Rac1 Activity Is Modulated by Huntingtin and Dysregulated in Models of Huntington\u27s Disease

BACKGROUND: Previous studies suggest that Huntingtin, the protein mutated in Huntington\u27s disease (HD), is required for actin based changes in cell morphology, and undergoes stimulus induced targeting to plasma membranes where it interacts with phospholipids involved in cell signaling. The small GTPase Rac1 is a downstream target of growth factor stimulation and PI 3-kinase activity and is critical for actin dependent membrane remodeling. OBJECTIVE: To determine if Rac1 activity is impaired in HD or regulated by normal Huntingtin. METHODS: Analyses were performed in differentiated control and HD human stem cells and HD Q140/Q140 knock-in mice. Biochemical methods included SDS-PAGE, western blot, immunoprecipitation, affinity chromatography, and ELISA based Rac activity assays. RESULTS: Basal Rac1 activity increased following depletion of Huntingtin with Huntingtin specific siRNA in human primary fibroblasts and in human control neuron cultures. Human cells (fibroblasts, neural stem cells, and neurons) with the HD mutation failed to increase Rac1 activity in response to growth factors. Rac1 activity levels were elevated in striatum of 1.5-month-old HD Q140/Q140 mice and in primary embryonic cortical neurons from HD mice. Affinity chromatography analysis of striatal lysates showed that Huntingtin is in a complex with Rac1, p85alpha subunit of PI 3-kinase, and the actin bundling protein alpha-actinin and interacts preferentially with the GTP bound form of Rac1. The HD mutation reduced Huntingtin interaction with p85alpha. CONCLUSIONS: These findings suggest that Huntingtin regulates Rac1 activity as part of a coordinated response to growth factor signaling and this function is impaired early in HD

Huntingtin associates with the actin cytoskeleton and alpha-actinin isoforms to influence stimulus dependent morphology changes

One response of cells to growth factor stimulus involves changes in morphology driven by the actin cytoskeleton and actin associated proteins which regulate functions such as cell adhesion, motility and in neurons, synaptic plasticity. Previous studies suggest that Huntingtin may be involved in regulating morphology however, there has been limited evidence linking endogenous Huntingtin localization or function with cytoplasmic actin in cells. We found that depletion of Huntingtin in human fibroblasts reduced adhesion and altered morphology and these phenotypes were made worse with growth factor stimulation, whereas the presence of the Huntington\u27s Disease mutation inhibited growth factor induced changes in morphology and increased numbers of vinculin-positive focal adhesions. Huntingtin immunoreactivity localized to actin stress fibers, vinculin-positive adhesion contacts and membrane ruffles in fibroblasts. Interactome data from others has shown that Huntingtin can associate with alpha-actinin isoforms which bind actin filaments. Mapping studies using a cDNA encoding alpha-actinin-2 showed that it interacts within Huntingtin aa 399-969. Double-label immunofluorescence showed Huntingtin and alpha-actinin-1 co-localized to stress fibers, membrane ruffles and lamellar protrusions in fibroblasts. Proximity ligation assays confirmed a close molecular interaction between Huntingtin and alpha-actinin-1 in human fibroblasts and neurons. Huntingtin silencing with siRNA in fibroblasts blocked the recruitment of alpha-actinin-1 to membrane foci. These studies support the idea that Huntingtin is involved in regulating adhesion and actin dependent functions including those involving alpha-actinin

Autophagy Activation by Transcription Factor EB (TFEB) in Striatum of HDQ175/Q7 Mice

Background: Mutant huntingtin (mHTT) is encoded by the Huntington’s disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy. Objective: To examine if increasing TFEB protein levels in HD mouse striatum induces autophagy and influences mHTT levels. Methods: We introduced cDNA encoding TFEB with an HA tag (TFEB-HA) under the control of neuron specific synapsin 1 promoter into the striatum of 3 month old HDQ175/Q7 mice using adeno-associated virus AAV2/9. The levels of exogenous TFEB were analyzed using qPCR and Western blot. Proteins involved in autophagy, levels of huntingtin, and striatal-enriched proteins were examined using biochemical and/or immunohistochemical methods. Results: In HD mice expressing TFEB-HA, HA immunoreactivity distributed throughout the striatum in neuronal cell bodies and processes and preferentially in neuronal nuclei and overlapped with a loss of DARPP32 immunoreactivity. TFEB-HA mRNA and protein were detected in striatal lysates. There were increased levels of proteins involved with autophagosome/lysosome activity including LAMP-2A, LC3II, and cathepsin D and reduced levels of mutant HTT and the striatal enriched proteins DARPP32 and PDE10A. Compared to WT mice, HDQ175/Q7 mice had elevated levels of the ER stress protein GRP78/BiP and with TFEB-HA expression, increased levels of the astrocyte marker GFAP and pro-caspase 3. Conclusion: These results suggest that TFEB expression in the striatum of HDQ175/Q7 mice stimulates autophagy and lysosome activity, and lowers mHTT, but may also increase a neuronal stress response

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

The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival

Human huntingtin (Htt) contains 3144 amino acids and has an expanded polyglutamine region near the NH2-terminus in patients with Huntington’s disease. While numerous binding partners have been identified to NH2-terminal Htt, fewer proteins are known to interact with C-terminal domains of Htt. Here we report that kalirin, a Rac1 activator, is a binding partner to C-terminal Htt. Kalirin and Htt co-precipitated from mouse brain endosomes and co-localized at puncta in NRK and immortalized striatal cells and primary cortical neurons. We mapped the interaction domains to kalirin674-1272 and Htt2568-3144 and determined that the interaction between kalirin and Htt was independent of HAP1, a known interactor for Htt and kalirin. Kalirin precipitated with mutant Htt was more abundant than with wild-type Htt and had a reduced capacity to activate Rac1 when mutant Htt was present. Expression of Htt2568-3144 caused cytotoxicity, partially rescued by co-expressing kalirin674-1272 but not other regions of kalirin. Our study suggests that the interaction of kalirin with the C-terminal region of Htt influences the function of kalirin and modulates the cytotoxicity induced by C-terminal Htt

Focused Ultrasound-Mediated Disruption of the Blood–Brain Barrier for AAV9 Delivery in a Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is a monogenic neurodegenerative disorder caused by a cytosine–adenine–guanine (CAG) trinucleotide repeat expansion in the HTT gene. There are no cures for HD, but the genetic basis of this disorder makes gene therapy a viable approach. Adeno-associated virus (AAV)-miRNA-based therapies have been demonstrated to be effective in lowering HTT mRNA; however, the blood–brain barrier (BBB) poses a significant challenge for gene delivery to the brain. Delivery strategies include direct injections into the central nervous system, which are invasive and can result in poor diffusion of viral particles through the brain parenchyma. Focused ultrasound (FUS) is an alternative approach that can be used to non-invasively deliver AAVs by temporarily disrupting the BBB. Here, we investigate FUS-mediated delivery of a single-stranded AAV9 bearing a cDNA for GFP in 2-month-old wild-type mice and the zQ175 HD mouse model at 2-, 6-, and 12-months. FUS treatment improved AAV9 delivery for all mouse groups. The delivery efficacy was similar for all WT and HD groups, with the exception of the zQ175 12-month cohort, where we observed decreased GFP expression. Astrocytosis did not increase after FUS treatment, even within the zQ175 12-month group exhibiting higher baseline levels of GFAP expression. These findings demonstrate that FUS can be used to non-invasively deliver an AAV9-based gene therapy to targeted brain regions in a mouse model of Huntington’s disease

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