Sanyal Anwesha, Scanavachi Gustavo, Somerville Elliott, Saminathan Anand, Nair Athul, Oikonomou Athanasios, Hatzakis Nikos S, Kirchhausen Tom
Department of Cell Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, 200 Longwood Ave, Boston, MA 02115, USA.
bioRxiv. 2024 Jan 1:2023.12.30.573738. doi: 10.1101/2023.12.30.573738.
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.
内吞途径既是细胞内分子摄取的重要途径，也是致病物质的潜在进入点。细胞毒性聚集体在细胞间的传播，如帕金森病（PD）中α-突触核蛋白（α-syn）的聚集体，就体现了这种双重性。在这里，我们使用了一种源自人诱导多能干细胞（iPSC）的诱导神经元模型（iNs），该模型易在内源性α-syn的晚期内体和溶酶体中聚集介导下死亡，由内化的α-syn预形成纤维（PFFs）引发。亲本iPSC或其他非神经元细胞未观察到这种PFF介导的死亡。使用活细胞光学显微镜观察报告内体-溶酶体损伤的生物传感器读数，我们发现，无论内化的PFFs是否存在，iNs中高达约10%的晚期内体和溶酶体表现出自发性的组成性穿孔。亲本iPSC和非神经元细胞中不存在这种损伤，这与通过体积聚焦离子束扫描电子显微镜（FIB-SEM）在iNs和小鼠脑CA1锥体神经元中直接观察到的一部分内溶酶体的限制膜上的纳米孔造成的部分损伤相对应，而在培养的iPSC或其他非神经元细胞以及小鼠肝脏和皮肤中未发现这种损伤。我们认为，iNs和一般神经元中受损的限制膜是胞质α-syn进入被困在内体-溶酶体腔中的PFFs的主要通道，从而引发PFF介导的α-syn聚集。值得注意的是，通过使用阿匹莫德或空泡菌素-1抑制内体磷脂酰肌醇-3-磷酸/磷脂酰肌醇5-激酶（PIKfyve激酶）消除iNs中固有的内体-溶酶体穿孔，显著减少了PFF诱导的α-syn聚集，尽管PFFs继续进入内体-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。我们的结果揭示了神经元中存在令人惊讶的固有穿孔内体-溶酶体，强调了它们在由内化PFFs诱导的有毒α-syn聚集体形成过程中的关键早期参与。这一发现为采用PIKfyve激酶抑制作为对抗突触核蛋白病的潜在治疗策略提供了基础。
