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.

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.

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.