VCP regulates early tau seed amplification via specific cofactors

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: APEX2; Cofactors; Disaggregase; Seeding; Tau; VCP; p97.

Fig. 1

VCP identified by proximity labeling from tau aggregation. A Schematic of the TMT-MS study performed for proteomics. B VCP was the most significant hit enriched in the early tau aggregation proteome. Unpaired t- test, P value < 0.000001

Fig. 2

Reduction of VCP inhibits tau seeding. A The tau biosensor seeding assay is based on exposure of cells to exogenous tau seeds. This triggers intracellular aggregation of tau RD (P301S)-clover/cerulean that is detected by FRET. B Timeline of siRNA treatment for VCP knockdown (KD) in biosensors. C KD of VCP reduced tau seeding. Error bars represent S.D. Representative graph from n = 3 independent experiments, with each data point derived from technical triplicate. P value **** < 0.0001; One-Way ANOVA with a 95% confidence interval. D VCP KD increased uptake of tau fibrils labeled with AF-647, measured by flow cytometry. Error bars represent S.E.M (n = 3). P value ** 0.0029; Unpaired t-test with a 95% confidence interval

Fig. 3

Acute exposure of inhibitors differentially impacts tau aggregation. A Timeline of experiment: 1 h exposure of tau biosensor cells to inhibitors, followed by 4 h of 25 nM tau before washout. B ML-240 increased tau seeding. P values: ns = 0.4353, **** < 0.0001 (C) NMS-873 dose-dependently decreased tau seeding. P values: ** 0.0030, **** < 0.0001 (D) Proteasome inhibitor, MG132, increased tau seeding. P values: ns = 0.8492, * 0.0415, ** 0.0024, **** < 0.0001. Error bars represent S.D. Representative data of n = 3 independent experiments, with each data point derived from technical triplicate. E Fluorescence microscopy confirmed the effects of VCP and proteasome inhibition on tau seeding. Scale bar = 50 μm. F Compound treatment did not change tau-AF-647 uptake as measured by flow cytometry. Error bars represent S.E.M (n = 3). P values: ns = 0.9960, 0.9992, 0.1742, in order of bars on the graph. One-Way ANOVA with a 95% confidence interval

Fig. 4

ML-240 induces Gal3 puncta formation. v2L biosensors overexpressing mRuby3-galectin3 were treated with compounds for 1 h prior to addition of tau fibrils and media replacement after 4 h. A Both ML-240 (3 µM) and LLOMe (1 mM) induced Gal3 puncta with no effects of NMS-873 (3 µM). Co-treatment of ML-240 and NMS-873 also induced Gal3 puncta. Representative images of n = 3 independent experiments taken for 5 h of compound alone incubation. Scale bar = 25 μm. B ML-240 increased tau seeding ~ 5x; LLOMe increased seeding by ~ 2x. NMS-873 suppressed the tau seeding enhanced by ML-240. Representative data of n = 3 independent experiments, with each data point derived from technical triplicate. Error bars represent S.D. P values: **** 0.005, * 0.0331, *** 0.0007, ** 0.0017, *** 0.0001 in order of the bars on the graph. One-Way ANOVA with a 95% confidence interval

Fig. 5

VCP inhibition impacts tau seeding early in the process. A Timeline of compound and tau treatments at different time points in the seeding process. B ML-240 increased tau aggregation ~ 16 to 25-fold, but only when administered < 8 h after seed exposure. Representative images are shown in the right panel. Scale bar = 50 μm. P values: **** < 0.0001, ns (16hr) = 0.5892, ns (24hr) = 0.4340, ns (48hr) = 0.3569. (C) NMS-873 decreased tau seeding by ~50%, but only when administered <8h after seed exposure. Representative images are shown in the right panel. Scale bar = 50μm. P values: ** 0.0039, *** 0.0004, ns (16hr) = 0.0788, ns (24hr) = 0.8695, ns (48hr) = 0.0547. Error bars represent S.D. Representative data of n=3 independent experiments, with each data point derived from technical triplicate. One-Way ANOVA with a 95% confidence interval

Fig. 6

VCP inhibitors differentially impact tau seeding in human neurons. A Differentiated iPSC-derived human neurons were transduced with tau RD (P301S)-clover/ruby lentivirus for 48 h followed by seeding in the presence or absence of 100 nM VCP inhibitors. B ML-240 enhanced tau seeding in neuronal biosensors whereas C NMS-873 suppressed tau seeding. Error bars represent S.D. Representative data of n = 3 independent experiments. Each dot represents an image taken per condition, with 4 different locations captured per well, for a total of 5 wells per condition. P value: **** < 0.0001; Paired t-test with a 95% confidence interval

Fig. 7

ML-240 enhances seeding by human brain lysates. A ML-240 increased seeding by AD and CBD brain samples onto v2L P301S tau biosensors and (B) by AD brain sample onto WT tau (3R/4R) biosensors. C ML-240 increased seeding of FTLD type A brain lysate onto TDP-43 biosensors. Error bars represent S.D. Representative data of n = 3 independent experiments, with each data point derived from technical triplicate. P value: **** < 0.0001, *** 0.0008; One-Way ANOVA with a 95% confidence interval. D Representative microscopy images showing effects of ML-240 on tauopathy lysates and TDP-43 seeding. Scale bar = 50 μm

Fig. 8

VCP cofactors differentially regulate tau seeding. VCP cofactors were either knocked out via CRISPR/Cas9 (A-D) or knocked down via siRNA (E–G) in v2L biosensors prior to exposure to increasing amounts of tau fibrils. A Knockout of FAF2 increased tau seeding whereas knockout of B ATXN3, C NSFL1C, and D UBE4B reduced tau seeding. P values: FAF2 (*** 0.0001, **** < 0.0001); ATXN3 (**** < 0.0001); NSFL1C (*** 0.0002); UBE4B (**** < 0.0001). E Knockdown of NGLY1, F NPLOC4, and G OTUB1, decreased tau seeding. P values: NGLY1(**** < 0.0001); NPLOC4 (**** < 0.0001); OTUB1 (*** 0.0004, **** < 0.0001, *** 0.0001). Graphs are representative of n = 3 independent experiments, with each data point derived from technical triplicate. Error bars represent S.D. Some error bars are too small to be visible. H Cofactor KO did not affect tau uptake. P values: ns = 0.9819, 0.9988, 0.9956, 0.9928, in order of bars on the graph. I Cofactor KD did not affect tau uptake. P values: ns = 0.9795, 0.1856, 0.3928, in order of bars on the graph. Error bars represent S.E.M (n = 3). One-Way ANOVA with a 95% confidence interval

Fig. 9

VCP regulation of tau seeding. VCP regulates endolysosomal integrity, which governs the amount of tau seeds escaping into the cytoplasm. VCP then acts on tau seeds that enter the cytoplasm to promote or inhibit either their degradation or amplification. Disaggregase activity of VCP removes monomer for degradation. This might occur at the end of filaments, which would decrease seeding, or from within, which could fragment fibrils and promote seeding. Chemical inhibitors and cofactors bias the process towards different paths