PDF(Pubmed)
Abstract：
The endocytic pathway is both an essential route of molecular uptake in cells and a potential entry point for pathology-inducing cargo. The cell-to-cell spread of cytotoxic aggregates, such as those of α-synuclein (α-syn) in Parkinson\'s Disease (PD), exemplifies this duality. Here we used a human iPSC-derived induced neuronal model (iNs) prone to death mediated by aggregation in late endosomes and lysosomes of endogenous α-syn, seeded by internalized pre-formed fibrils of α-syn (PFFs). This PFF-mediated death was not observed with parental iPSCs or other non-neuronal cells. Using live-cell optical microscopy to visualize the read out of biosensors reporting endo-lysosome wounding, we discovered that up to about 10% of late endosomes and lysosomes in iNs exhibited spontaneous constitutive perforations, regardless of the presence of internalized PFFs. This wounding, absent in parental iPSCs and non-neuronal cells, corresponded to partial damage by nanopores in the limiting membranes of a subset of endolysosomes directly observed by volumetric focused ion beam scanning electron microscopy (FIB-SEM) in iNs and in CA1 pyramidal neurons from mouse brain, and not found in iPSCs or in other non-neuronal cells in culture or in mouse liver and skin. We suggest that the compromised limiting membranes in iNs and neurons in general are the primary conduit for cytosolic α-syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated α-syn aggregation. Significantly, eradicating the intrinsic endolysosomal perforations in iNs by inhibiting the endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase (PIKfyve kinase) using Apilimod or Vacuolin-1 markedly reduced PFF-induced α-syn aggregation, despite PFFs continuing to enter the endolysosomal compartment. Crucially, this intervention also diminished iN death associated with PFF incubation. Our results reveal the surprising presence of intrinsically perforated endo-lysosomes in neurons, underscoring their crucial early involvement in the genesis of toxic α-syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.
摘要：
胞吞途径既是细胞中分子摄取的重要途径，也是病理诱导货物的潜在切入点。细胞毒性聚集体的细胞间扩散，如帕金森病（PD）中的α-突触核蛋白（α-syn）说明了这种双重性。在这里，我们使用了人类iPSC衍生的诱导神经元模型（iNs），该模型易于通过内源性α-syn的晚期内体和溶酶体中的聚集介导死亡。由内化的预先形成的α-syn原纤维（PFF）接种。在亲本iPSC或其他非神经元细胞中未观察到这种PFF介导的死亡。使用活细胞光学显微镜观察生物传感器的读数，报告内溶酶体受伤，我们发现，iN中高达约10％的晚期内体和溶酶体表现出自发的本构穿孔，无论是否存在内在化的PFF。这个伤人,在亲本iPSC和非神经元细胞中不存在，对应于通过体积聚焦离子束扫描电子显微镜（FIB-SEM）在iN和小鼠大脑的CA1锥体神经元中直接观察到的内溶酶体子集的限制膜中的纳米孔的部分损伤，并且在iPSCs或培养物中的其他非神经元细胞或小鼠肝脏和皮肤中没有发现。我们建议iN和神经元中受损的限制膜通常是细胞溶质α-syn进入内溶酶体腔内的PFF的主要管道，启动PFF介导的α-syn聚集。重要的是,通过抑制内体磷脂酰肌醇-3-磷酸/磷脂酰肌醇5-激酶（PIKfyve激酶），使用阿吡莫德或Vuacolin-1显着减少PFF诱导的α-syn聚集，根除iN的内在内溶酶体穿孔，尽管PFF继续进入内溶酶体区室。至关重要的是，这种干预也减少了与PFF孵育相关的iN死亡。我们的结果揭示了在神经元中存在的内在穿孔的内在溶酶体，强调它们在由内化PFF诱导的毒性α-syn聚集体的形成中的关键早期参与。这一发现为采用PIKfyve激酶抑制作为潜在的治疗策略来对抗突触核蛋白病提供了基础。
