Pathophysiology of adaptor protein 5 complex related hereditary spastic paraplegia

轴突病的特征在于神经元投射纤维的变性。在遗传性痉挛性截瘫（HSP）中，皮质脊髓束的纤维主要受到影响，这导致进行性痉挛性步态障碍。HSP具有广泛的遗传异质性，迄今已鉴定出50多个不同的基因座（SPG）。在这里，我们提出了三种不同的HSP亚型，即SPG 11，SPG 15和SPG 48，这是密切相关的最近确定的衔接蛋白复合物5（AP 5）的细胞缺陷的特点。SPG 15中突变的Zfyve 26和SPG 11中突变的Spatacsin都可以与AP 5复合物共免疫沉淀，而与SPG 48相关的AP 5 Z1是AP 5复合物本身的亚基。基于细胞系中的敲除实验，推测AP 5复合物参与蛋白质转运至溶酶体。我们产生的SPG 15基因敲除小鼠表现出进行性步态障碍伴共济失调，这与小脑皮质神经元丢失和浦肯野细胞丢失有关。作为一个提示内溶酶体缺陷SPG 15基因敲除小鼠，我们观察到Lamp 1阳性脑组分向更高的密度，改变溶酶体酶活性的脑提取物和细胞内自发荧光物质的积累Lamp 1阳性囊泡结构的转变。通过比较SPG 15对照细胞和SPG 15敲除细胞中的内溶酶体蛋白，我们可以缩小潜在缺陷的范围。由于自发荧光物质的积累在我们实验室产生的SPG 11敲除小鼠中也很明显，因此这些小鼠将被纳入我们的分析中。为了更好地描绘可能的AP 5依赖性和非依赖性缺陷，我们建议另外产生SPG 48敲除小鼠。通过比较不同的小鼠模型和分析SPG 15-，SPG 11-和SPG 48-缺陷细胞的内溶酶体系统，我们希望了解AP 5相关HSP的发病机制和AP 5功能。

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.