Neurodegenerative tauopathies are caused by the 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 stimulate Hsp70 ATPase activity, as JD mutations that block this interaction abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abolished its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

Identification of aggresome-associated proteins by mass spectrometry

DS1 and DS10 cells were grown to confluency in two T300s per condition. Cells were harvested, pelleted, and washed prior to storage as 4x0.5 T300 pellets at -80°C. For each condition, three pellets were thawed on ice, and each was lysed by trituration in 1 mL ice-cold PBS with 0.25% Triton-X containing cOmplete™ mini EDTA-free protease inhibitor cocktail tablet (Roche) at a concentration of 10% w/vol, followed by a 15-minute incubation on ice. Aggresomes and nuclei were collected by centrifuging at 1000xg for 15 minutes, followed by resuspension in 400 μL lysis buffer. An Omni-Ruptor 250 probe sonicator was then used at 30% power for thirty 3-second pulses to partially dissolve the pellets. Samples were centrifuged at 250xg for 5 minutes, and the supernatant was set aside as Fraction B. Pellets were re-homogenized in an additional 400 μL lysis buffer, and sonication and centrifugation was repeated. The final supernatant was added to the previous Fraction B (800 μL volume total). A Bradford assay (Bio-Rad) with BSA standard curve was performed, and the protein concentrations were calculated for the nine fractions. Protein concentrations were normalized to 1.1 μg/μL. 72 μL of 10% sarkosyl was added to 650 μL of each sample in ultra-centrifuge tubes (Beckman Coulter), and samples were rotated end-over-end at room temperature for one hour. Samples were then spun at 186,000xg for 60 minutes, supernatant was set aside, and pellets were washed with 1 mL lysis buffer prior to an additional 30-minute 186,000xg spin. Final pellets were resuspended in 30 μL PBS containing 2% SDS and 2% BME by boiling and trituration. 5 μL of Fraction B supernatants and pellets were loaded onto NuPAGE 10% Bis-Tris gels (Life Technologies) and were run at 150 V for 60 minutes. Gels were washed 1x with water and were then stained with SimplyBlue SafeStain (Life Technologies). Images of gels were captured using a digital Syngene imager.

For LC-MS/MS-based detection of proteins, 20 μL re-suspended Fraction B pellets were run 1 cm onto an Any kD Mini-Protean TGX gel (Bio-Rad) followed by Coomassie Blue staining. Whole lanes were excised using ethanol-washed razor blades, and gel samples were cut into 1 mm chunks. Gel pieces were reduced with DTT and alkylated with iodoacetamides (Sigma-Aldrich) and were then digested overnight with trypsin (Promega). Next, excised proteins were subjected to solid-phase extraction cleanup with Oasis HLB plates (Waters). The processed samples were then analyzed by LC-MS/MS using a Q Exactive mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). Samples were injected onto a 180 μm i.d., 15-cm long column packed with reverse-phase material ReproSil-Pur C18-AQ, 1.9 μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted with a gradient from 1-28% buffer B (80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.08% formic acid in water) over 60 minutes. The mass spectrometer could acquire up to 20 MS/MS spectra for each full spectrum obtained. Raw mass spectrometry data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3^40,41. Peptide identification was performed using the X!Tandem and open MS search algorithm (OMSSA) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended^42,43. Fragment and precursor tolerances of 20 ppm and 0.1 Da were specified, and three miscleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification, and oxidation of Met was set as a variable modification.

Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index software⁴⁴. Finally, spectral counts were added across triplicates. Proteins with a spectral count greater than 5 in DS10, but not identified in DS1, were reported. To calculate enrichment of proteins in the DS10 samples vs DS1 samples, the spectral counts were first negative log10 transformed. False Discovery Rate (FDR) analysis was performed using the multiple unpaired t-tests analysis in Prism (GraphPad). The original FDR method of Benjamini and Hochberg was applied, with the desired FDR set to 1.5%. Differences are reported as DS10 spectral index – DS1 spectral index. The -log10(q-value) is reported. Gene ontology (GO) analysis was conducted via the Metascape gene annotation and analysis resource⁴⁵.

Tau Aggregate Degradation Time Courses

OFF1::DS10 cells were treated with two rounds of the indicated pooled gRNA lentivirus and were maintained in 24-well plates to assess viability. Two weeks later, a time course examining the decay of tau aggregate seeding activity in the various non-lethal knockouts was performed as follows: Confluent 24-wells were resuspended in 1 mL media, and 3.5 μL cells were re-plated into 200 μL total volume in 96-well plates. Tau RD-YFP expression was turned off using 30 ng/mL doxycycline for 1 day, 2 days, 3 days, 4 days, or 5 days. After five days, cells reached confluency and were passaged onto coverslips. 48 hours later, at which point tauRD-YFP expression reached its maximum, cells were fixed, and cells containing or lacking inclusions were manually counted. Six replicates of 150+ cells were counted per condition, and averages were calculated. One-way analysis of variance with Bonferroni’s multiple comparison test was used to assess statistical significance relative to non-target controls.