Targeted Degradation of Aberrant Tau in Frontotemporal Dementia Patient-Derived Neuronal Cell Models
Tauopathies are a family of neurodegenerative diseases characterized by a shared pathology of aberrant forms of tau protein accumulation leading to neuronal death in focal areas of the brain. Positron emission tomography (PET) tracers that bind to tau aggregates are used to aid diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01- 175 was designed to engage both tau and Cereblon (CRBN), a substrate receptor for the Cullin-4 RING E3 ubiquitin ligase family member (CRL4CRBN), to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, which recapitulate disease phenotypes of tau accumulation, insolubility and toxicity. Furthermore, QC-01-175 had minimal effect on tau levels in neurons from healthy controls, indicating specificity for degradation of disease-relevant forms of tau. QC-01-175 also rescued vulnerability to stress in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau species formed in ex vivo FTD patient-derived neurons are amenable to targeted protein degradation, representing an important advance towards the development of a tau targeted therapeutic.
Sample Processing Protocol
Sample preparation TMT LC-MS3 mass spectrometry. A152T neurons at 6 weeks of differentiation were treated with DMSO vehicle, 1 μM of degrader QC-01-175 or 1 μM negative control QC-03-075 in biological triplicates for 4 h, or pre-treated for 30 min with 10 μM MLN4924 followed by 1 μM QC-01-175 addition for 3.5 h, in biological duplicates. Neuronal cells were washed in PBS (Corning VWR, Radnor PA, USA) and collected at 3000xg centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, protease and phosphatase inhibitors (Roche) were added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 0.25 – 2 mg.mL-1. A micro-BCA assay (Pierce) was used to determine final protein concentration in the cell lysates. 100 μg of protein for each sample were reduced and alkylated as previously described (Donovan et al. 2018). Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein; Fisher Scientific) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 22 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein; Promega) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. The sample channels were combined at a 1:1:1:1:1:1:1:1:1:1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters, Milford MA, USA) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted 4 volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid, and acidified (pH 2−3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse phase HPLC (Agilent LC1260, Santa Clara CA, USA) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on an EasySpray ES803 75 μm inner diameter microcapillary column (Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6–27% acetonitrile in 1.0% formic acid with a flow rate of 350 nL/min. Each analysis used an MS3-based TMT method as described previously (McAlister et al. 2014). The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10. The Advanced Peak Detection (APD) algorithm was disabled.
Data Processing Protocol
LC-MS data analysis. Proteome Discoverer 2.2 (Thermo Fisher Scientific) was used for .RAW file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot human database (September 2016) with both the forward and reverse sequences. Database search criteria are as follows: tryptic with two missed cleavages, a precursor mass tolerance of 2 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of cysteine (57.0211 Da), static TMT labelling of lysine residues and N-termini of peptides (229.163 Da), variable oxidation of methionine (15.9951 Da), variable phosphorylation of serine, threonine and tyrosine (79.966 Da) and variable acetylation (42.011 Da), Methionine-loss (131.040 Da) or methionine-loss + acetylation (83.030 Da) of the protein N-terminus. TMT reporter ion intensities were measured using a 0.003 Da window around the theoretical m/z for each reporter ion in the MS3 scan. Peptide spectral matches with poor quality MS3 spectra were excluded from quantitation (summed signal-to-noise across 11 channels < 200 and precursor isolation specificity < 0.5), and resulting data was filtered to only include proteins that had a minimum of 2 unique peptides identified. Reporter ion intensities were normalized and scaled using in-house scripts in the R framework (Team RCR: A Language and Environment for Statistical Computing http://www.R-project.org/; accessed Nov. 1, 2017). Statistical analysis was carried out using the limma package within the R framework (Ritchie et al. 2015).
Eric Fischer, Dana-Farber Cancer Institute
Eric Fischer, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA., Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA ( lab head )
Silva MC, Ferguson FM, Cai Q, Donovan KA, Nandi G, Patnaik D, Zhang T, Huang HT, Lucente DE, Dickerson BC, Mitchison TJ, Fischer ES, Gray NS, Haggarty SJ. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. Elife. 2019 8 PubMed: 30907729