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

遗传性痉挛性截瘫是一组遗传和临床异质性单基因运动神经元疾病。皮质脊髓束变性是导致下肢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病例中高达40%。Spastin的微管切断活性已被广泛研究。然而，它与ER的精确相互作用仍然难以捉摸。多种证据支持Spastin在ER内的定位，以及与ER成形蛋白的相互作用。很少有人知道如何Spastin相关的ER结构的变化可能会影响其功能。我们的初步研究结果表明，Spastin对商店操作的钙进入（SOCE）的直接影响。因此，我们推测Spastin与ER组分的直接相互作用调节ER功能和细胞Ca 2+稳态。由于Spastin是HSP中最常见的突变蛋白，因此我们的目标是确定HSP的人类神经元模型中致病性SOCE活性的影响。为了研究这些假说，我们提出了破译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作用。在下一步中，我们将剖析Spastin依赖性SOCE对HSP病理学的影响。由于人类皮质神经元难以在人类完整大脑中研究，因此iPSC技术将用于产生和研究SPG4依赖的SOCE对神经元中HSP病理学的影响。具体来说，我们将扩大Spastin的作用，SOCE使用hiPSC皮层神经元的研究，描绘形态，电生理和转录的影响受损的SOCE在HSP和探索SOCE损伤作为一个共同的致病机制的行动在HSP。

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.