Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that <i>spg11</i> and <i>zfyve26</i> KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. <b>Abbreviations</b>: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type

遗传性痉挛性截瘫（Hereditary spastic paraplegia, HSP）是一组遗传异质性疾病，以皮质脊髓轴突变性引发的下肢痉挛为核心特征。SPG11与SPG15具有相似的临床病程，二者共同构成了最常见的常染色体隐性遗传性痉挛性截瘫亚型。其对应的两种蛋白均参与巨自噬（macroautophagy，亦称自噬）及自噬性溶酶体再生（autophagic lysosome reformation, ALR）过程。本研究发现，*spg11*与*zfyve26*基因敲除（knockout, KO）小鼠在相同时间进程内出现运动功能障碍，该表型与神经元内自发荧光物质蓄积增加及进行性神经元丢失密切相关。与自噬性溶酶体再生功能受损的表型一致，饥饿处理后的基因敲除小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）中管形成事件减少，且敲除小鼠脑组织切片的神经元内溶酶体数量降低。为验证两种蛋白作用于同一分子通路，我们同时敲除两个基因后未观察到病理表型加重。
进一步研究显示，将磷脂酰肌醇磷酸化为磷脂酰肌醇-4-磷酸（phosphatidylinositol-4-phosphate, PtdIns4P）的PI4K2A（phosphatidylinositol 4-kinase type 2 alpha），可在基因敲除小鼠而非野生型（wild type, WT）脑组织分离得到的自发荧光沉积物中蓄积。在出现症状前的基因敲除小鼠神经元的自噬溶酶体上，即可检测到PI4K2A水平升高。免疫标记实验进一步表明，饥饿处理后的基因敲除MEFs中，LAMP1阳性结构上的PtdIns4P水平更高。作为磷脂招募的自噬性溶酶体再生关键效应分子，网格蛋白（clathrin）与DNM2/动力蛋白2（dynamin 2）与LAMP1阳性结构的结合也出现增强。
由于PI4K2A过表达会损害自噬性溶酶体再生，而敲低PI4K2A则可增加管形成事件，因此我们认为PI4K2A可调控自噬溶酶体上的磷酸肌醇水平，进而影响自噬性溶酶体再生下游效应分子的招募。综上，PI4K2A可能在SPG11与SPG15相关疾病的发病机制中发挥重要作用。

<b>缩写说明</b>：ALR：自噬性溶酶体再生（autophagic lysosome reformation）；AP-5：衔接蛋白复合物5（adaptor protein complex 5）；BFP：蓝色荧光蛋白（blue fluorescent protein）；dKO：双基因敲除（double knockout）；EBSS：Earle平衡盐溶液（Earle’s balanced salt solution）；FBA：足基角（foot base angle）；GFP：绿色荧光蛋白（green fluorescent protein）；HSP：遗传性痉挛性截瘫（hereditary spastic paraplegia）；KO：基因敲除（knockout）；LAMP1：溶酶体相关膜蛋白1（lysosomal-associated membrane protein 1）；MAP1LC3B/LC3：微管相关蛋白1轻链3β（microtubule-associated protein 1 light chain 3 beta）；MEF：小鼠胚胎成纤维细胞（mouse embryonic fibroblast）；SQSTM1/p62：自噬底物衔接蛋白p62（sequestosome 1）；PI4K2A：磷脂酰肌醇4-激酶2α型（phosphatidylinositol 4-kinase type 2 alpha）；PtdIns3P：磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate）；PtdIns4P：磷脂酰肌醇-4-磷酸（phosphatidylinositol-4-phosphate）；RFP：红色荧光蛋白（red fluorescent protein）；SPG：痉挛性截瘫基因（spastic paraplegia gene）；TGN：反式高尔基网络（trans-Golgi network）；WT：野生型（wild type）