Background Neuroinflammation may contribute to the pathogenesis of Huntington’s disease, given evidence of activated microglia and elevated levels of inflammatory molecules in disease gene carriers, even those many years from symptom onset. We have shown previously that monocytes from Huntington’s disease patients are hyper-reactive to stimulation in a manner dependent on their autonomous expression of the disease-causing mutant HTT protein. To date, however, whether human microglia are similarly hyper-responsive in a cell-autonomous manner has not been determined. Methods Microglial-like cells were derived from human pluripotent stem cells (PSCs) expressing mutant HTT containing varying polyglutamine lengths. These included lines that are otherwise isogenic, such that any observed differences can be attributed with certainty to the disease mutation itself. Analyses by quantitative PCR and immunofluorescence microscopy respectively of key genes and protein markers were undertaken to determine whether Huntington’s disease PSCs differentiated normally to a microglial fate. The resultant cultures and their supernatants were then assessed by various biochemical assays and multiplex ELISAs for viability and responses to stimulation, including the release of pro-inflammatory cytokines and reactive oxygen species. Conditioned media were applied to PSC-derived striatal neurons, and vice versa, to determine the effects that the secretomes of each cell type might have on the other. Results Human PSCs generated microglia successfully irrespective of the expression of mutant HTT. These cells, however, were hyper-reactive to stimulation in the production of pro-inflammatory cytokines such as IL-6 and TNFα. They also released elevated levels of reactive oxygen species that have neurotoxic potential. Accompanying such phenotypes, human Huntington’s disease PSC-derived microglia showed increased levels of apoptosis and were more susceptible to exogenous stress. Such stress appeared to be induced by supernatants from human PSC-derived striatal neurons expressing mutant HTT with a long polyglutamine tract. Conclusions These studies show, for the first time, that human Huntington’s disease PSC-derived microglia are hyper-reactive due to their autonomous expression of mutant HTT. This provides a cellular basis for the contribution that neuroinflammation might make to Huntington’s disease pathogenesis.

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.

The huntingtin (HTT) protein in its mutant form is the cause of the inherited neurodegenerative disorder, Huntington’s disease. Beyond its effects in the central nervous system, disease-associated mutant HTT causes aberrant phenotypes in myeloid-lineage innate immune system cells, namely monocytes and macrophages. Whether the wild-type form of the protein, however, has a role in normal human macrophage function has not been determined. Here, the effects of lowering the expression of wild-type (wt)HTT on the function of primary monocyte-derived macrophages from healthy, non-disease human subjects were examined. This demonstrated a previously undescribed role for wtHTT in maintaining normal macrophage health and function. Lowered wtHTT expression was associated, for instance, with a diminished release of induced cytokines, elevated phagocytosis and increased vulnerability to cellular stress. These may well occur by mechanisms different to that associated with the mutant form of the protein, given an absence of any effect on the intracellular signalling pathway predominantly associated with macrophage dysfunction in Huntington’s disease.

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.

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by the expansion of the CAG repeat in exon 1 of the huntingtin (HTT) gene, which results in a mutant protein with an extended polyglutamine tract. Inflammation occurs in both the brain and the periphery of HD patients and mouse models, with increases in brain and/or plasma levels of neurotoxic TNFα and several other proinflammatory cytokines. TNFα promotes the generation of many of these cytokines, such as IL6, which raises the possibility that TNFα is central to the inflammatory milieu associated with HD. A number of mouse studies have reported that the suppression of chronic immune activation during HD has beneficial consequences. Here, we investigated whether TNFα contributes to the peripheral inflammation that occurs in the R6/2 mouse model, and whether the in vivo blockade of TNFα, via etanercept treatment, can modify disease progression. We found that etanercept treatment normalised the elevated plasma levels of some cytokines. This did not modify the progression of certain behavioural measures, but slightly ameliorated brain weight loss, possibly related to a reduction in the elevated striatal level of soluble TNFα.

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the huntingtin (HTT) gene. CAG repeat length explains around half of the variation in age at onset (AAO) but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FANCD2- and FANCI-associated nuclease 1 (FAN1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed AAO and slower progression of HD, suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilizing effects are FAN1 concentration and CAG repeat length-dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders.

Background While the central nervous system is considered to be the primary site of Huntington’s disease pathology, the systemic innate immune system may be a modifier of disease progression. Myeloid cells from Huntington’s disease patients exhibit numerous functional abnormalities, and use of RNA deep-sequencing to perform whole transcriptome analysis of primary cultures demonstrates significant transcriptional differences in Huntington’s disease monocytes in their basal, unstimulated state. Human Huntington’s disease monocytes are intrinsically abnormal and transcriptional dysregulation is a key mechanism underlying innate immune dysfunction in Huntington’s disease. This hyper-excitable phenotype of Huntington’s disease myeloid cells is corrected upon HTT lowering, but of further note HTT lowering in monocytes from unaffected, non-gene carriers results in a similar reduction in inflammatory cytokine production levels to that seen in Huntington’s disease ones, suggesting a role for wild-type HTT in myeloid cell function. Aim To investigate the mechanisms of altered transcriptional regulation that are associated with HD myeloid cell hyper-reactivity, and the role of wild-type HTT in normal myeloid cell responses Methods RNA-sequencing was performed for whole transcriptome analysis of primary monocytes isolated from HD patients and control subjects cultured with and without a proinflammatory stimulus. ChIP-seq was undertaken to identify changes in histone modifications or transcription factor binding that are associated with altered transcription. Glucan particle-mediated delivery of anti-HTT siRNA was used to investigate the effects of HTT lowering on a range of myeloid cell phenotypes. Results That Huntington’s disease myeloid cells have a proinflammatory phenotype in the absence of stimulation is consistent with a priming effect of mutant huntingtin, whereby basal dysfunction leads to an exaggerated inflammatory response when a stimulus is encountered. The molecular mechanisms underpinning these changes may be associated with effects of mHTT on specific histone modifications and transcription factors to increase the expression of particular genes associated with inflammation. The effects of the disease-associated form of HTT on myeloid cells may be an extension of a normal role of the wild-type form in these cells. The observation that HTT lowering reduces inflammatory cytokine production from control myeloid cells has now been confirmed in additional healthy, non-HD cohorts. Together with effects on other aspects of myeloid cell function, this confirms a novel role for wild-type HTT in immune system function under resting conditions. Conclusions A novel role for wild-type HTT in immune system function exists under resting conditions. Further research is demonstrating the underlying characteristic of this newly discovered role for HTT and how it may be changed in the disease state to cause a hyper-reactive phenotype. Support Medical Research Council, Rosetrees Trust, Brain Research Trust

Background The corticostriatal (CS) pathway, comprising layer V cortical projection neurons (CPN) and medium spiny neurons (MSN), is one of the first brain pathways to succumb to Huntington’s disease (HD) pathology. As a result, disrupted CS connectivity is evident and contributes to the motor and cognitive symptoms experienced by HD patients. Aims The aim of this work is to investigate the CS pathway using a purely human tissue-derived in vitro system. Methods This project utilizes two familial iPSC lines; the control line, with 20/20 HTT CAG repeat lengths (20Q), and a juvenile HD line, with 20/73 CAG repeats (73Q). These lines were differentiated in parallel to either MSNs or CPNs, and co-cultured in microfluidic chambers to physically recapitulate the human CS pathway. Results High-resolution fluorescence microscopy has revealed the formation of CS synapses within MFC co-cultures, complimented by live cell imaging with calcium binding dye Fluo4, which demonstrates the successful transmission of calcium between neuronal populations within MFCs. CPN cultures show a HD phenotype in their cytoskeletal dynamics, as axon projection efficiency is drastically reduced in 73Q CPNs compared to 20Q. Furthermore, 73Q MSNs exhibit enhanced cell death after BDNF-withdrawal compared to 20Q cultures. Finally, the intrinsic membrane properties of iPSC-derived MSNs also differ with disease state, as 73Q MSNs are hyper-excitable, with an extended latency to fire and extended refractory period. Conclusion These results provide a novel insight into the human CS pathway and suggest subtle differences in both the development and function of the CS pathway in HD.

Background Pluripotent stem cell technology is allowing the study of disease-relevant cell types of human origin, and is accompanied by much interest in their application for the screening of disease phenotypes and potential therapeutics. Here, we have generated and characterised induced pluripotent stem cells (iPSCs) from HD gene carriers differentiated into disease-specific striatal medium spiny neurons (MSNs) by high content imaging to investigate the effects of increasing HTT CAG repeat length on neuronal function. Aims To adapt a protocol for the directed differentiation of iPSCs to MSNs to a high throughput format, in order to carry out a broad phenotypic screen of HD-related phenotypes using high content imaging. Methods The iPSC lines we used were derived from closely related individuals, three of whom were suffering from juvenile HD and their unaffected mother. This created a unique familial allelic series with increasing HTT CAG repeat lengths of 20, 56, 67 and 73 CAGs. High content imaging was carried out at differentiation day 36 using the Opera Phenix High Content Screening System and data analysed using Columbus software (Perkin Elmer). Results We report here significant phenotypic differences in endpoint total cell number and culture composition between control and HD lines with the latter accumulating fewer cells over time and a reduction in the proliferative precursor pool. Interestingly, there were no significant differences in the proportions of CTIP2+ cells, neurons or MSNs generated. However, in terms of pathology, we find evidence of enhanced cell death, reduced DARPP-32 expression within MSNs and mHTT inclusion formation in the longest (73 CAG) length line. Conclusions We find no differences in holistic measurements of neuronal differentiation between control and HD lines suggesting that the pathway to neuronal development is unaffected at this stage. However, differences in endpoint cell number and HD-related pathologies were found which could be used as potential endpoints for high throughput drug screening. Support Takeda, Cerevance Ltd, UCLH Biomedical Research Centre, Medical Research Council

Neurodegenerative diseases, characterised by the progressive and selective neuronal death in the central nervous system, are frequently accompanied by an activated immune system. In Huntington's disease (HD), clinical and animal studies show evidence of immune activity, along with hyper-reactive monocyte/macrophage responses, while application of immunosuppressive regimens have imparted beneficial effects to HD mice. These findings suggest a contributory role of the immune system in HD pathology, with immune-based interventions offering a potential therapeutic strategy. Herein, we show that peripheral and CNS immune system activity increased with disease progression in HD mouse models and defined the phenotype of the immune response. Additionally, the depletion of monocytes and macrophages in vivo, via clodronate liposome treatment, revealed a major contributory role of these innate immune cells to the chronic inflammatory milieu observed during the course of the disease. This suggests that peripheral immunomodulatory strategies targeting monocytes and macrophages could be relevant for HD.

Huntington disease (HD) neuropathology has a devastating effect on brain structure and consequently brain function; neuroimaging provides a means to assess these effects in gene carriers. In this chapter we first outline the unique utility of structural imaging in understanding HD and discuss some of the acquisition and analysis techniques currently available. We review the existing literature to summarize what we know so far about structural brain changes across the spectrum of disease from premanifest through to manifest disease. We then consider how these neuroimaging findings relate to patient function and nonimaging biomarkers, and can be used to predict disease onset. Finally we review the utility of imaging measures for assessment of treatment efficacy in clinical trials.

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington’s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.

Background A number of neurodegenerative diseases, characterised by progressive and selective death of neurons in the CNS, are accompanied by activation of the peripheral immune system. In Huntington’s disease (HD), clinical and animal studies show elevated immune factors that are hallmarks of immune activity and the use of immunosuppressive regimens have shown beneficial effects in HD mice. These results suggest a contributory role of the immune system in HD pathology, with immune based interventions offering potential therapeutic strategy to disease. Aim To assess peripheral and central nervous system (CNS) immune system activity in HD mouse model during disease course to determine if/when peripheral immunomodulation will be relevant for HD treatment. Method R6/2 mice were investigated pre and post-symptomatic stages for immune activity in the brain and periphery through the assessment of gene expression and protein levels of interleukin (IL)−1β, IL-6, IL-10, IL-17 and tumour necrosis factor (TNF)α cytokines. Gene and cell surface (flow cytometry) expression of monocyte and macrophage activation (CD40 and OX40l) and T cell activation (OX40 and CD25) markers were also measured. Results At 14 weeks old (late-stage disease) cytokines and cell surface markers are elevated in several peripheral compartments as well as the brain. At 8 weeks (pre-symptomatic stage) however, immune activity is detectable in the periphery but not in the brain. Conclusion Immune activity in the periphery precedes immune activation in the CNS suggesting the peripheral immune system may promote activation of the CNS immune system during HD, possibly through the secretion of pro-inflammatory factors that cross the blood brain barrier. Additionally, myeloid immune cells in the CNS and periphery display differing activated phenotypes during late stage HD with microglial cells presenting a predominantly M1 phenotype while monocyte/macrophages show a M2 predominating phenotype. Funding Medical Research Council and the CHDI Foundation

Background Mitochondrial dysfunction is a known component of HD pathogenesis, but the precise mechanisms and temporal order of events linked to mHTT inclusion formation and neurodegeneration are unclear. For example, observations from clinical samples and in vitro models implicate respiratory impairment and enhanced glycolysis in HD cells. Aim To identify early signs of mitochondrial dysfunction and perturbed bioenergetics in a human HD neuronal model, and better understand the interaction between the proteotoxic environment of mHTT-expressing cells and their metabolic capacity. Methods We are using a human neural stem cell (NSC) line (ReNcell VM) that over-expresses huntingtin (HTT) exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71Q and 129Q), and can be rapidly differentiated into mixed populations of neurons and glia in various culture formats. The pathogenic HTT exon 1 lines develop both intranuclear and cytoplasmic mHTT-containing inclusions, as well as accumulating a diffuse aggregated form of mHTT in a time and poly Q-dependent manner. We have correlated with this measures of oxygen consumption rate and extracellular acidification rates using an XF96 Seahorse Bioanalyser, mitochondrial volume and basal ΔΨm by live confocal imaging and potential impairment individual respiratory chain complexes using in vitro assays. Results Despite no overt reduction in cell viability in the mHTT exon 1 expressing lines we have uncovered sub-pathological alterations to mitochondrial function. We report differences in key respiratory parameters (e.g. basal and maximal respiration) in line with a significant decrease in basal mitochondrial ΔΨm in mHTT exon 1 cells. Here, we will present our findings correlating the inclusion phenotypes and mitochondrial dysfunction in the presence of mHTT exon 1. Conclusions We have established a robust and disease-relevant in vitro cellular model of HD that has revealed potentially important early changes to neuronal bioenergetics and mitochondrial function. Using this system we aim to not only identify novel pathways in disease pathogenesis that may be amenable to therapeutic targeting, but also to simultaneously develop a novel in vitro cellular platform for high-throughput compound screening.