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

遗传性痉挛性截瘫（HSP）是一种进行性的痉挛性步态障碍，由皮质脊髓束纤维的长度依赖性退行性变引起。可以区分不同的遗传HSP实体（SPG）。 SPG 48由AP 5 Z1突变引起。其基因产物显示与另外5种蛋白质共沉淀，其中Spatacsin和Spastizin与HSP亚型SPG 11和SPG 15相关。这些蛋白质后来被鉴定为一种新型衔接蛋白复合物5（AP-5）的亚基，AP 5 Z1的基因产物作为其亚基，Spatacsin和Spastizin作为支架的可能组分。最近的证据支持AP-5作为retromer的备用系统的作用，以促进跨膜蛋白再循环到trans-Golgi网络（TGN），其中阳离子非依赖性甘露糖-6-磷酸受体（CI-MPR）和分拣蛋白作为可能的货物受体。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，其特征在于神经元内自发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显示高尔基体的结构改变和自噬通量的阻断。此外，溶酶体从自体溶酶体的再循环，也称为自噬溶酶体重组（ALR）的过程，受到损害。然而，AP-5的破坏为什么会导致高尔基体结构改变、一些高尔基体相关蛋白在溶酶体中积累、在应激条件下自噬通量受损以及ALR受损，这仍然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，Spg 48 KO小鼠的MEF和浦肯野细胞中的Pi 4k 2a丰度和分布发生了变化。由于Pi 4k 2a是产生PI（4）P的关键酶，PI（4）P参与各种膜运输和信号传导事件，包括胞吐和自噬，因此我们假设PI（4）P相关磷脂信号传导的改变可能是不同缺陷的共同基础。因此，我们将比较在AP-5破坏后Pi 4k 2a及其产物PI（4）P的亚细胞分布。我们将进一步研究Pi 4k 2a的过表达、敲低或敲除如何影响自噬、溶酶体功能和ALR。此外，我们将研究Pi 4k 2a的敲低或药理学抑制是否可以恢复AP-5破坏后观察到的缺陷。为了了解Pi 4k 2a的再分布如何与AP-5相关，将AP-5定位在活细胞中至关重要。因此，我们将产生具有AP-5的荧光标记亚基的细胞克隆，并在稳态和诱导自噬和ALR时可视化AP-5。为了获得关于可能的货物受体的进一步线索，我们将进行下拉测定和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.