Pathophysiology of adaptor protein 5 complex related hereditary spastic paraplegia

轴突病以神经元投射纤维变性为特征。遗传性痉挛性截瘫（HSP）主要影响皮质脊髓束纤维，导致进行性痉挛性步态障碍。HSP的特点是广泛的遗传异质性，迄今已鉴定出50多个不同的位点（SPGs）。在这里，我们提出表征三种不同HSP亚型的细胞缺陷，即SPG11， SPG15和SPG48，它们与最近发现的adaptor protein complex 5 （AP5）密切相关。SPG15中突变的Zfyve26和SPG11中突变的Spatacsin都能与AP5复合体共免疫沉淀，而与SPG48相关的AP5Z1本身是AP5复合体的一个亚基。基于细胞系的敲低实验，推测AP5复合体参与了蛋白质向溶酶体的转运。spg15基因敲除小鼠表现出进行性步态障碍伴共济失调，这与皮层神经元丢失和小脑浦肯野细胞丢失有关。作为SPG15基因敲除小鼠内溶酶体缺陷的提示，我们观察到lamp1阳性脑部分向高密度转移，脑提取物中溶酶体酶活性的改变以及lamp1阳性囊泡结构中细胞内自身荧光物质的积累。我们建议通过比较spg15对照细胞和敲除细胞中的内溶酶体蛋白来缩小潜在缺陷。由于在我们实验室产生的spg11敲除小鼠中也明显存在自体荧光物质的积累，因此这些小鼠将被纳入我们的分析。为了更好地描述可能的ap5依赖性和非依赖性缺陷，我们建议另外生成spg48敲除小鼠。通过比较不同小鼠模型，分析SPG15-、SPG11-和spg48缺陷细胞的内溶酶体系统，我们希望了解AP5相关HSP和AP5功能的发病机制。

Axonopathies are characterized by the degeneration of neuronal projection fibers. In hereditary spastic paraplegia (HSP) fibers of the corticospinal tract are primarily affected, which results in a progressive spastic gait disorder. HSP is characterized by a broad genetic heterogeneity with more than 50 different loci (SPGs) identified to date. Here we propose to characterize the cellular defects in three different HSP subtypes, namely SPG11, SPG15, and SPG48, which are closely connected to the recently identified adaptor protein complex 5 (AP5). Both Zfyve26 mutated in SPG15 and Spatacsin mutated in SPG11 can be coimmunoprecipitated with the AP5 complex, while AP5Z1 associated with SPG48 is a subunit of the AP5 complex itself. Based on knockdown experiments in cell lines it has been speculated that the AP5 complex is involved in protein transport to the lysosome. SPG15-knockout-mice generated by us show a progressive gait disorder with ataxia, which is associated with cortical neuron loss and loss of Purkinje cells in the cerebellum. As a hint for an endolysosomal defect in SPG15 knockout mice we observed a shift of Lamp1-positive brain fractions towards higher densities, alterations of lysosomal enzyme activities in brain extracts and the accumulation of intracellular autofluorescent material in Lamp1-positive vesicular structures. We know propose to narrow down the underlying defect by comparing endolysosomal proteins in SPG15-control and -knockout cells. As accumulation of autofluorescent material is also evident in SPG11-knockout-mice generated in our lab, these mice will be included in our analyses. To better delineate possible AP5-dependent and -independent defects, we propose to generate SPG48-knockout-mice in addition. By comparing the different mouse models and analysis of the endolysosomal system of SPG15-, SPG11- and SPG48-deficient cells we hope to get insights into the pathogenesis of AP5 associated HSP and AP5 functions.