Mechanisms of HIPK2 in neurodegeneration

项目摘要 肌萎缩侧索硬化症（ALS）或Lou Gehrig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， 脊髓运动神经元和上运动神经元及其轴突在侧柱的进行性损失 脊髓在过去的10年里，这一过程的分子机制有了巨大的扩展。 由于发现了与家族性ALS和ALS有因果关系的基因突变， （FALS）和散发性ALS（SALS）。这些“ALS疾病基因”的特征表明， 通过泛素-蛋白酶体途径（蛋白质稳态）的蛋白质稳态可能有助于 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的蛋白质的积累，其破坏正常的神经元 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触发内质网（ER）应激诱导的细胞凋亡。 细胞死亡，导致神经退行性变，其机制尚不清楚。这项建议 重点关注ER应激的IRE 1 α通路下游的神经元细胞死亡机制。我们证明了 突变SOD 1蛋白诱导的内质网应激激活高度保守的激酶HIPK 2 （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， HIPK 2作用于IRE 1 α-ASK 1的下游和JNK的上游，以促进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HIPK 2 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。使用 磷酸化HIPK 2特异性抗体，我们表明HIPK 2在脊髓中的激活先于症状发作 在SOD 1G 93 A小鼠中。重要的是，SOD 1G 93 A; Hipk 2-/-小鼠中HIPK 2的缺失减轻了神经变性，延迟了神经元的凋亡。 疾病发作和生存期。最后，我们将HIPK 2在内质网应激中的发现扩展到人类， 使用大量来自FALS和SALS患者的脊髓组织的疾病。这些结果一起 支持HIPK 2是IRE 1 α通路下游的重要靶点的假设， ALS中ER应激诱导的神经元细胞死亡我们提出了三个多学科的目标，以调查 HIPK 2在ALS中ER应激诱导的细胞死亡机制中的强大但以前未被认识的作用。结果 这些研究不仅解决了理解ALS疾病机制的主要挑战， 还将为开发潜在的治疗靶点以减轻ALS中的神经元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.