Mouse husbandry
Mice were housed in standard conditions on a 12-h light–dark cycle and provided with water and standard chow ad libitum. In some experiments, as documented in the main text, mice were provided with water infused with an azido-amino acid or 0.1% methionine, 0.35% cysteine (low methionine) chow (Envigo, TD.160659). All animal procedures were approved by the Administrative Panel on Laboratory Animal Care at Stanford University. All experiments used male mice. No power analysis was performed to determine sample sizes. Randomization and blinding were not performed and were generally not relevant to the experiments reported herein.
Mouse sources
All transgenic mouse lines were obtained from The Jackson Laboratory. Besides the BONCAT lines generated in this study, the generation of which is described below, the transgenic lines used in this study included B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (The Jackson Laboratory, 005359), B6.C-Tg(CMV-cre)1Cgn/J (The Jackson Laboratory, 006054), C57BL/6-Gt(ROSA)26Sortm1(CAG-GFP,-Mars*L274G)Esm/J (The Jackson Laboratory, 028071) and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (The Jackson Laboratory; 007914). Homozygous cre lines were bred to homozygous BONCAT lines or the Ai14 reporter line to generate offspring heterozygous for the cre driver and heterozygous for the BONCAT transgene or Ai14 reporter transgene. Wild-type C57BL/6 mice used for ageing-related AAV transduction experiments were obtained from the National Institute of Aging colony. Wild-type C57BL/6 used for non-ageing-related AAV transduction experiments or used as background controls were obtained from The Jackson Laboratory.
Transgenic mouse generation
The new BONCAT models, PheRS(T413G) and TyrRS(Y43G), introduced in this paper were generated in collaboration with The Jackson Laboratory. sgRNAs (ACTGGAGTTGCAGATCACGA and GCAGATCACGAGGGAAGAGG) were designed to insert a cassette encoding a CMV-IE enhancer/chicken β-actin/rabbit β-globin hybrid promoter (CAG) followed by a floxed stop cassette containing 3×SV40 polyadenylation signals, an eGFP sequence, a viral 2A oligopeptide (P2A) self-cleaving peptide, which mediates ribosomal skipping, and either the gene encoding PheRS(T413G) or TyrRS(Y43G) into the Gt(ROSA)26Sor locus. gRNA, the cas9 mRNA and a donor plasmid were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs with well-recognized pronuclei. Injected embryos were transferred to pseudopregnant females. Surviving embryos were transferred to pseudopregnant females. Resulting progeny were screened by DNA sequencing to identify correctly targeted pups, which were then bred to C57BL/6J mice for germline transmission. This colony was backcrossed to C57BL/6J mice for at least three generations. Sperm was cryopreserved at The Jackson Laboratory. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes. The PheRS(T413G) model (C57BL/6J-Gt(ROSA)26Sorem2(CAG-GFP,-Farsa*T413G)Msasn/J) and the TyrRS(Y43G) model (C57BL/6J-Gt(ROSA)26Sorem3(CAG-GFP,-Yars1*Y43G)Msasn/J) are available for purchase from The Jackson Laboratory (stocks 033734 and 033735, respectively).
Biological replicates
All mice were maintained as individual biological replicates except for the neuron-to-microglia protein-transfer experiments. In the neuron-to-microglia protein-transfer experiment, enriched labelled neuronal protein from microglia was expected to be relatively minimal. Therefore, to ensure detection by LC–MS, the brains of 3 mice, for a total of 900,000 microglia, were pooled to generate a single biological replicate.
Background controls
Background controls are necessary for all BONCAT studies to account for proteins that are nonspecifically enriched by the DBCO pull-down method or nonspecifically labelled with fluorescent alkynes. Background control mice, wild-type mice genetically incapable of incorporating the NCAA, were treated identically with the NCAA relative to their BONCAT counterparts. In all of our experiments, background controls represent the same biological material (brain region, mouse age, protein fraction and/or cell type) as the labelled sample under analysis. In the protein degradation study, background samples for each labelled age and region were derived from the same respective age and region of background control mice. In the protein aggregation study, background samples for each labelled aged aggregate sample were derived from aggregates isolated from aged background control mice. In the microglia study, background samples for each microglia sample derived from labelled mice were from microglia isolated from background control mice.
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