Shing Kai, Sapp Ellen, Boudi Adel, Liu Sophia, Seeley Connor, Marchionini Deanna, DiFiglia Marian, Kegel-Gleason Kimberly B
Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129.
CHDI Management/CHDI Foundation, New York, NY 10001.
bioRxiv. 2023 Jul 20:2023.01.26.525697. doi: 10.1101/2023.01.26.525697.
Expansion of a triplet repeat tract in exon1 of the HTT gene causes Huntington's disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. No disease modifying treatments exist but lowering mutant huntingtin (mHTT) by gene therapy is a promising approach to treat Huntington's disease (HD). It is not clear when lowering should be initiated, how much lowering is necessary and for what duration lowering should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model we analyzed whole body mHtt lowering initiated at different ages and sustained for different time-periods. Subcellular fractionation (density gradient ultracentrifugation), protein chemistry (gel filtration, western blot, and capillary electrophoresis immunoassay), liquid chromatography and mass spectrometry of lipids, and bioinformatic approaches were used to test effects of mHTT transcriptional lowering. mHTT protein in cytoplasmic and synaptic compartments of the caudate putamen, which is most affected in HD, was reduced 38-52%. Little or no lowering of mHTT occurred in nuclear and perinuclear regions where aggregates formed at 12 months of age. mHtt transcript repression partially or fully preserved select striatal proteins (SCN4B, PDE10A). Total lipids in striatum were reduced in LacQ140 mice at 9 months and preserved by early partial mHtt lowering. The reduction in total lipids was due in part to reductions in subclasses of ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), which are known to be important for white matter structure and function. Lipid subclasses phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses other than ceramide were preserved by early mHtt lowering. Pathway enrichment analysis of RNAseq data imply a transcriptional mechanism is responsible in part for changes in myelin lipids, and some but not all changes can be rescued by mHTT lowering. Our findings suggest that early and sustained reduction in mHtt can prevent changes in levels of select striatal proteins and most lipids but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.
亨廷顿基因(HTT)外显子1中三联体重复序列的扩增会导致亨廷顿舞蹈症(HD)。突变的HTT蛋白(mHTT)存在众多异常相互作用,具有多种多形性效应。目前尚无疾病修饰治疗方法,但通过基因疗法降低突变型亨廷顿蛋白(mHTT)是治疗亨廷顿舞蹈症(HD)的一种有前景的方法。目前尚不清楚何时应开始降低mHTT水平、需要降低多少以及降低多长时间才能取得疗效。此外,尚未评估降低mHTT对脑脂质的影响。我们使用一种可诱导mHtt的小鼠模型,分析了在不同年龄开始并持续不同时间段的全身mHtt降低情况。采用亚细胞分级分离(密度梯度超速离心)、蛋白质化学方法(凝胶过滤、蛋白质免疫印迹和毛细管电泳免疫分析)、脂质的液相色谱和质谱分析以及生物信息学方法来测试mHTT转录降低的效果。在HD中受影响最严重的尾状核壳核的细胞质和突触区室中的mHTT蛋白减少了38 - 52%。在12月龄时形成聚集体的核区和核周区,mHTT几乎没有降低或降低程度很小。mHtt转录抑制部分或完全保留了某些纹状体蛋白(SCN4B、PDE10A)。LacQ140小鼠在9个月时纹状体中的总脂质减少,早期部分降低mHtt可使其得到保留。总脂质的减少部分归因于神经酰胺(Cer)、鞘磷脂(SM)和单半乳糖基二酰甘油(MGDG)亚类的减少,已知这些物质对白质结构和功能很重要。LacQ140小鼠中脂质亚类磷脂酰肌醇(PI)、磷脂酰丝氨酸(PS)和双甲基磷脂酸(BisMePA)也发生了变化。除神经酰胺外的所有亚类水平通过早期降低mHtt得以保留。RNAseq数据的通路富集分析表明,转录机制部分导致了髓磷脂脂质的变化,部分但并非所有变化可通过降低mHTT得到挽救。我们的研究结果表明,早期且持续降低mHtt可以防止某些纹状体蛋白水平和大多数脂质的变化,但长期来看,一种错误折叠且抗降解的mHTT形式会阻碍一些益处。