Skeletal muscle has recently arisen as a novel regulator of Central Nervous System (CNS) function and aging, secreting bioactive molecules known as myokines with metabolism-modifying functions in targeted tissues, including the CNS. Here we report the generation of a novel transgenic mouse with enhanced skeletal muscle lysosomal and mitochondrial function via targeted overexpression of Transcription Factor E-B (TFEB). We have discovered that the resulting geroprotective benefits in skeletal muscle reduces neuroinflammation and accumulation of tau-associated pathological hallmarks in a mouse model of tau pathology. Muscle TFEB overexpression also significantly ameliorates proteotoxicity, reduces neuroinflammation and promotes transcriptional remodeling of the aging CNS, preserving cognition and memory in aging mice. Our results implicate the maintenance of skeletal muscle function throughout aging to direct regulation of CNS health and disease, and suggest that skeletal-muscle originating factors may act as novel therapeutic targets against age-associated neurodegenerative disorders.

150 µg of lysate was reduced with DTT, alkylated with IAA and digested at 1 µg trypsin to 50 µg protein ratio for 16 hours at 37°C. Digests were acidified with 200 mM HCl, cleaned with MCX columns, dried with a vacuum concentrator, and reconstituted in 0.1% formic acid in water. One µg of each sample with 150 fmol of Pierce Retention Time Calibrant was loaded onto a 30 cm fused silica picofrit 75 µm column and 3.5 cm 150 µm fused silica Kasil1 frit trap loaded with 3 µm Reprosil-Pur C18 reverse-phase resin analyzed with a Thermo Easy nano-LC 1000. The PRTC mixture is used to assess the quality of the column before and during analysis. Four of these quality control runs are analyzed prior to any sample analysis and then after every six sample runs another quality control run is analyzed. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. The 40-minute QC gradient consists of a 0 to 16% B in 5 minutes, 16 to 35% B in 20 minutes, 35 to 75% B in 1 minute, 75 to 100% B in 5 minutes, followed by a wash of 9 minutes and a 30-minute column equilibration. The 110-minute sample LC gradient consists of a 2 to 7% for 1 minute, 7 to 14% B in 35 minutes, 14 to 40% B in 55 minutes, 40 to 60% B in 5 minutes, 60 to 98% B in 5 minutes, followed by a 9-minute wash and a 30-minute column equilibration. Peptides were eluted from the column with a 50°C heated source and electrosprayed into a Thermo Orbitrap Fusion Lumos Mass Spectrometer with the application of a distal 3 kV spray voltage. For the quality control analysis, a cycle of one 120,000 resolution full-scan mass spectrum (350-2000 m/z) followed by a data-independent (DIA) MS/MS spectra on the loop count of 76 DIA MS/MS spectra using an inclusion list at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with an 8 m/z overlapping isolation window. For the sample digest, first a chromatogram library of 6 independent injections is analyzed from a pool of all samples. For each injection a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 100 m/z (400-500 m/z, 500-600 m/z…900-1000 m/z) followed by a DIA MS/MS spectra on the loop count of 26 at 30,000 resolution, AGC target of 4e5, 60 sec maximum injection time, 33% normalized collision energy with a 4 m/z overlapping isolation window. The chromatogram library data is used to quantify proteins from individual sample runs. These individual runs consist of a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 350-2000 m/z, AGC target of 4e5, 100 ms maximum injection time followed by a DIA MS/MS spectra on the loop count of 76 at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with an overlapping 8 m/z isolation window. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher XCalibur data system. Thermo RAW files were converted to mzML format using Proteowizard (version 3.0.19045) using vendor peak picking and demultiplexing. Chromatogram spectral libraries were created using default settings (10 ppm tolerances, trypsin digestion, HCD b- and y-ions) of Walnut in EncyclopeDIA (version 0.6.14) using the Uniprot mouse canonical FASTA. Quantitative spectral libraries were created by mapping spectral to the chromatogram spectral library using EncyclopeDIA requiring a minimum of 3 quantitative ions and filtering peptides at a 1% FDR using Percolator 3.01. The quantitative spectral library is imported into Skyline-daily 4.2.1.19058 with the Uniprot mouse canonical FASTA as the background proteome to map peptides to proteins. The mzML data is imported and all data is TIC normalized.

There are 16 male mouse skeletal muscle (quadriceps) samples - 9 samples are non-trangenic and 7 samples are cTFEB;HSACre bigenic mice that overexpress Transcription Factor E-B (TFEB). Samples are either 6 months (young) or 14 months (old). One reference (sample labeled "MM") is also included in the data which is a pool of all the samples.