DOI: 10.25345/c5gt5ft24
title: MassIVE MSV000097012 - Altered mechanical properties of astrocytes lacking MLC1; implications for the leukodystrophy MLC
title: Loss of function of the astrocyte protein MLC1 causes Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), a leukodystrophy characterized by white matter edema and slow neurological deterioration. MLC1 dysfunction leads to swelling of perivascular astrocyte endfeet and an impaired attachment of endfeet to blood vessels. In isolated primary astrocytes, loss of MLC1 impairs recovery of astrocytes from cell swelling. MLC1 modulates gating of mechanosensitive ion channels involved in volume regulation, but the cellular function of MLC1 is not completely understood. The cytoskeleton plays a crucial role in cell volume regulation, and interactions between the cytoskeleton and cell membrane affect the properties of mechanosensitive ion channels. Therefore, we investigated whether primary Mlc1-null astrocytes from mice show a disruption in their mechanical properties. Proteomic analysis confirmed dysregulation of several cytoskeleton-related pathways in Mlc1-null astrocytes. Total protein extracts derived from Wild type and Mlc1-null astrocyte pellets were lysed in a buffer containing 1 percent Triton X-100, 0.5percent sodium deoxycholate, 150 mm NaCl, 10 mm Hepes (pH 7.4) and protease (Sigma-Aldrich, P8340) and phosphatase (Roche Diagnostics, Mannheim, Germany) inhibitor cocktail. Lysates were passed through a 26-gauge needle, incubated on ice for 20 min and centrifuged at 14.000 rpm for 20 min at 4 degree. Samples obtained were analyzed in triplicate: 50 ug of cell lysates derived from each sample were sonicated in an ultrasonic bath for 20 min, incubated in 30 mM TCEP for 10 min and then in 20 mM iodoacetamide for 30 min in the dark at RT. Proteins were precipitate in a mix of acetone, methanol, and ethanol mixture (25, 25, 50) over night at -20 degree. The protein pellet was centrifuged and digested with 12 ng/ul trypsin (Promega Corporation, WI, United States) in 25 mM ammonium bicarbonate and 1 M urea at 37 degree overnight. Formic acid was added to a final concentration of 0.1 percent to stop the enzymatic digestion and an aliquot of the resulting peptide solution was injected in an Ultimate 3000 UHPLC (Dionex, Thermo Fisher Scientific Waltham, Massachusetts, USA) coupled with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Peptides were desalted on a trap column (Acclaim PepMap 100 C18, Thermo Fisher, Scientific Waltham, Massachusetts, USA) and then separated on a 45-cm-long silica capillary (Silica Tips FS 360-75-8, New Objective, MA, United States), packed in-house with a C18, 1.9 um, 100 A resin (Michrom BioResources, CA, United States). The analytical separation ran for 180 min using a gradient of buffer A (5 percent acetonitrile and 0.1 percent formic acid) and buffer B (95 percent acetonitrile and 0.1 percent formic acid). The gradient started with 5 percent of buffer B, which rose to 6 percent in 5 min, to 32 percent in 130 min, to 55 percent in 25 min, and up to 80 percent in 4 min and then washing and equilibrating steps were added until a total 180 min run time. Full scan MS data were acquired in the 350-1,550 m over z mass range in the Orbitrap with 120K resolution. Data-dependent acquisition was performed in top-speed mode (3 s long maximum total cycle): the most intense precursors with charge greater than 1 were selected through a monoisotopic precursor selection (MIPS) filter, quadrupole isolated and fragmented by 30 percent higher-energy collision dissociation (HCD). Fragment ions were analyzed in the ion trap with rapid scan rate. Raw data were analyzed by the software Proteome Discoverer 2.4 (Thermo Fisher Scientific) using the reviewed version of SwissProt mouse (taxonomy 10090) database containing 17090 sequences. Spectral matches were filtered using Percolator node, with 1percent false discovery rate (FDR) based on q values. Only master proteins were considered and specific trypsin cleavages with two miss-cleavages were admitted. Cysteine carbamydomethylation was set as static modification, while methionine oxidation and N-acetylation on protein terminus were set as variable modifications. 10 ppm and 0.6 Da tolerance were considered for MS and MSMS data assignment, respectively. Quantification was based on precursor ion intensity of unique and razor peptides and abundance values were normalized on total peptide amount. Quantitative results were analyzed by Proteome Discoverer: protein ratio calculation was based on pairwise ratio. Proteins were filtered considering only those quantified at least twice in one group. Of this group, proteins were considered up or down regulated in Mlc1 null vs wild type if log2 ratio