Deciphering the physiological role of Spastin for the regulation of Store Operated Calcium Entry and its pathological consequences in iPSC Derived SPG4 Neurons

遗传性痉挛截瘫是一组遗传性和临床异质性的单基因运动神经元病。皮质脊髓束退行性变是导致肢体痉挛的常见结构特征。SPG4突变，也称为SPAST，是HSP最常见的原因，在所有常染色体显性HSP病例中发现高达40%。痉挛S的微管切断活动已经得到了广泛的研究。然而，它与内质网的精确相互作用仍然难以捉摸。多条证据支持Spastin在内质网中的定位，以及与内质网形成蛋白的相互作用。关于内质网结构中与Spastin相关的变化如何影响其功能，人们知之甚少。我们的初步研究结果表明，Spastin对商店操作的钙内流(SOCE)有直接影响。因此，我们假设Spastin与ER组分的直接相互作用调节ER功能和细胞内钙稳态。由于Spastin是HSP中最常见的突变蛋白，此外，我们的目标是在HSP的人类神经元模型中确定致病SOCE活性的影响。为了研究这些假说，我们建议破译Spastin在SOCE中的分子功能。我们将研究Spastin对细胞钙稳态的剂量依赖性影响。在下一步，我们将剖析Spastin依赖的SOCE在HSP病理中的作用。由于人类皮质神经元很难在完整的人脑中进行研究，因此将利用IPSC技术产生并研究SPG4依赖的SOCE对神经元HSP病理的影响。具体地说，我们将利用HPSC皮层神经元扩大对Spastin在SOCE中作用的研究，描述HSP中SOCE受损的形态、电生理和转录效应，并探索SOCE损伤在HSP中的常见致病作用机制。

Hereditary Spastic Paraplegia is a genetically and clinically heterogeneous group of monogenic motor neuron disease. Corticospinal tract degeneration is the common structural feature leading to spasticity of the lower limbs. Mutations in SPG4, also referred as SPAST, are the most frequent cause of HSP and found in up to 40% of all autosomal dominant HSP cases. Spastin´s microtubule severing activity has been extensively studied. However, its precise interaction with the ER remains elusive. Multiple lines of evidence support the localization of Spastin within the ER, and interaction with the ER shaping proteins. Very little is known about how Spastin-associated changes in ER structure may affect its function. Our preliminary findings indicate a direct effect of Spastin on Store-operated Calcium entry (SOCE). Therefore, we hypothesize that direct interaction of Spastin with ER component modulates ER function and cellular Ca2+ homeostasis. Since Spastin is the most commonly mutated protein in HSP, additionally we aim to determine effects of pathogenic SOCE activity in a human neuronal model of HSP. To investigate these hypotheses, we propose to decipher the molecular function of Spastin in SOCE. We will investigate Spastin dose dependent effects on cellular calcium homeostasis. In the next step we will dissect Spastin-dependent SOCE effects on HSP pathology. Since human cortical neurons are difficult to study in human intact brains, iPSC technology will be used to generate and investigate SPG4-dependnet SOCE effects on HSP pathology in neurons. Specifically, we will expand the study of Spastin’s role in SOCE using hiPSC cortical neurons, delineate morphological, electrophysiological and transcriptional effects of impaired SOCE in HSP and explore SOCE impairment as a common pathogenic mechanism of action in HSP.