Mechanisms of HIPK2 in neurodegeneration

项目概要 肌萎缩侧索硬化症 (ALS)，或称卢伽雷氏病，是一种成人发病的神经退行性疾病， 影响上、下运动神经元。 ALS 患者的主要临床特征包括肌肉萎缩和 脊髓运动神经元和上运动神经元及其侧柱轴突逐渐丧失 脊髓。在过去的十年里，这一现象的分子机制得到了巨大的扩展。 由于发现与家族性 ALS 存在因果关系的基因突变，该疾病已成为毁灭性疾病 (FALS) 和散发性 ALS (SALS)。这些“ALS 疾病基因”的特征表明，功能障碍 通过泛素-蛋白酶体途径（蛋白质稳态）实现蛋白质稳态可能有助于 ALS 的发病机制和疾病进展。与遗传数据一致，一个关键的病理特征 FALS 和 SALS 是运动神经元中错误折叠蛋白质的积累，会破坏正常的神经元 功能，包括轴突运输、线粒体生物能量学、基因表达和突触连接。 错误折叠蛋白的持续积累最终引发内质网（ER）应激诱导 细胞死亡，通过我们知之甚少的机制导致神经退行性变。这个提议 重点研究 ER 应激 IRE1α 通路下游的神经元细胞死亡机制。我们表明 通过药物或突变 SOD1 蛋白诱导的 ER 应激会激活高度保守的激酶 HIPK2 （同源结构域相互作用蛋白激酶 2）促进神经元细胞死亡。生化证据表明 HIPK2 作用于 IRE1α-ASK1 下游和 JNK 上游，促进 ER 应激介导的细胞死亡。在 此外，蛋白质组学、磷酸肽图谱和诱变进一步表明 ER 应激激活 HIPK2 通过促进激酶结构域内特定丝氨酸和苏氨酸残基的磷酸化。使用 磷酸化 HIPK2 特异性抗体，我们发现脊髓中的 HIPK2 激活先于症状出现 在 SOD1G93A 小鼠中。重要的是，SOD1G93A;Hipk2-/- 小鼠中 HIPK2 的缺失可减轻神经退行性变，延迟 疾病发作并延长生存期。最后，我们将 HIPK2 在 ER 应激中的发现扩展到人类 使用来自 FALS 和 SALS 患者的大量脊髓组织来研究疾病。综合起来，这些结果 支持以下假设：HIPK2 是 IRE1α 通路下游的重要靶标，可促进 ALS 中内质网应激诱导的神经元细胞死亡。我们提出三个多学科目标来调查 HIPK2 在 ALS 内质网应激诱导的细胞死亡机制中发挥着强有力的作用，但之前并未得到重视。结果 这些研究不仅将解决理解 ALS 疾病机制的重大挑战， 还将提供新的方向来开发潜在的治疗靶点，以减轻 ALS 中的神经元细胞死亡。

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is an adult-onset neurodegenerative disease that affects upper and lower motor neurons. The key clinical features in ALS patients include muscle wasting, and progressive loss of spinal motor neurons and upper motor neurons and their axons in the lateral columns of the spinal cord. The past 10 years have witnessed a tremendous expansion in the molecular mechanisms of this devastating disease thanks to the discoveries of genetic mutations that are causally linked to both familial ALS (FALS) and sporadic ALS (SALS). Characterizations of these “ALS disease genes” suggest that dysfunctions in protein homeostasis via the ubiquitin-proteasome pathways (proteostasis) might contribute to the pathogenesis and disease progression in ALS. Consistent with the genetic data, a key pathological feature in FALS and SALS is accumulation of misfolded proteins in motor neurons, which disrupts normal neuronal functions, including axonal transport, mitochondrial bioenergetics, gene expression, and synaptic connectivity. Persistent accumulation of misfolded proteins eventually triggers endoplasmic reticulum (ER) stress-induced cell death, which leads to neurodegeneration through mechanisms that are poorly understood. This proposal focuses on the neuronal cell death mechanism downstream of the IRE1α pathway of ER stress. We show that ER stress, induced pharmacologically or by mutant SOD1 proteins, activates a highly conserved kinase HIPK2 (homeodomain interacting protein kinase 2) to promote neuronal cell death. Biochemical evidence shows that HIPK2 acts downstream of IRE1α-ASK1 and upstream of JNK to promote ER stress-mediated cell death. In addition, proteomics, phospho-peptide mapping and mutagenesis further show that ER stress activates HIPK2 by promoting phosphorylation on specific Serine and Threonine residues within the kinase domain. Using phospho-HIPK2-specific antibodies, we show that HIPK2 activation in the spinal cord precedes symptom onset in SOD1G93A mice. Importantly, loss of HIPK2 in SOD1G93A;Hipk2-/- mice mitigates neurodegeneration, delays disease onset and prolongs survival. Finally, we have extended our findings of HIPK2 in ER stress to human disease using a large number of spinal cord tissues from FALS and SALS patients. Together, these results support the hypothesis that HIPK2 is an essential target in the downstream of IRE1α pathway that promotes ER stress-induced neuronal cell death in ALS. We propose three multidisciplinary Aims to investigate the robust, yet previously unappreciated role of HIPK2 in ER stress-induced cell death mechanism in ALS. Results from these studies will not only address a major challenge in understanding disease mechanism in ALS, they will also provide new directions to develop potential therapeutic targets to mitigate neuronal cell death in ALS.