Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling
Tau is a scaffolding protein which serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). We have recently shown that amyloid-, the second hallmark of AD, induces de novo protein synthesis of tau. Importantly, this activation was found to be tau-dependent, raising the question of whether FTD-tau by itself affects protein synthesis. To investigate this, we applied non-canonical amino acid labelling to visualise and identify newly synthesised proteins in the K369I tau transgenic K3 mouse model of FTD. This revealed that protein synthesis was massively decreased in neurons containing pathologically phosphorylated tau, a finding confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH-MS proteomics, we identified changes in 247 proteins of the de novo proteome of K3 mice. These included decreased synthesis of the ribosomal proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. Together, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.
Sample Processing Protocol
One brain hemisphere from each mouse was snap-frozen after removing the cerebellum samples were extracted in radioimmunoprecipitation assay (RIPA) buffer (Cell Signalling, #9806) as previously described (Bodea et al, 2017). For quantitative mass spectrometry, AHA-labelled proteins were first purified using BONCAT. 250 μg of extract from each sample was alkylated with iodoacetamide and then incubated with 100 μM DIBO-biotin (Click Chemistry Tools, A112) overnight at 4°C. Biotinylated proteins were purified using 100 μg of streptavidin-coated Dynabeads (ThermoFisher, 11205D), with the beads first being washed in IP wash buffer (0.1% SDS and 0.05% Tween in Tris-buffered saline (TBS) and then in TBS. Sample preparation information dependent acquisition (IDA) and nano-liquid chromatography tandem mass spec (nano LC MS/MS) and SWATH-MS analysis BONCAT-purified proteins bound to beads from 5 WT and 5 K3 samples were placed in Triethylammonium bicarbonate (TEAB) buffer and subsequently reduced with DTT, followed by alkylation with iodoacetamide. Samples were then digested with 80 ng of trypsin overnight. Peptides were then transferred to a new tube acidified with formic acid. Peptides were then diluted in loading buffer (2% acetonitrile, 97.9% water, 0.1% formic acid) and subjected to 1D-IDA nanoLC MS/MS analysis (IDA-LC–MS/MS) and SWATH-MS. Data acquisition via 1D IDA Each sample was injected onto a reverse-phase trap column (Halo-C18, 160Å, 2.7µm, 200 µm x 2 cm) for pre-concentration and desalted with loading buffer. The peptide trap was then switched into line with the analytical column (Halo-C18, 160Å, 2.7µm, 150 µm x 10cm). Peptides were eluted from the column using linear solvent gradients of 5-35% of mobile phase B (99.9% acetonitrile, 0.1% formic acid). After the peptide elution, the column was cleaned with 90% mobile phase B and then equilibrated with 95% mobile phase A (99.9% water, 0.1% formic acid). The reverse phase nano-LC eluent was subject to positive ion nano-flow electrospray analysis in an information dependant acquisition mode (IDA). A time of flight (TOF)-MS survey scan was acquired (m/z 350-1,500, 0.25 s) with the 10 most intense multiply charged ions (2+ to 5+; exceeding 150 counts per second) in the survey scan being sequentially subjected to MS/MS analysis. MS/MS spectra were accumulated for 50 ms in the mass range m/z 100–1,500 with rolling collision energy. Data acquisition via independent acquisition (SWATH-MS) Each sample was prepared as above with the reverse phase nano-LC eluent being subjected to positive ion nano-flow electrospray analysis in a data independent acquisition mode (SWATH). For SWATH MS, m/z window sizes were determined based on precursor m/z frequencies (m/z 400–1,250) in previous IDA data (SWATH variable window acquisition, 60 windows in total). In SWATH mode, first a TOF-MS survey scan was acquired (m/z 350-1,500, 0.05 s) then the 60 predefined m/z ranges were sequentially subjected to MS/MS analysis. MS/MS spectra were accumulated for 60 ms in the mass range m/z 350-1,500 with rolling collision energy optimised for lowed m/z in m/z window +10%. To minimize instrument condition caused bias, SWATH data were acquired in random order for the samples with one blank run between every sample injection.
Data Processing Protocol
Data processing of IDA-data The ten data files generated by IDA-MS analysis were collectively searched with ProteinPilot (v5.0) (Sciex) using the ParagonTM algorithm in thorough mode. UniProt database (171218_Mouse_unipt_Reviewed.fasta, Ref: http://www.uniprot.org) containing 16,944 mouse proteins (Mus musculus) and microtubule-associated protein tau (human) (UniProtKB - P10636) was used for searching the data. Carbamidomethylation of Cys residues was selected as a fixed modification. An Unused Score cut-off was set to 1.3 (95% confidence for identification), and global protein FDR of 1%. Resultant data files were used for the generation of an ion-library with further modifications as described in the following section. Data processing for SWATH-MS quantification ID-IDA result files generated in this study were converted as an ion-library file using PeakView (v2.1) (Sciex). Peptides containing the carbamidomethylation (N-term, Cysteine) and Oxidation (M) were manually retained to include some of the most frequently observed peptide modifications in the ion library. The modified master Ion library and the SWATH data files were imported into PeakView (v2.1). SWATH data were extracted using following parameters: The top 6 most intense fragments of each peptide were extracted from the SWATH data sets. Modified peptides (as mentioned above) were included and shared peptides were excluded. After data processing, peptides with confidence > 99% and FDR < 1% were used for quantitation. Following the SWATH data extraction, proteins that were also identified in PBS treated samples were manually excluded from the final list to reflect the proteins that had been exclusively quantified in AHA-treated samples. The extracted protein peak areas were compared between WT and K3 treated samples using APAF in-house statistical analysis program. The protein peak areas were log transformed, normalised to the total protein peak area for each run and subjected to one sample t-test to compare relative protein peak area between the sample groups.