Native Mutant Huntingtin in Human Brain

Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean‐Paul; Young, Anne B.; Wexler, Nancy S.; DiFiglia, Marian

doi:10.1074/jbc.m111.286609

Cited by 23 publications

(10 citation statements)

References 52 publications

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“…Consequently, research has been directed at truncated forms [41] instead of the full-length protein. However, recent studies have started to examine the presence of the native full-length protein in human brain [42], leading to the generation of more physiological models of HD pathology [43,44] and suggesting that full length HTT may also be pathogenic in HD [45,46], thus boosting pharmaceutical research into drugs augmenting HTT clearance. The development of the assay is driven by the necessity to quantify in a precise and sensitive way the full length HTT protein in multiple biological matrices.…”

Section: Discussionmentioning

confidence: 99%

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

et al. 2013

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BackgroundHuntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.ResultsAn ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.ConclusionsThe method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

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Section: Discussionmentioning

confidence: 99%

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

et al. 2013

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“…Signal was detected using Super Signal West Pico Chemiluminescent kit (Pierce #34080) and a CCD imaging system (Alpha Inno-tech) or Hyperfilm ECL (GE Healthcare #28906839) and densitometry was determined using ImageJ software (NIH). Primary antibodies were rabbit polyclonal anti-HTT antibody Ab1 (Sapp et al, 2012) (1:2000 in blocking buffer) and mouse monoclonal anti-GAPDH antibody (1:2000 in blocking buffer; Sigma #MAB374). Secondary antibodies were peroxidase-labeled anti-rabbit IgG (1:2500 in blocking buffer, Jackson Immunoresearch #711035152) or anti-mouse IgG (1:5000 in blocking buffer, Jackson Immu- noresearch #715035150).…”

Section: Methods Detailsmentioning

confidence: 99%

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

Didiot

Ferguson

et al. 2018

Cell Reports

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SUMMARY Huntington’s disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ~50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA sub-cellular localization might play a role in important neuronal regulatory mechanisms.

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“…The lysates were centrifuged at 20,000 g at 4°C for 20 min, and the supernatants were used for western-blots. The HTT antibodies 2B7 ( Weiss et al, 2009 ) (1:1000), ab1 ( Sapp et al, 2012 ) (1:3000) and MW1 ( Ko et al, 2001 ) (1:1000) have been described previously. The antibody S830 ( Sathasivam et al, 2001 ) (1:10000) is a kind gift from Dr Gillian Bates.…”

Section: Methodsmentioning

confidence: 99%

A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

Yao

Cui

Al‐Ramahi

et al. 2015

eLife

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Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.DOI: http://dx.doi.org/10.7554/eLife.05449.001

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Native Mutant Huntingtin in Human Brain

Cited by 23 publications

References 52 publications

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

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