FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat.

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.

Human Huntington's disease pluripotent stem cell-derived microglia develop normally but are abnormally hyper-reactive and release elevated levels of reactive oxygen species.

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.

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.

RNA-Seq of Huntington's disease patient myeloid cells reveals innate transcriptional dysregulation associated with proinflammatory pathway activation.

Innate immune activation beyond the central nervous system is emerging as a vital component of the pathogenesis of neurodegeneration. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The systemic innate immune system is thought to act as a modifier of disease progression; however, the molecular mechanisms remain only partially understood. Here we use RNA-sequencing to perform whole transcriptome analysis of primary monocytes from thirty manifest HD patients and thirty-three control subjects, cultured with and without a proinflammatory stimulus. In contrast with previous studies that have required stimulation to elicit phenotypic abnormalities, we demonstrate significant transcriptional differences in HD monocytes in their basal, unstimulated state. This includes previously undetected increased resting expression of genes encoding numerous proinflammatory cytokines, such as IL6 Further pathway analysis revealed widespread resting enrichment of proinflammatory functional gene sets, while upstream regulator analysis coupled with Western blotting suggests that abnormal basal activation of the NFĸB pathway plays a key role in mediating these transcriptional changes. That HD 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 once a stimulus is encountered. These data advance our understanding of mutant huntingtin pathogenesis, establish resting myeloid cells as a key source of HD immune dysfunction, and further demonstrate the importance of systemic immunity in the potential treatment of HD and the wider study of neurodegeneration.

Prion-mediated neurodegeneration is associated with early impairment of the ubiquitin-proteasome system.

Prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of misfolded prion protein (PrP(Sc)) in the brain. The critical relationship between aberrant protein misfolding and neurotoxicity currently remains unclear. The accumulation of aggregation-prone proteins has been linked to impairment of the ubiquitin-proteasome system (UPS) in a variety of neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington's diseases. As the principal route for protein degradation in mammalian cells, this could have profound detrimental effects on neuronal function and survival. Here, we determine the temporal onset of UPS dysfunction in prion-infected Ub(G76V)-GFP reporter mice, which express a ubiquitin fusion proteasome substrate to measure in vivo UPS activity. We show that the onset of UPS dysfunction correlates closely with PrP(Sc) deposition, preceding earliest behavioural deficits and neuronal loss. UPS impairment was accompanied by accumulation of polyubiquitinated substrates and found to affect both neuronal and astrocytic cell populations. In prion-infected CAD5 cells, we demonstrate that activation of the UPS by the small molecule inhibitor IU1 is sufficient to induce clearance of polyubiquitinated substrates and reduce misfolded PrP(Sc) load. Taken together, these results identify the UPS as a possible early mediator of prion pathogenesis and promising target for development of future therapeutics.

Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models.

Inflammation is a growing area of research in neurodegeneration. In Huntington's disease (HD), a fatal inherited neurodegenerative disease caused by a CAG-repeat expansion in the gene encoding huntingtin, patients have increased plasma levels of inflammatory cytokines and circulating monocytes that are hyper-responsive to immune stimuli. Several mouse models of HD also show elevated plasma levels of inflammatory cytokines. To further determine the degree to which these models recapitulate observations in HD patients, we evaluated various myeloid cell populations from different HD mouse models to determine whether they are similarly hyper-responsive, as well as measuring other aspects of myeloid cell function. Myeloid cells from each of the three mouse models studied, R6/2, HdhQ150 knock-in and YAC128, showed increased cytokine production when stimulated. However, bone marrow CD11b(+) cells did not show the same hyper-responsive phenotype as spleen and blood cells. Furthermore, macrophages isolated from R6/2 mice show increased levels of phagocytosis, similar to findings in HD patients. Taken together, these results show significant promise for these mouse models to be used to study targeting innate immune pathways identified in human cells, thereby helping to understand the role the peripheral immune system plays in HD progression.

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry.

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic ß-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. ß-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated ß-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded ß-sheet-rich proteins accumulate.

An exploratory double-blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease.

AIMS: Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntington's disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS: This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS: Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS: Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation.

Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.

BACKGROUND: Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. RESULTS: An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. CONCLUSIONS: The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

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.

Wild-type huntingtin regulates human macrophage function.

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.

Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington's disease by etanercept treatment.

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.

In vivo neutralization of the protagonist role of macrophages during the chronic inflammatory stage of Huntington's disease.

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.

Structural imaging in premanifest and manifest Huntington disease.

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.

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells.

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.

Disruption of immune cell function by mutant huntingtin in Huntington's disease pathogenesis.

Innate immune dysfunction is increasingly recognised as a key characteristic of neurodegenerative disease. In the fatal inherited neurological disorder, Huntington's disease, altered innate immune cell function and increased inflammation are observed in the brain and the periphery of disease gene carriers many years before symptom onset, suggesting a potentially early and important role in disease pathogenesis. This is due, at least in part, to the intrinsic effects of the disease-causing protein, mutant huntingtin, expressed in innate immune cells themselves. Understanding whether such innate immune dysfunction in Huntington's disease can be targeted to slow the onset and/or the progression of the disease has significant therapeutic implications and is the subject of much current research.

Differential actions of IL-1 alpha and IL-1 beta in glial cells share common IL-1 signalling pathways.

The cytokine interleukin (IL)-1 plays important roles in peripheral and central inflammation via the actions of two ligands IL-1 alpha and IL-1beta that bind to the IL-1 type I receptor (IL-1RI) and trigger identical responses. However, some recent evidence suggests that IL-1alpha and IL-1beta may have differential actions in the CNS. The aim of this study was to characterise the molecular mechanisms responsible for their differential actions in the brain. We show that, while IL-1alpha and IL-1beta induce identical IL-1 signalling pathways, IL-1beta is significantly more potent than IL-1alpha in stimulating IL-6 release in primary mixed glia. These data suggest that the differential effects of IL-1alpha and IL-1beta on glial cells are mediated by alternative pathways to the classical IL-1 signalling cascade.

Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington's Disease T Lymphocytes.

Huntington's disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington's disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington's disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington's disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington's disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington's disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington's disease innate immune system should not be extended to include the adaptive immune system.