels of HTT exon 1 expression. No overt cell-death phenotypes were seen but subtle differences between control and mutant lines were observed. To complement the HTT exon 1 model, an induced pluripotent stem cell (iPSC) model was generated from an HD family who carried a range of CAG-repeat length mutations. These cells were differentiated into medium spiny neurons (MSNs) and both models were used to study the trafficking of huntingtin within cells; whilst differences were observed between the trafficking of control and mutant HTT exon 1, these were not apparent in the MSNs which express full-length HTT

Mislocalization of Nucleocytoplasmic Transport Proteins in Human Huntington’s Disease PSC-Derived Striatal Neurons

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT). Disease progression is characterized by the loss of vulnerable neuronal populations within the striatum. A consistent phenotype across HD models is disruption of nucleocytoplasmic transport and nuclear pore complex (NPC) function. Here we demonstrate that high content imaging is a suitable method for detecting mislocalization of lamin-B1, RAN and RANGAP1 in striatal neuronal cultures thus allowing a robust, unbiased, highly powered approach to assay nuclear pore deficits. Furthermore, nuclear pore deficits extended to the selectively vulnerable DARPP32 + subpopulation neurons, but not to astrocytes. Striatal neuron cultures are further affected by changes in gene and protein expression of RAN, RANGAP1 and lamin-B1. Lowering total HTT using HTT-targeted anti-sense oligonucleotides partially restored gene expression, as well as subtly reducing mislocalization of proteins involved in nucleocytoplasmic transport. This suggests that mislocalization of RAN, RANGAP1 and lamin-B1 cannot be normalized by simply reducing expression of CAG-expanded HTT in the absence of healthy HTT protein

Nomenclature of Genetic Movement Disorders:Recommendations of the International Parkinson and Movement Disorder Society Task Force – An Update

In 2016, the Movement Disorder Society Task Force for the Nomenclature of Genetic Movement Disorders presented a new system for naming genetically determined movement disorders and provided a criterion-based list of confirmed monogenic movement disorders. Since then, a substantial number of novel disease-causing genes have been described, which warrant classification using this system. In addition, with this update, we further refined the system and propose dissolving the imaging-based categories of Primary Familial Brain Calcification and Neurodegeneration with Brain Iron Accumulation and reclassifying these genetic conditions according to their predominant phenotype. We also introduce the novel category of Mixed Movement Disorders (MxMD), which includes conditions linked to multiple equally prominent movement disorder phenotypes. In this article, we present updated lists of newly confirmed monogenic causes of movement disorders. We found a total of 89 different newly identified genes that warrant a prefix based on our criteria; 6 genes for parkinsonism, 21 for dystonia, 38 for dominant and recessive ataxia, 5 for chorea, 7 for myoclonus, 13 for spastic paraplegia, 3 for paroxysmal movement disorders, and 6 for mixed movement disorder phenotypes; 10 genes were linked to combined phenotypes and have been assigned two new prefixes. The updated lists represent a resource for clinicians and researchers alike and they have also been published on the website of the Task Force for the Nomenclature of Genetic Movement Disorders on the homepage of the International Parkinson and Movement Disorder Society (https://www.movementdisorders.org/MDS/About/Committees--Other-Groups/MDS-Task-Forces/Task-Force-on-Nomenclature-in-Movement-Disorders.htm). © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre‐clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non‐human or non‐neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra‐nuclear inclusions in a polyQ length‐dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction