Naphade Swati, Embusch Alexander, Madushani Kuruwitage Lakshika, Ring Karen L, Ellerby Lisa M
The Buck Institute for Research on Aging, Novato, CA, United States.
California Institute of Regenerative Medicine, San Francisco, CA, United States.
Front Neurosci. 2018 Feb 5;11:736. doi: 10.3389/fnins.2017.00736. eCollection 2017.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT) protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs) as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116) neural stem cells (NSCs) prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases), MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs). Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-β) pathway as the top dysregulated pathway in HD. TGF-β was significantly upregulated in HD-NSCs and addition of TGF-β to HD-NSCs was found to be neuroprotective. To determine if TGF-β regulated MMP and TIMP activity, C116- and HD-NSCs were exogenously treated with recombinant TGF-β. TIMP-1 levels were found to be elevated in response to TGF-β treatment, representing a potential mechanism through which elevated TGF-β levels confer neuroprotection in HD. Studying the mechanism of action of MMPs and TIMPs, and their interactions with mHTT in human isogenic patient-derived NSCs elucidates new mechanisms of HD neurotoxicity and will likely provide novel therapeutics for treatment of HD.
亨廷顿舞蹈症（HD）是一种常染色体显性神经退行性疾病，其特征为进行性运动障碍、精神症状和认知障碍。HD由CAG重复序列扩增引起，该扩增导致突变型亨廷顿蛋白（mHTT）N端编码一段多聚谷氨酰胺残基。mHTT的蛋白水解产生有毒片段，主要在纹状体和皮质中引起神经毒性和大量神经元细胞死亡。抑制mHTT裂解可降低神经元毒性，提示mHTT蛋白水解参与HD发病机制。我们实验室之前针对已知人类蛋白酶进行的一项无偏向性siRNA筛选确定基质金属蛋白酶（MMPs）是mHTT蛋白水解和毒性的调节因子。为了进一步研究HD中MMP的激活情况，我们使用了由HD患者来源的诱导多能干细胞制备的同基因HD及对照校正（C116）神经干细胞（NSCs），来研究MMP及其内源性抑制剂在这个高度相关的模型系统中的作用。我们发现，与对照C116-NSCs相比，HD-NSCs中MMP-2和MMP-9（明胶酶）、MMP-3/10及MMP-14的表达和活性发生了改变。MMP活性失调伴随着基质金属蛋白酶内源性抑制剂（称为基质金属蛋白酶组织抑制剂，TIMPs）水平的相应变化。具体而言，我们观察到HD-NSCs中TIMP-1和TIMP-2水平降低，提示MMPs表达和活性改变的部分原因是这些内源性抑制剂丰度较低。免疫荧光分析显示，HD-NSCs细胞核或聚集体中MMP/TIMP定位增加，提示其与mHTT可能存在相互作用。在HD-NSCs中发现TIMP-1与mHTT聚集体在离散的点状结构中相关联。这些事件共同导致HD中神经毒性增加。之前对这些NSCs的表征显示，转化生长因子β（TGF-β）通路是HD中失调最严重的通路。TGF-β在HD-NSCs中显著上调，并且发现向HD-NSCs中添加TGF-β具有神经保护作用。为了确定TGF-β是否调节MMP和TIMP活性，我们用重组TGF-β对外源处理C116-和HD-NSCs。发现TGF-β处理后TIMP-1水平升高，这代表了TGF-β水平升高在HD中赋予神经保护作用的一种潜在机制。研究MMPs和TIMPs的作用机制及其与人类同基因患者来源NSCs中mHTT的相互作用，阐明了HD神经毒性的新机制，并可能为HD治疗提供新的疗法。
