Studying the pathophysiology of REEP1 and REEP2-associated axonal disorders

遗传性痉挛性截瘫（HSPs）包括一种不断增长和异质性的疾病，主要影响皮质运动神经元，从而导致进行性痉挛性步态障碍。病理学上，热休克蛋白可归类为轴突病，其特征是中枢神经系统长轴突的枯死变性，而细胞体的完整性通常在病程中保持较长时间。SPG31基因与常染色体显性遗传的热休克蛋白有关，并编码er驻留蛋白REEP1。在以前的工作中，我们可以证明REEP1通过其Reticulon结构域诱导膜曲率，并且REEP1敲除小鼠皮质运动神经元细胞体中的内质网在结构上发生了改变。REEP2是在HSP中突变的REEP蛋白家族的另一个成员（SPG72）。与REEP1一样，它具有n端网状结构域，主要在神经元中表达。在这里，我们建议比较和研究REEP1和REEP2在膜形成、内质网结构和内质网功能方面的作用。为此，我们还生成了REEP2敲除小鼠。为了能够详细分析内质网的结构，我们进一步建立了一个报告菌株，在内质网的管腔内表达tdTomato蛋白，该菌株将与REEP1-和reep2敲除小鼠交配。在先进的光学显微镜的帮助下，我们将能够研究活培养的原代神经元或从REEP1或REEP2敲除小鼠获得的组织样本轴突中的内质网。除了分析内质网结构外，我们还将讨论内质网功能（如未折叠蛋白反应、分泌途径或细胞内钙稳态）是否会在REEP1或REEP2或两者均被破坏时受到损害。据预测，HSP相关的SPG31变异要么导致变异蛋白缺失，要么破坏Reticulon结构域，而保持Reticulon结构域完整的REEP1变异表现为显性遗传性运动神经病变（dHMN5）。因此，我们也建立了HMN相关spg31突变的REEP1敲入小鼠模型。与我们的REEP1敲除小鼠进行比较，有望使我们能够解决在HMN中观察到的突变是否通过直接毒性作用起作用。

Hereditary Spastic Paraplegias (HSPs) comprise a growing and heterogeneous group of diseases primarily affecting cortical motoneurons which thus lead to a progressive spastic gait disorder. Pathologically, HSPs can be classified as axonopathies and are characterized by a dying-back degeneration of the long axons of the central nervous system while the integrity of the cell body is often preserved long into the disease course. The gene SPG31 is associated with autosomal-dominantly inherited HSP and encodes the ER-resident protein REEP1. In previous works we could show that REEP1 induces membrane curvature via its Reticulon domain and that the ER in cell bodies of cortical motoneurons of REEP1-knockout mice is structurally altered. REEP2 is another member of the REEP family of proteins mutated in HSP (SPG72). Like REEP1 it is characterized by an N-terminal Reticulon domain and is predominantly expressed in neurons. Here, we propose to compare and study the roles of REEP1 and REEP2 for membrane shaping, ER structure and the consequences for the functionality of the ER. For this purpose we also generated REEP2 knockout mice. To be able to analyze the ER structure in detail we further established a reporter strain, which expresses the tdTomato protein within the lumen of the ER, which will be mated with REEP1- and REEP2-knockout mice. With the help of advanced light microscopy we will thus be able to study the ER in axons of live cultured primary neurons or tissue samples obtained from REEP1 or REEP2 knockout mice. Apart from the analysis of the ER structure we will also address, whether ER functions such as the unfolded protein response, the secretory pathway or intracellular calcium homeostasis are compromised upon disruption of REEP1 or REEP2 or both. While HSP associated SPG31 variants are predicted to either result in the absence of the variant protein or to destroy the Reticulon domain, REEP1 variants, which leave the Reticulon domain intact, manifest as a dominant hereditary motoneuropathy (dHMN5). Therefore we also established a REEP1 knockin mouse model for a HMN associated SPG31mutation. Comparison with our REEP1 knockout mouse will hopefully allow us to address whether mutations observed in HMN act via a direct toxic effect.