The role of AP-5 in the endolysosomal system, autophagy and autophagic lysosome reformation

遗传性痉挛性截瘫（HSP）的特征是一种进行性痉挛性步态障碍，由皮质脊髓束纤维的长度依赖性死背变性引起。可以区分不同的遗传HSP实体（SPGs）。SPG48是由AP5Z1突变引起的。其基因产物与另外5个蛋白共沉淀，其中Spatacsin和Spastizin与热休克亚型SPG11和SPG15相关。这些蛋白后来被鉴定为一种新型接头蛋白复合物5 （AP-5）的亚基，其亚基是AP5Z1的基因产物，Spatacsin和Spastizin可能是支架的成分。最近的证据支持AP-5作为逆转录物的备份系统，促进跨膜蛋白再循环到反式高尔基网络（TGN），而阳离子非依赖性甘露糖-6-磷酸受体（CI-MPR）和sortilin可能是货物受体。我们可以证明AP-5亚基的KO小鼠发展为进行性痉挛性步态障碍，其特征是神经元内自身荧光物质的进行性积累和轴突变性。KO小鼠胚胎成纤维细胞在应激条件下表现出高尔基体结构改变和自噬通量阻滞。此外，从自噬溶酶体中回收溶酶体的过程也被称为自噬溶酶体重组（ALR），受到损害。然而，为什么AP-5的破坏会导致高尔基结构改变，溶酶体中一些高尔基相关蛋白的积累，应激条件下自噬通量受损和ALR受损，这是一个悬而未决的问题。我们第一个资助期的初步数据表明，Pi4k2a在Spg48 KO小鼠的mef和浦肯野细胞中的丰度和分布发生了变化。由于Pi4k2a是产生PI(4)P的关键酶，而PI(4)P参与多种膜运输和信号事件，包括胞吐和自噬，我们假设PI(4)P相关磷脂信号的改变可能是不同缺陷的共同基础。因此，我们将比较AP-5被破坏后Pi4k2a及其产物PI(4)P的亚细胞分布。我们将进一步研究Pi4k2a过表达、敲低或敲除对自噬、溶酶体功能和ALR的影响。此外，我们将研究Pi4k2a的敲低或药理学抑制是否可以恢复AP-5破坏时观察到的缺陷。为了了解Pi4k2a的再分配与AP-5的关系，有必要在活细胞中定位AP-5。因此，我们将生成带有荧光标记的AP-5亚基的细胞克隆，并可视化AP-5在稳态和诱导自噬和ALR时的状态。为了获得关于可能的货物受体的进一步线索，我们将进行下拉试验和TurboID方法来确定在基线和饥饿时与AP-5亚基相互作用的蛋白质。

Hereditary spastic paraplegia (HSP) is characterized as a progressive spastic gait disorder that arises from a length-dependent dying back degeneration of corticospinal tract fibers. Distinct genetic HSP entities (SPGs) can be distinguished. SPG48 is caused by mutations in AP5Z1. Its gene product was shown to co-precipitate with 5 additional proteins, among them Spatacsin and Spastizin, which are associated with HSP subtypes SPG11 and SPG15. The proteins were later identified as the subunits of a novel adaptor protein complex 5 (AP-5) with the gene product of AP5Z1 as its subunit and Spatacsin and Spastizin as likely components of the scaffold. Recent evidence supports a role of AP-5 as a backup system of retromer to promote the recycling of transmembrane proteins to the trans-Golgi network (TGN) with the cation independent mannose-6-phosphate receptor (CI-MPR) and sortilin as likely cargo receptors. We could show that KO mice for the subunit of AP-5 develop a progressive spastic gait disorder characterized by progressive accumulation of intraneuronal autofluorescent material and axon degeneration. KO mouse embryonic fibroblasts displayed structural alterations of the Golgi and a block in the autophagic flux under stressed conditions. Moreover, the recycling of lysosomes from autolysosomes, a process also known as autophagic lysosome reformation (ALR), was compromised. Still it is an open question, why the disruption of AP-5 results in the altered Golgi structure, accumulation of some Golgi-related proteins in lysosomes, impaired autophagic flux at stressed conditions and compromised ALR. Our preliminary data from the first funding period suggest that the Pi4k2a abundance and distribution are altered in MEFs and in Purkinje cells of Spg48 KO mice. Because Pi4k2a is a key enzyme for the production of PI(4)P, which is involved in a variety of membrane trafficking and signaling events including exocytosis and autophagy, we hypothesize that alterations of PI(4)P-related phospholipid signaling could be the common basis of the different defects. Therefore, we will compare the subcellular distribution of Pi4k2a and its product PI(4)P upon disruption of AP-5. We will further study how the overexpression, knockdown or knockout of Pi4k2a affects autophagy, lysosome function and ALR. In addition, we will investigate whether the knockdown or the pharmacological inhibition of Pi4k2a can restore defects observed upon disruption of AP-5. To understand how the redistribution of Pi4k2a relates to AP-5, it will be essential to localize AP-5 in living cells. Therefore, we will generate cell clones with a fluorescently tagged subunit of AP-5 and visualize AP-5 at steady state and upon induction of autophagy and ALR. To get further clues about possible cargo receptors we will perform pull-down assays and a TurboID approach to determine proteins interacting with the subunit of AP-5 at baseline and upon starvation.