DnaJC7 specifically regulates tau seeding

Abstract

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

Figure 1.. A proteomic approach to identify tau aggregate interactors.

A) Tau aggresomes were partially purified from DS1 and DS10 HEK293 cells expressing tauRD-YFP. Detergent fractionation enabled generation of a sarkosyl-insoluble fraction containing tau aggresomes. Aggregates were recovered from the pellet by running protein on an SDS-PAGE gel and then extracting protein from individual lanes from the gel to be analyzed via LC-MS/MS. B) Volcano plot showing proteins enriched in the sarkosyl-insoluble fraction as a fold enrichment from cells expressing tauRD-YFP aggregates (DS10, dark blue dots) over cells expressing tauRD-YFP that does not form aggregates (DS1, gray dots). The red line indicates an FDR of 1.5%. GO term enrichment analyses of biological processes is also shown for select GO terms: orange dots, chaperone-mediated protein folding (chaperone folding); green dots, regulation of ubiquitination (Ub regulation); teal dots, ubiquitin-dependent protein catabolic process (Ub degradation). C) Spectral indices for a selection of the proteins identified only in the DS10 insoluble fraction. Viable knockouts are shown as green bars. Non-Viable knockouts are shown as black bars. Error bars represent the SEM of three extracted protein SDS-PAGE gel bands.

Figure 2.. DnaJC7 KO uniquely extends tau seed lifespan in dividing cells.

A) Schematic showing the HEK293 OFF1::DS10 system. A selection of the hits from the proteomics screen were knocked out in these cells. The cells are then allowed to grow with tau expression turned OFF for 0–5 days before resuming tau expression. Error bars represent the SEM of six technical replicates. B) The persistence of tau aggregates in OFF1::DS10 cells with the indicated knockout was quantified following 3 (orange bars) or 5 (purple bars) days of repressed tau expression. Error bars represent the SEM of six technical replicates. C) Confocal microscopy images showing tau aggregate organization in the DnaJC7 KO and nontargeting control cells following 0 or 5 days of repression of tau expression. D) Extended time course for tau aggregate clearance in the OFF1::DS10 system with DnaJC7 KO (orange) and the nontargeting (purple) and untreated (green) controls. Error bars represent SEM of six technical replicates. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001

Figure 3.. A targeted genetic screen for modifiers of transient tau seeding identifies specific JDPs.

A) Schematic showing the HEK293 tau biosensor system consisting of tauRD fused to either mCerulean3 or mClover3 fluorescent proteins. The base biosensor cells had each JDP individually knocked out to generate 50 distinct cell lines. Recombinant, sonicated tau fibrils (seeds) are added to the cells to induce seeding of the tauRD constructs, which is detected as a FRET signal via flow cytometry 48 hours after treatment. B) Representative data showing the effects of the individual knockouts of JDPs on tau seeding in biosensor cells, quantified as FRET signal via flow cytometry. Cells were seeded with a dose titration of sonicated tau fibrils. Knockout of DnaJC7 (ΔC7, orange) and DnaJB6 (ΔB6, green) are highlighted. The remaining JDP knockouts are denoted as ΔJDP (gray). Each batch of knockout cell lines was normalized to the DnaJC7 KO seeding signal and then all batches are plotted together. The seeding assay for the DnaJC7 KO and the nontargeting control (Non-Target, purple) were repeated ten times. C) Extended time course harvesting of the tau seeding assay for DnaJB6 KO, DnaJC7 KO, and nontargeting control cells harvested at 24 h (24H, dashed lines) and 48 h (48H, solid lines) timepoints. Coloring as in B). Error bars represent SEM of three technical replicates. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001

Figure 4.. DnaJC7 knockout increases tau seeding across multiple seed sources.

Intracellular A) naked or B) lipofectamine-mediated tau seeding in nontargeting control (Non-Target, purple), DnaJC7 KO (ΔC7, orange), and DnaJB6 KO (ΔB6, green) tau biosensor cells. Cells were seeded with sonicated recombinant tau fibrils (rTau); brain lysates from patients with Alzheimer’s Disease (AD), Progressive Supranuclear Palsy (PSP), or Corticobasal Degeneration (CBD); and cell lysates of DS1, DS9, or DS10 cells. 100 nM or 10 nM of recombinant tau were added for naked and lipo seeding, respectively. 25 μL or 5 μL of patient brain or lysate or 20 μg or 5 μg cell lysate were added for naked and lipo seeding, respectively. NT denotes no treatment with seeds. Error bars represent SEM of three technical replicates. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001

Figure 5.. DnaJC7 regulates tau seeding in multiple experimental approaches.

A) Rescue of DnaJC7 KO in tau biosensor cells with either wildtype (WT C7, green) or HPQ mutant (HPQ C7, dark blue) DnaJC7 constructs. The Ruby fluorophore alone (Ruby Control, orange) and a vehicle control (Vehicle Control, purple) were also added to the DnaJC7 KO cells. B) Overexpression of DnaJC7 constructs in control tau biosensor cells. The cells were transfected with the same constructs as in A). C) Model of DnaJC7 with domains colored as follows: TPR1, green; TPR2A, teal; TPR2B, dark blue; JD, orange. Positions of ALS-associated mutations are shown as purple spheres. D) Rescue of DnaJC7 KO in tau biosensor cells with ALS-associated mutants of DnaJC7 and WT control, sorted by domain location. Rescue with the WT DnaJC7 construct is shown in all domains as a grey, dashed line. Error bars represent SEM of three technical replicates. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001