Batra Sushobhna, Vaquer-Alicea Jaime, Manon Victor A, Kashmer Omar M, Lemoff Andrew, Cairns Nigel J, White Charles L, Diamond Marc I
Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX.
bioRxiv. 2023 Aug 30:2023.08.30.555637. doi: 10.1101/2023.08.30.555637.
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 reactions typically take days, yet seeding into the complex cytoplasmic milieu can happen within hours. A cellular machinery might regulate this process, but potential players are unknown.
We used proximity labeling to identify factors that control seed amplification. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity upon seeded intracellular tau aggregation. We identified valosin containing protein (VCP/p97) 5h after seeding. Mutations in VCP underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We utilized tau biosensors, a cellular model for tau aggregation, to study the effects of VCP on tau seeding.
VCP knockdown reduced tau seeding. However, distinct chemical inhibitors of VCP and the proteasome had opposing effects on aggregation, but only when given <8h of seed exposure. ML-240 increased seeding efficiency ~40x, whereas NMS-873 decreased seeding efficiency by 50%, and MG132 increased seeding ~10x. We screened VCP co-factors in HEK293 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. Reduction of FAF2 and UBXN6 increased tau seeding.
VCP uses distinct cofactors to determine seed replication efficiency, consistent with a dedicated cytoplasmic processing complex that directs seeds towards dissolution 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，并作为其自身在细胞质中复制的特定模板。播种反应通常需要数天，但播种到复杂的细胞质环境中可能在数小时内发生。一种细胞机制可能调节这一过程，但潜在的参与者尚不清楚。
我们使用邻近标记来识别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，以在细胞内播种tau蛋白聚集时重建过氧化物酶活性。播种后5小时，我们鉴定出含缬酪肽蛋白（VCP/p97）。VCP的突变是两种神经退行性疾病——多系统蛋白病和空泡性tau蛋白病的基础，但其机制作用尚不清楚。我们利用tau生物传感器（一种tau蛋白聚集的细胞模型）来研究VCP对tau蛋白播种的影响。
VCP敲低降低了tau蛋白播种。然而，VCP和蛋白酶体的不同化学抑制剂对聚集有相反的作用，但仅在种子暴露8小时内给药时如此。ML-240使播种效率提高约40倍，而NMS-873使播种效率降低50%，MG132使播种增加约10倍。我们通过基因敲除或敲低在HEK293生物传感器细胞中筛选VCP辅助因子。ATXN3、NSFL1C、UBE4B、NGLY1和OTUB1的减少降低了tau蛋白播种，NPLOC4的减少也降低了tau蛋白播种，而NPLOC4还独特地增加了可溶性tau蛋白水平。FAF2和UBXN6的减少增加了tau蛋白播种。
VCP使用不同的辅助因子来确定种子复制效率，这与一个专门的细胞质加工复合体一致，该复合体指导种子进行溶解与扩增。
