Batra Sushobhna, Vaquer-Alicea Jaime, 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
Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, UT Southwestern Medical Center, 6124 Harry Hines Blvd, Dallas, TX, NS8.334, United States.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, United States.
Mol Neurodegener. 2025 Jan 7;20(1):2. doi: 10.1186/s13024-024-00783-z.
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. Seeding into the complex cytoplasmic milieu happens within hours, implying the existence of unknown factors that regulate this process.
We used proximity labeling to identify proteins that control seed amplification within 5 h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5 h 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 pharmacological manipulations of VCP.
VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on seeding in HEK293T tau biosensor cells and human neurons: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors only functioned when administered within 8 h 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 cytoplasmic processing complex centered 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出来，进入相邻或相连的细胞，并作为其自身在细胞质中复制的特定模板。在数小时内就会发生播种到复杂的细胞质环境中，这意味着存在调节这一过程的未知因素。
我们使用邻近标记来鉴定在种子暴露后5小时内控制种子扩增的蛋白质。我们将分裂的APEX2与tau重复结构域（RD）的C末端融合，以便在细胞内tau蛋白播种聚集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原纤维或脑匀浆中，并在对VCP进行各种基因和药理学操作后，测量对细胞摄取和细胞内tau蛋白聚集诱导的影响。
VCP基因敲低减少了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播种有相反的影响：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小时内给药时起作用，表明VCP在种子处理的早期起作用。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。降低ATXN3、NSFL1C、UBE4B、NGLY1和OTUB1会减少tau蛋白播种，NPLOC4也是如此，它还独特地增加了可溶性tau蛋白水平。相比之下，降低FAF2会增加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。这与以VCP为中心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。
