Mechanisms of HIPK2 in neurodegeneration

项目总结 肌萎缩侧索硬化症(ALS)，或卢格里克病，是一种成人起病的神经退行性疾病 影响上半部和下部运动神经元。ALS患者的主要临床特征包括肌肉萎缩，以及 延髓外侧柱脊髓运动神经元和上运动神经元及其轴突进行性丢失 脊髓。在过去的10年里，人们见证了这一分子机制的巨大扩展。 由于发现了与家族性肌萎缩侧索硬化症有因果联系的基因突变，导致了毁灭性的疾病 (FALS)和散发性ALS(SALS)。这些“肌萎缩侧索硬化症疾病基因”的特征表明 在蛋白质动态平衡中，通过泛素-蛋白酶体途径(蛋白稳定期)可能有助于 肌萎缩侧索硬化的发病机制和疾病进展。与遗传数据一致，这是一种关键的病理特征 FALS和SALS是错误折叠的蛋白质在运动神经元中堆积，扰乱正常神经元 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触发内质网(ER)应激诱导 细胞死亡，通过鲜为人知的机制导致神经退化。这项建议 重点研究内质网应激下IRE1α通路下游的神经细胞死亡机制。我们证明了 药物或突变的SOD1蛋白诱导的内质网应激激活高度保守的HIPK2 (同源结构域相互作用蛋白激酶2)促进神经细胞死亡。生化证据表明， HIPK2作用于IRE1JNK1下游和α上游，促进内质网应激介导的细胞死亡。在……里面 此外，蛋白质组学、磷酸肽图谱和突变进一步表明，内质网应激激活HIPK2 通过促进激酶结构域中特定丝氨酸和苏氨酸残基的磷酸化。vbl.使用 磷酸化HIPK2特异性抗体，我们表明HIPK2在脊髓中的激活在症状出现之前 在SOD1G93A小鼠中。重要的是，SOD1G93A中HIPK2的缺失；Hipk2-/-小鼠减轻了神经退行性变，延迟 疾病发作并延长生存时间。最后，我们将HIPK2在内质网应激中的发现推广到人类 疾病使用大量来自FALS和SALS患者的脊髓组织。总而言之，这些结果 支持HIPK2是IRE1α途径下游的重要靶点的假说 内质网应激诱导ALS神经细胞死亡。我们提出了三个多学科目标来研究 HIPK2在肌萎缩侧索硬化症内质网应激诱导的细胞死亡机制中的强大作用，但以前未被认识到。结果 这些研究不仅将解决在理解ALS疾病机制方面的主要挑战，他们 也将为开发潜在的治疗靶点以减轻ALS神经细胞死亡提供新的方向。

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.