背景神经退行性tau蛋白病可能基于病理性tau组件的接种而进展,从一个细胞中释放出聚集体,获得进入相邻或连接的小区,并作为其自身在细胞质中复制的特定模板。体外接种反应通常需要几天,然而,播种到复杂的细胞质环境发生在几个小时内,牵涉到一个由未知玩家组成的机器,在急性期控制这个过程。方法我们使用邻近标记来确定在种子暴露后5小时内控制种子扩增的因素。我们将split-APEX2融合到tau重复结构域(RD)的C端,以在接种细胞内tau聚集后5h重建过氧化物酶活性。含有Valosin的蛋白质(VCP/p97)是最高的。VCP包含两种神经退行性疾病的显性突变,多系统蛋白病和空泡tau蛋白病,但其机制作用尚不清楚。我们使用永生化细胞和人类神经元来研究VCP对tau接种的影响。我们将细胞暴露于细胞培养基中的原纤维或脑匀浆,并测量了VCP的各种遗传和化学操作后对细胞内tau聚集的摄取和诱导的影响。结果VCP敲低减少tau接种。化学抑制剂对HEK293Ttau生物传感器细胞和人类神经元的聚集具有相反的作用:ML-240增加了接种效率,而NMS-873减少了它。这些抑制剂仅在种子暴露后8小时内给药时有效,表明VCP在种子加工早期的作用。我们通过基因敲除或敲除在HEK293T生物传感器细胞中筛选了30个VCP辅因子。减少ATXN3,NSFL1C,UBE4B,NGLY1和OTUB1减少了tau播种,NPLOC4也是如此,它也独特地增加了可溶性tau水平。相比之下,FAF2的减少增加了tau播种。结论化学抑制剂和辅因子减少对tau接种的不同作用表明VCP调节了该过程。这与基于VCP的专用细胞质处理复合物一致,该复合物可将种子急剧导向降解与扩增。
UNASSIGNED: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase.
UNASSIGNED: We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (
VCP/p97) was the top hit.
VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of
VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP.
UNASSIGNED: VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30
VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding.
UNASSIGNED: Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.