Analysis of mutant and total huntingtin expression in Huntington’s disease murine models

Fodale, Valentina; Pintauro, Roberta; Daldin, Manuel; Altobelli, Roberta; Spiezia, Maria Carolina; Bisbocci, Monica; Macdonald, Douglas; Bresciani, Alberto

doi:10.1038/s41598-020-78790-5

Cited by 18 publications

(20 citation statements)

References 36 publications

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“…We then probed the same lysates for MAB2166 antibody (which detects both Htt and mHtt) or polyQ antibody to detect mHtt. We found diminished levels of Htt protein in the Q175DN mice striatum, consistent with earlier reports ( 39 , 40 ), but a further diminishment of Htt and decrease in polyQ mHTT in the Q175DN-SUMO1KO striatum ( Fig. 4 A , C , and D ).…”

Section: Resultssupporting

confidence: 92%

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

Ramírez-Jarquín

Sharma

Zhou

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the in vivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32–positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

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Section: Resultssupporting

confidence: 92%

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

Ramírez-Jarquín

Sharma

Zhou

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Interestingly, Massai and colleagues, who reported a decrease in tHtt in human peripheral blood mononuclear cells, utilized an antibody which targets amino acids 1844-2131 of the Htt protein [28] , which is much closer to the 3' terminal than those antibodies used in either of our reported studies. However, it is important to note that the antibody used by Massai and colleagues, 4E10, has also been shown to recognize a 1-573 amino acid N-terminal fragment of Htt in a poly-Q dependent manner, suggesting potential reactivity with one or more additional Htt epitopes [32] . This discrepancy may also contribute to the variation in study ndings.…”

Section: Discussionmentioning

confidence: 99%

“…This discrepancy may also contribute to the variation in study ndings. Without the existence of a calibration set of recombinant tHtt proteins of varying fragment lengths, the measure of tHtt protein, which may be cleaved by numerous proteases, remains a relative quantitative assay, and the potential to compare across assay and antibody platforms is limited [9,32,33] .…”

Section: Discussionmentioning

confidence: 99%

Salivary Huntingtin protein is uniquely associated with clinical features of Huntington’s Disease

Parkin

Corey–Bloom

Snell

et al. 2022

Preprint

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IntroductionMeasuring Huntingtin (Htt) protein in peripheral cells represents an essential step in biomarker discovery for Huntington’s Disease (HD), however to date, investigations into the salivary expression of Htt has been lacking.MethodIn the current study, we quantified total Htt (tHtt) and mutant Htt (mHtt) protein in matched blood and saliva samples using single molecule counting (SMC) immunoassays: 2B7-D7F7 (tHtt) and 2B7-MW1 (mHtt). Matched samples, and clinical data, were collected from 95 subjects: n=19 manifest HD, n=34 premanifest HD (PM), and n=42 normal controls (NC). ResultsTotal Htt and mHtt levels were not correlated in blood and saliva. Plasma tHtt was significantly associated with age, and participant sex; whereas salivary mHtt was significantly correlated with age, CAG repeat length and CAP score. Plasma and salivary tHtt did not differ across cohorts. Salivary and plasma mHtt were significantly increased in PM compared to NC; salivary mHtt was also significantly increased in HD compared to NC. Only salivary tHtt and mHtt were significantly correlated with clinical measures.Conclusions Salivary Htt is uniquely associated with clinical measures of HD and offers significant promise as a relevant, non-invasive HD biomarker. Its use could be immediately implemented into both translational and clinical research applications.

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“…First, we fixed the 2B7 antibody, directed against the N17 portion of HTT, as the capture antibody. This strategy was previously published by us and others to be successful [4,6,9,[17][18][19]. Furthermore, the 2B7 antibody [13] has been used as the SMC capture antibody in combination with MW1 in the CHDI HTT 040 assay for mHTT quantification in clinical trials [20].…”

Section: Testing Of Htt Polyglutamine Length-independent Antibodiesmentioning

confidence: 99%

“…There also seems to be a correlation between the amount of mHTT protein in the CSF and the disease stage [4,5,7]. The quantification of HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wildtype HTT together) is expected to overcome some limitations of mHTT detection, namely its sensitivity to protein length, polyglutamine length, and conformation [4,6,[8][9][10][11]. Such an assay will provide a measure of the effect of HTT-lowering interventions on the overall HTT levels in select biosamples.…”

Section: Introductionmentioning

confidence: 99%

Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay

Fodale

Pintauro

Daldin

et al. 2022

JHD

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Background: The use of biomarkers has become a major component of clinical trial design. In Huntington’s disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein. Objective: We aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples. Methods: An ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples. Results: A novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF. Conclusion: We have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

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Analysis of mutant and total huntingtin expression in Huntington’s disease murine models

Cited by 18 publications

References 36 publications

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

Salivary Huntingtin protein is uniquely associated with clinical features of Huntington’s Disease

Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay

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