Heat shock promotes inclusion body formation of mutant huntingtin (mHtt) and alleviates mHtt-induced transcription factor dysfunction

Chen, Justin Y.; Parekh, Miloni; Seliman, Hadear; Bakshinskaya, Dariya; Dai, Wei; Kwan, Kelvin Y.; Chen, Kuang Yu; Liu, Alice Y.‐C.

doi:10.1074/jbc.ra118.002933

Cited by 22 publications

(48 citation statements)

References 84 publications

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“…Co-immunoprecipitation results showed that ZnT3 protein was not detected in aggregates, suggesting that down-regulation of ZnT3 does not come from the recruit of mHtt. Additionally, mHtt affects gene expression via altering the activity of transcription factors or abnormally interacting with ones [74][75][76]. Two major transcriptional pathways, namely CRE and Sp1mediated transcription, have been extensively studies in HD [53].…”

Section: Discussionmentioning

confidence: 99%

Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Sp1 to ZnT3 promoter

Niu

Yang

Wang

et al. 2020

Preprint

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Background Synaptic dysfunction caused by mutant huntingtin greatly contributes to Huntington’s disease (HD) pathogenesis. HD patients show cognitive impairment as well as uncontrolled movements. Vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. However, whether does mutant huntingtin affect zinc homeostasis in the brain or not? This will be of great significance for further revealing the pathogenesis of HD. Methods N171-HD82Q transgenic mice and cultured BHK cells expressing N-terminal mutant huntingtin fragment containing 160 glutamines (160Q BHK cells) were used to investigate the effect of mutant huntingtin on zinc homeostasis and its molecular mechanisms. Results Herein, we have demonstrated that the density of synaptic vesicular zinc decreases in the cortex, striatum and hippocampus of N171-82Q mice. Given that vesicular zinc concentration depends on the abundance of zinc transporter 3 (ZnT3) on the membrane of synaptic vesicles, ZnT3 expression is detected in the brain of N171-82Q mice and 160Q BHK cells. Mutant huntingtin leads to a dramatical decrease in ZnT3 mRNA and protein levels in the three brain regions of these mice aged from 14 to 20 weeks. Significantly, Sp1 activates ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Nevertheless, mutant huntingtin inhibits the binding of Sp1 to the promoter of ZnT3 gene and down-regulates ZnT3 expression. Furthermore, the overexpression of Sp1 ameliorates inhibition of ZnT3 gene transcription by mutant huntingtin. Conclusions Collectively, this first study to reveal a significant loss of synaptic vesicular zinc and ZnT3 expression caused by mutant huntingtin in the early stage of HD. Our findings have revealed the molecular mechanism underlying this change. Mutant huntingtin inhibits the binding of Sp1 to ZnT3 gene promoter to reduce ZnT3 expression. The imbalance of vesicular zinc homeostasis may be closely associated with synaptic dysfunction and cognitive deficits in HD. This work sheds novel mechanistic insights into the pathogenesis of HD and promises a potential therapeutic strategy for HD.

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Section: Discussionmentioning

confidence: 99%

Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Sp1 to ZnT3 promoter

Niu

Yang

Wang

et al. 2020

Preprint

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“…Inhibition of HSP90 by chemical inhibitor or knockdown leads to degradation of mHtt and mAtax-3. Heat shock and HSP70 are known to enhance mHtt aggregation and provide cytoprotection (42). Sorbitol, a chemical chaperone has been shown to promote mHtt aggregation and reduce cytotoxicity of mHtt.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular chaperones of HSP family (HSC70, HSP70, HSP90) are known to affect mutant protein aggregation. HSC70 has been shown to suppress protein aggregation whereas HSP70 and heat shock are reported to enhance protein aggregation (38)(39)(40)42). We hypothesized that OPTN may be promoting aggregation of mutant proteins by modulating the function of HSP70 family chaperones.…”

Section: How Does Optineurin Promote Mutant Protein Aggregation?mentioning

confidence: 97%

“…Genetic studies in yeast have shown that cytosolic HSP70 (Ssa family) is required for mutant Huntingtin aggregation (41). Recent studies suggest that aggregation of mutant proteins to form inclusion bodies is facilitated by chaperones of HSP70 family that leads to reduction of diffusible mutant protein (which is more toxic) resulting in enhanced cell survival (42).…”

Section: Introductionmentioning

confidence: 99%

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Optineurin promotes aggregation of mutant huntingtin and mutant ataxin-3, and reduces cytotoxicity of aggregates

Moharir¹,

Ak²,

Swarup³

2020

Preprint

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Optineurin (OPTN), a cytoplasmic adaptor protein involved in cargo selective autophagy of bacteria, damaged mitochondria and mutant protein aggregates, is frequently seen in pathological structures containing protein aggregates, associated with several neurodegenerative diseases. However, the function of OPTN in these protein aggregates is not known. Here, we have explored the role of OPTN in mutant protein aggregation and in cytoprotection from toxicity of mutant proteins. Mutant huntingtin (mHtt) and mutant ataxin-3 (mAtax-3) showed reduced formation of aggregates in Optn-/- mouse embryonic fibroblasts as compared with wild type cells. Co-expression of OPTN enhanced aggregate formation by mHtt and mAtax-3 in Optn-/- cells. C-terminal domain of OPTN (412-577 amino acids) was necessary and sufficient to promote aggregate formation by these mutant proteins. The E478G mutant of OPTN, defective in ubiquitin-binding and autophagy, was also able to promote aggregation of mHtt and mAtax-3. OPTN and its C-terminal domain form a complex with the chaperone HSP70 known to promote mutant protein aggregation. Overexpression of mHtt or mAtax-3 induced more cell death in Optn-/- cells compared with wild type cells. Importantly, compared to wild type cells, Optn-deficient cells having mHtt or mAtax-3 aggregates showed higher level of cell death in neuronal (N2A) and non-neuronal cells. Our results show that OPTN promotes formation of mutant huntingtin and mutant ataxin-3 aggregates, and this function of OPTN might be mediated through interaction with HSP70 chaperones. Our results also show that OPTN reduces cytotoxicity caused by these mutant protein aggregates.

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“…The effects of transient heat shock in driving mHtt IB formation is dependent on the production of HSP chaperones 9 , proteins widely acknowledged to play important roles in protein folding, structuring, and quality control 11,12 . Here, we used osmolytes as tools to further assess the role of protein structuring in driving the assembly and aggregation of the intrinsically disordered polyQ-Htt exon1 protein into IBs.…”

mentioning

confidence: 99%

Osmolytes dynamically regulate mutant Huntingtin aggregation and CREB function in Huntington’s disease cell models

Aravindan

Chen

Choudhry

et al. 2020

Sci Rep

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Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to repress CREB-reporter gene activity. This repression is mitigated either by stabilizing osmolytes, which deplete diffuse mHtt or by urea, which negates protein–protein interaction. Our results show that stabilizing polyol osmolytes promote mHtt aggregation, alleviate CREB dysfunction, and promote survival under stress to support the hypothesis that lower molecular weight entities of disease protein are relevant pathogenic species in neurodegeneration.

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Heat shock promotes inclusion body formation of mutant huntingtin (mHtt) and alleviates mHtt-induced transcription factor dysfunction

Cited by 22 publications

References 84 publications

Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Sp1 to ZnT3 promoter

Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Sp1 to ZnT3 promoter

Optineurin promotes aggregation of mutant huntingtin and mutant ataxin-3, and reduces cytotoxicity of aggregates

Osmolytes dynamically regulate mutant Huntingtin aggregation and CREB function in Huntington’s disease cell models

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