Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.
Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.
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DOI：
10.1093/braincomms/fcaa231
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发表时间：
2021
影响因子：
4.8
通讯作者：
Bates GP
中科院分区：
文献类型：
--
作者：
Landles C;Milton RE;Jean A;McLarnon S;McAteer SJ;Taxy BA;Osborne GF;Zhang C;Duan W;Howland D;Bates GP
亨廷顿病是由CAG/聚谷氨酰胺重复扩增引起的。突变的CAG重复序列经历了躯体不稳定，导致大脑中有数百个CAG；亨廷顿病的遗传修饰者已经表明，躯体不稳定是发病年龄和疾病进展的主要驱动因素。随着CAG重复序列的扩大，外显子1不与外显子2剪接的可能性增加，导致编码全长亨廷顿蛋白的两个转录本以及高致病性和易聚集的外显子1亨廷顿蛋白。针对亨廷顿蛋白基因或转录本的策略是治疗开发的主要焦点。为了更好地了解亨廷顿蛋白的分子发病机制，以及评估亨廷顿蛋白降低方法在临床前研究和临床试验中的影响，追踪亨廷顿蛋白所有异构体的水平是至关重要的。亨廷顿蛋白可溶形式和聚集形式的亨廷顿蛋白生物测定在均相时间分辨荧光和中尺度发现平台上被广泛使用，但这些方法不区分外显子1亨廷顿蛋白和全长亨廷顿蛋白。此外，它们经常被用来在高度扩张的聚谷氨酰胺区域的背景下量化亨廷顿蛋白水平，目前还没有适当的蛋白质标准。在这里，我们致力于开发新的亨廷顿蛋白生物检测方法，以确保所有可溶的亨廷顿蛋白亚型都能被区分开来。我们利用了具有CAG190 CAG的ZQ175亨廷顿病小鼠模型，CAG190 CAG重复大小的蛋白质标准不可用。最初，6种抗体的30种组合在三个技术平台上进行了测试：均相时间分辨荧光、放大发光邻近均相分析和中间尺度发现，并采用分类策略选择最佳分析方法。我们发现，如果没有多谷氨酰胺长度匹配的标准，绝大多数可溶性突变亨廷顿蛋白的分析不能用于定量目的，因为高度扩张的多谷氨酰胺束降低了分析性能。我们的新检测方法与现有的方法相结合，提供了一个跟踪工具包：总可溶性突变亨廷顿蛋白、可溶性外显子1亨廷顿蛋白、可溶性突变亨廷顿蛋白(不包括外显子1亨廷顿蛋白)和总可溶性全长亨廷顿蛋白(突变体和野生型)。还开发了几种跟踪疾病进展的新型聚集分析方法。这些选定的分析方法可以用来比较亨廷顿病小鼠模型中亨廷顿蛋白亚型的水平，并确定这些亚型对遗传或治疗操作的反应。Landles等人。据报道，他们已经开发出新的生物检测方法，提供了一种工具包，用于在三个生物检测平台上检测亨廷顿病小鼠模型组织裂解物中可溶的和聚集的HTT蛋白亚型。
Huntington’s disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington’s disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1 huntingtin protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of huntingtin protein-lowering approaches in preclinical studies and clinical trials. Huntingtin protein bioassays for soluble and aggregated forms of huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 huntingtin protein and full-length huntingtin protein. In addition, they are frequently used to quantify huntingtin protein levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel huntingtin protein bioassays to ensure that all soluble huntingtin protein isoforms could be distinguished. We utilized the zQ175 Huntington’s disease mouse model that has ∼190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyglutamine-length-matched standard, the vast majority of soluble mutant huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant huntingtin protein, soluble exon 1 huntingtin protein, soluble mutant huntingtin protein (excluding the exon 1 huntingtin protein) and total soluble full-length huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare the levels of huntingtin protein isoforms in a wide variety of mouse models of Huntington’s disease and to determine how these change in response to genetic or therapeutic manipulations. Landles et al. report that they have developed novel bioassays to provide a tool kit for the detection of soluble and aggregated isoforms of the HTT protein in tissue lysates from mouse models of Huntington’s disease on three bioassay platforms.
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影响因子：
3.7
作者：
Menalled LB;Kudwa AE;Miller S;Fitzpatrick J;Watson-Johnson J;Keating N;Ruiz M;Mushlin R;Alosio W;McConnell K;Connor D;Murphy C;Oakeshott S;Kwan M;Beltran J;Ghavami A;Brunner D;Park LC;Ramboz S;Howland D
通讯作者：
Howland D
DOI：
10.1073/pnas.1221891110
发表时间：
2013-02-05
影响因子：
11.1
作者：
Sathasivam, Kirupa;Neueder, Andreas;Bates, Gillian P.
通讯作者：
Bates, Gillian P.
DOI：
10.1073/pnas.96.8.4604
发表时间：
1999-04-13
影响因子：
11.1
作者：
Scherzinger, E;Sittler, A;Wanker, EE
通讯作者：
Wanker, EE
影响因子：
3.7
作者：
Macdonald, Douglas;Tessari, Michela A.;Munoz-Sanjuan, Ignacio
通讯作者：
Munoz-Sanjuan, Ignacio
DOI：
10.1097/nen.0b013e318040b2c8
发表时间：
2007-04-01
影响因子：
3.2
作者：
Schilling, Gabriele;Klevytska, Alexandra;Borchelt, David R.
通讯作者：
Borchelt, David R.