Abstract
BackgroundNeuroinflammation 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.MethodsMicroglial-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.ResultsHuman 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.ConclusionsThese 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.
Highlights
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
They released elevated levels of reactive oxygen species that have neurotoxic potential. Accompanying such phenotypes, human Huntington’s disease pluripotent stem cells (PSCs)-derived microglia showed increased levels of apoptosis and were more susceptible to exogenous stress. Such stress appeared to be induced by supernatants from human PSCderived striatal neurons expressing mutant HTT with a long polyglutamine tract
Huntington’s disease PSCs show no deficits in their ability to differentiate to mature microglia PSC lines of various HTT polyglutamine lengths were differentiated to a microglial fate by inducing tissue macrophage-like characteristics in cells of a myeloid lineage [42]
Summary
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. Huntington’s disease (HD) is a fatal, autosomal dominant neurodegenerative disorder characterised by progressive cognitive, psychiatric and motor impairments, and widespread neuronal degeneration throughout the brain [1] It is caused by a CAG triplet repeat expansion in exon 1 of the gene encoding huntingtin (HTT), resulting in a pathogenic expanded stretch of thirty six or more glutamine residues in the N-terminal region of the HTT protein [2]. Systemic immune cells express mutant HTT in a manner that correlates with the disease burden of HD patients [3] and various immune system abnormalities have been shown in Huntington’s disease gene carriers [4] These include altered levels of circulating innate immune proteins that correlate with disease progression and can be detected years before disease onset [5,6,7,8]. Suggests that the innate immune system may play a disease-modifying role in HD pathogenesis [9,10,11,12,13,14]
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