Colum Connolly

Age-dependent neurovascular abnormalities and altered microglial morphology in the YAC128 mouse model of Huntington disease.

Central nervous system (CNS) inflammatory processes including microglial activation have been implicated in the pathogenesis of neurodegenerative diseases such as Huntington Disease (HD). We report age-dependent changes in striatal microglial morphology and vasculature in the YAC128 mouse model of HD. Decreases in microglial ramification along with a decrease in vessel diameter and increased vessel density and length suggest the presence of microgliosis and proangiogenic activity in YAC128 mice. Our hypothesis for this study was that the changes in microglial morphology and perturbations in vasculature may be involved in the pathogenesis of HD and that peripheral challenge with the bacterial endotoxin, lipopolysaccharide (LPS), will exacerbate these microglial and vascular changes as well as the HD phenotype in YAC128 mice at 12 months. Chronic peripheral LPS (1mg/kg) potentiated microglial activation indicated by an increase in microglial cell body size and retraction of processes. This potentiation in microglial activation with chronic peripheral LPS challenge was paralleled with vascular remodeling including dilatation, increased vessel wall thickness, increased BBB permeability and fibrinogen deposition in YAC128 striatum. Although peripheral LPS caused an increase in microglial activation and degenerative changes in cerebrovasculature, the phenotypic hallmarks of HD in YAC128 mice such as motor coordination deficits and decreased striatal volume were not exacerbated by chronic peripheral LPS exposure. This study identifies age-dependent increases in microglial activation and angiogenesis in YAC128 at 12 months. Peripheral inflammation induced by chronic LPS causes similar changes but does not influence the HD phenotype in YAC128 mice.

A SNP in the HTT promoter alters NF-[kappa]B binding and is a bidirectional genetic modifier of Huntington disease

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case–based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Forkhead box protein p1 is a transcriptional repressor of immune signaling in the CNS: implications for transcriptional dysregulation in Huntington disease

Forkhead box protein p1 (Foxp1), a transcription factor showing highly enriched expression in the striatum, has been implicated in central nervous system (CNS) development, but its role in the mature brain is unknown. In order to ascertain functional roles for Foxp1 in the CNS, we have identified gene targets for Foxp1 both in vitro and in vivo using genome-wide expression microarrays and chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-seq) assays. We found that mouse Foxp1 overexpression in striatal cells elicited expression changes of genes related to immune signaling, transcriptional regulation and a manually curated Huntingtons disease (HD)-signaling pathway. Similar results were found when the gene expression data set was integrated with Foxp1-binding data determined from ChIP-seq analysis. In vivo lentiviral-mediated overexpression of human FOXP1 in the context of mutant huntingtin (Htt) protein resulted in a robust downregulation of glial cell-associated, immune genes, including those encoding a variety of cytokines and chemokines. Furthermore, Foxp1-induced expression changes were significantly negatively correlated with those changes elicited by mutant Htt protein in several different HD mouse models, and most significantly in post-mortem caudate from human HD subjects. We finally show that Foxp1 interacts with mutant Htt protein in mouse brain and is present in nuclear Htt aggregates in the striatum of R6/1 transgenic mice. These findings implicate Foxp1 as a key repressor of immune signaling in the CNS and suggest that the loss of Foxp1-mediated gene regulation in HD contributes to the immune dysfunction in this disease. We further suggest that Foxp1-regulated pathways might be important mediators of neuronal-glial cell communication.

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 Huntingtons 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.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients

Significance Chronic neuroinflammation and oxidative stress are likely complicit in driving disease progression in Huntingtons disease (HD). Here, we describe the mechanism of action of a unique chemical scaffold that is highly selective for activation of NRF2, the master transcriptional regulator of cellular antiinflammatory and antioxidant defense genes. The use of this scaffold revealed that NRF2 activation responses were muted in HD patient-derived neural stem cells, suggesting increased susceptibility of this critical renewable cell population to oxidative stress in HD brain. However, pharmacological activation of NRF2 was able to repress inflammatory responses in mouse microglia and astrocytes, the principal cellular mediators of neuroinflammation, and in blood monocytes from HD patients. Our results suggest multiple protective benefits of NRF2 activation for HD patients. The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntingtons disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Enhanced immune response to MMP3 stimulation in microglia expressing mutant huntingtin

Huntingtons Disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. The YAC128 mouse model of HD expresses the full-length human huntingtin protein with 128 CAG repeats and replicates the phenotype and neurodegeneration that occur in HD. Several studies have implicated a role for neuroinflammation in HD pathogenesis. Studies on presymptomatic HD patients have illustrated microgliosis (activated microglia) in brain regions affected in HD. Mutant huntingtin expressing isolated primary monocytes (human HD patients) and primary macrophages (YAC128) are overactive in response to lipopolysaccharide (LPS) stimulation. In this study we demonstrate that cultured primary microglia (the resident immune cells of the brain cells) from YAC128 mice differentially express a wide number of cytokines compared to wildtype microglia cultures in response to LPS. Furthermore, this study outlines a direct interaction between mutant huntingtin and cytokine secretion in HD microglia. Increased cytokine release in YAC128 microglia can be blocked by cannabinoid activation or by mutant huntingtin knockdown with anti-sense oligonucleotide treatment. Matrix metalloprotease 3 (MMP3), an endogenous neuronal activator of microglia, also induces increased cytokine release from YAC128 microglia compared to wildtype microglia. We found elevated MMP levels in HD CSF, and MMP levels correlate with disease severity in HD. These data support a novel role for MMPs and microglial activation in HD pathogenesis. With an improved understanding of the specific cellular processes involved in HD neuroinflammation, novel therapeutic agents targeting these processes can be developed and hold great promise in the treatment of HD.