Landles Christian, Osborne Georgina F, Phillips Jemima, Canibano-Pico Maria, Nita Iulia M, Ali Nadira, Bobkov Konstantin, Greene Jonathan R, Sathasivam Kirupa, Bates Gillian P
Department of Neurodegenerative Disease, Huntington's Disease Centre, Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.
Rancho BioSciences, San Diego, CA 92127, USA.
Brain Commun. 2024 Nov 15;6(6):fcae410. doi: 10.1093/braincomms/fcae410. eCollection 2024.
Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the huntingtin (HTT) protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetic modifiers; therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the pre-mRNA can be alternatively processed to generate the transcript that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more and HTT1a is produced. The allelic series of knock-in mouse models, Q20, Q50, Q80, Q111, CAG140 and zQ175 with polyglutamine expansions of 20, 50, 80, 111, 140 and ∼190, can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23 and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the 'homogeneous time-resolved fluorescence' and 'Meso Scale Discovery' platforms. We tested 32 assays on each platform to detect 'full-length mutant HTT', HTT1a, 'total mutant HTT' (full-length HTT and HTT1a) and 'total full-length HTT' (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species-specific assays that detect full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, while and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG) are only 10% of wild-type, HTT-lowering strategies targeting full-length at sequences 3' to Intron 1 will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers and depletes levels of the HTT1a protein.
亨廷顿舞蹈症是一种遗传性神经退行性疾病，由CAG重复序列扩增引起，该序列编码亨廷顿蛋白（HTT）中的一段多聚谷氨酰胺序列。突变的CAG重复序列不稳定，在整个生命过程中会在特定的脑细胞和外周组织中扩增。已知参与DNA错配修复途径且作用于扩增的基因已被鉴定为基因修饰因子；因此，体细胞CAG重复序列扩增的速率决定了发病年龄和疾病进展速度。在CAG重复序列扩增的情况下，前体mRNA可以进行可变剪接，产生编码易于聚集且具有高度致病性的HTT1a蛋白的转录本。这可能是体细胞CAG重复序列扩增发挥致病作用的一种机制，因为CAG重复序列越长，产生的HTT1a就越多。携带20、50、80、111、140和约190个多聚谷氨酰胺扩增的敲入小鼠模型等位基因系列Q20、Q50、Q80、Q111、CAG140和zQ175，可用于模拟单个神经元内CAG重复序列扩增的分子和细胞后果。通过对皮质裂解物进行蛋白质免疫印迹分析，我们发现突变型HTT水平随CAG重复序列长度的增加而降低；在CAG140和zQ175皮质中，突变型HTT水平分别仅为野生型水平的23%和10%。为了确定检测全长HTT和HTT1a异构体的最佳生物测定方法，我们在“均相时间分辨荧光”和“Meso Scale Discovery”平台上研究了七种特性明确的抗体的两两组合。我们在每个平台上测试了32种测定方法，以检测“全长突变型HTT”、HTT1a、“总突变型HTT”（全长HTT和HTT1a）和“总全长HTT”（突变型和野生型）。这些测定方法均未重现蛋白质免疫印迹法测得的全长突变型HTT水平。我们建议使用检测全长突变型HTT、HTT1a或野生型HTT的异构体特异性和物种特异性测定方法，而不是同时检测多种异构体的方法。我们的发现，即随着CAG重复序列的扩增，全长突变型HTT水平降低，而HTT1a水平升高，对治疗策略具有重要意义。如果含有（CAG）的细胞中突变型HTT水平仅为野生型的10%，那么针对内含子1下游3'序列处全长HTT的降低HTT策略将主要降低野生型HTT，因为这些细胞中的突变型HTT水平已经很低。这些数据支持一种降低HTT1a蛋白水平并使其耗尽的治疗策略。
