Batra Sushobhna, Vaquer-Alicea Jaime Iii, Valdez Clarissa, Taylor Skyler P, Manon Victor A, Vega Anthony R, Kashmer Omar M, Kolay Sourav, Lemoff Andrew, Cairns Nigel J, White Charles L, Diamond Marc I
UT Southwestern: The University of Texas Southwestern Medical Center.
University of Exeter Faculty of Health and Life Sciences.
Res Sq. 2024 May 22:rs.3.rs-4307848. doi: 10.21203/rs.3.rs-4307848/v1.
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.
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.
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.
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.
神经退行性tau蛋白病可能基于病理性tau蛋白聚集体的播种而进展，即聚集体从一个细胞释放，进入相邻或相连的细胞，并在细胞质中作为自身复制的特定模板。体外播种反应通常需要数天，但在复杂的细胞质环境中的播种在数小时内就会发生，这意味着存在一个由未知参与者组成的机制在急性期控制这一过程。
我们使用邻近标记来识别在种子暴露后5小时内控制种子扩增的因素。我们将分裂型APEX2与tau重复结构域（RD）的C末端融合，以便在细胞内种子诱导的tau蛋白聚集5小时后重建过氧化物酶活性。含缬酪肽蛋白（VCP/p97）是最显著的发现。VCP存在导致两种神经退行性疾病（多系统蛋白病和空泡性tau蛋白病）的显性突变，但其作用机制尚不清楚。我们使用永生化细胞和人类神经元来研究VCP对tau蛋白播种的影响。我们将细胞暴露于细胞培养基中的纤维或脑匀浆中，并在对VCP进行各种基因和化学操作后，测量对细胞摄取和细胞内tau蛋白聚集诱导的影响。
VCP敲低减少了tau蛋白播种。化学抑制剂对HEK293T tau生物传感器细胞和人类神经元中的聚集有相反的影响：ML-240提高了播种效率，而NMS-873降低了播种效率。这些抑制剂仅在种子暴露后8小时内给药才有效，表明VCP在种子处理的早期阶段起作用。我们通过基因敲除或敲低在HEK293T生物传感器细胞中筛选了30种VCP辅助因子。ATXN3、NSFL1C、UBE4B、NGLY1和OTUB1的减少以及NPLOC4的减少均降低了tau蛋白播种，NPLOC4还独特地增加了可溶性tau蛋白水平。相比之下，FAF2的减少增加了tau蛋白播种。
化学抑制剂和辅助因子减少对tau蛋白播种的不同影响表明VCP调节这一过程。这与基于VCP的专门的细胞质加工复合体一致，该复合体将种子迅速导向降解与扩增。
