Inhibition of HIPK3 by AST487 Ameliorates Mutant HTT-Induced Neurotoxicity and Apoptosis via Enhanced Autophagy
Inhibition of HIPK3 by AST487 Ameliorates Mutant HTT-Induced Neurotoxicity and Apoptosis via Enhanced Autophagy
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AST487 抑制 HIPK3 通过增强自噬改善突变 HTT 诱导的神经毒性和细胞凋亡
DOI：
10.1007/s12264-021-00783-9
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发表时间：
2021-11
期刊：
影响因子：
--
通讯作者：
Fu Y
中科院分区：
文献类型：
--
作者：
Zhang X;Wen X;Al-Ramahi I;Botas J;Lu B;Fu Y
错误折叠和易于聚集的蛋白质的积累是许多神经退行性疾病的共同标志，降低这些蛋白质的水平可能为其中一些疾病的潜在治疗提供有希望的策略[1，2]。其中，亨廷顿病（HD）是由HTT（亨廷顿蛋白）基因突变引起的单基因疾病[3]，该基因编码具有扩展的多聚谷氨酰胺束（polyQ）的突变HTT蛋白（mHTT）。HD的单基因性质为mHTT和疾病病理学之间的因果关系提供了高置信度，使HD适用于测试减少致病蛋白的潜在有益作用。mHTT毒性功能的获得是HD的主要原因，降低mHTT蛋白水平已被证明可有效减轻其毒性。已知mHTT可通过自噬降解[4]，降低其蛋白水平可改善其下游毒性并治疗HD [5]。大量证据支持细胞凋亡在HD中的作用; mHTT可诱导细胞凋亡，进而促进神经元死亡[6]，因此调节自噬和细胞凋亡可能是减少神经元死亡的潜在手段。在表达诱导型mHTT N末端片段的转基因HD小鼠模型中关闭转基因可逆转神经病理学和运动缺陷[7]。其他遗传策略，如递送短发夹RNA、小干扰RNA、反义寡核苷酸[8]和CRISPR/Cas9介导的基因组编辑[9]可以减轻HD小鼠模型中的神经病理学。我们通过无偏遗传筛选鉴定了激酶HIPK 3（同源结构域相互作用蛋白激酶3）作为mHTT蛋白水平的新型调节剂[10]。通过突变敲除HIPK 3或失去其激酶活性可以通过自噬降低mHTT水平[10]。同时，HIPK 3是否有助于神经毒性以及抑制其激酶功能是否可以挽救HD相关表型仍然未知。在这项研究中，我们阐明了HIPK 3在HD发病机制中的潜在作用以及小分子HIPK 3抑制剂AST 487在HD模型中的可能治疗作用，包括HD小鼠原代神经元，人诱导多能干细胞（iPSC）衍生的神经元和HD苍蝇模型。我们首先研究了HIPK 3在HD中的潜在病理作用。在应激培养条件下，mHTT诱导的细胞毒性发生在从其原始基因座表达内源性mHTT蛋白的敲入小鼠模型（HD，Q表示多聚谷氨酰胺）的神经元中[11]。为了诱导细胞凋亡表型，将神经元培养在不含补充剂N2和B27的培养基中，并通过每个神经元中的Tuj 1（神经元特异性III类β-微管蛋白）面积测量其收缩[12]。敲低HIPK 3挽救了凋亡表型（图1A）。为了在人类神经元模型中进一步证实这一点，我们培养了源自人类iPSC的神经元。补充信息在线版本包含可在https://doi.org/10.1007/s12264-02100783-9获得的补充材料。
Accumulation of misfolded and aggregation-prone proteins is the common hallmark of many neurodegenerative disorders, and lowering the levels of these proteins may provide promising strategies for the potential treatment of some of these diseases [1, 2]. Among them, Huntington’s disease (HD) is a monogenic disease caused by mutation of the HTT (huntingtin) gene [3], which encodes the mutant HTT protein (mHTT) with an expanded polyglutamine tract (polyQ). The monogenetic nature of HD provides high confidence for the causal relationship between mHTT and disease pathology, making HD suitable for testing the potential beneficial effects of reducing disease-causing proteins. The gain of toxic function of mHTT is the major cause of HD, and lowering mHTT protein levels has been shown to effectively alleviate its toxicity. mHTT is known to be degraded by autophagy [4], and lowering its protein levels may ameliorate its downstream toxicity and treat HD [5]. Much evidence supports the role of apoptosis in HD; mHTT can induce apoptosis and then promote neuronal death [6], so the modulation of autophagy and apoptosis may be a potential means of reducing neuronal death. Turning off the transgene in a transgenic HD mouse model that expresses inducible mHTT N-terminal fragments reverses the neuropathology and motor deficits [7]. Other genetic strategies such as delivering short-hairpin RNAs, small interfering RNAs, antisense oligonucleotides [8], and CRISPR/Cas9-mediated genome editing [9] can attenuate the neuropathology in HD mouse models. We identified the kinase HIPK3 (homeodomain interacting protein kinase 3) as a novel modulator of mHTT protein levels from an unbiased genetic screen [10]. Knocking-down HIPK3 or loss of its kinase activity by mutagenesis lowers mHTT levels via autophagy [10]. Meanwhile, whether HIPK3 contributes to neurotoxicity and whether inhibiting its kinase function rescues HD-relevant phenotypes remained unknown. In this study, we elucidated the potential role of HIPK3 in HD pathogenesis and the possible therapeutic effects of the small-molecule HIPK3 inhibitor AST487 in HD models, including HD mouse primary neurons, human induced pluripotent stem cell (iPSC)-derived neurons, and HD fly models. We first investigated the potential pathological role of HIPK3 in HD. mHTT-induced cytotoxicity under stressed culture conditions occurred in neurons from a knock-in mouse model (HD, Q indicates polyglutamine) expressing endogenous mHTT protein from its original locus [11]. To induced apoptosis phenotypes, neurons were cultured in a medium without supplements N2 and B27, and their shrinkage was measured by the Tuj1 (neuronspecific class III beta-tubulin) area in each neuron [12]. Knocking down HIPK3 rescued the apoptotic phenotype (Fig. 1A). To further confirm this in a human neuronal model, we cultured neurons derived from human iPSCs Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s12264-02100783-9.
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影响因子：
2.5
作者：
Menalled, LB;Sison, JD;Chesselet, MF
通讯作者：
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影响因子：
15.9
作者：
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DOI：
10.1073/pnas.0501507102
发表时间：
2005-04-19
影响因子：
11.1
作者：
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通讯作者：
Davidson, BL
影响因子：
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作者：
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