Targeted brain metabolomics in two Lafora disease mouse models with or without PPP1R3C-deficiency

Published: 22 October 2024| Version 1 | DOI: 10.17632/vv9n57g87g.1
Contributors:
Felix Nitschke,
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Description

Targeted metabolomic profiles (protein-normalized relative abundances of brain metabolites) were obtained in two Lafora disease mouse models in the presence or absence of PPP1R3C (PTG). Genotypes and biological replicates are as follows: Cohort 1 WT1 (Epm2a+/+, Ppp1r3c+/+) n=9, LKO (Epm2a-/-, Ppp1r3c+/+) n=7, DKO1 (Epm2a-/-, Ppp1r3c-/-) n=10, PKO1 (Epm2a+/+, Ppp1r3c-/-) n=8; WT2 (Nhlrc1+/+, Ppp1r3c+/+) n=11, MKO (Nhlrc1-/-, Ppp1r3c+/+) n=9, DKO2 (Nhlrc1-/-, Ppp1r3c-/-) n=10, PKO2 (Nhlrc1+/+, Ppp1r3c-/-) n=6.

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Steps to reproduce

Mice: Epm2a-/-, herein termed LKO (B6.129-Epm2^atm1.1Kzy); Nhlrc1-/-, herein termed MKO (B6-Nhlrc1^tm1Bmin); Ppp1r3c-/-, herein termed PKO (B6.Cg-Ppp1r3c^tm1Adpr). (Cohort 1) 10 months old WT1, LKO, PKO1, and DKO1 (LKO and PKO double knockout) by crossing mice heterozygous for LKO and PKO; (Cohort 2) 10 months old WT2, MKO, PKO2, and DKO2 (MKO and PKO double knockout) by crossing mice heterozygous for MKO and PKO. Metabolites were extracted from microwave-fixed brain tissue, ground to a fine powder using a liquid nitrogen-cooled mortar and pistil, using cold 80% methanol. 70 mg tissue aliquots were resuspended well in 1 mL of the solvent. The suspension was diluted 1:5 in cold solvent, with extraction continuing in the cold by extensive intermittent vortexing prior to centrifugation of a defined volume (14,000 x g, 4degC, 10 min) and drying of 1mL of supernatant in a speedvac. The pellet was extracted in 1 mL 1 M NaOH, and protein determined using the Pierce BCA protein assay (Cat.# 23225). Protein values were later used for normalization of metabolic profiles. After submission of dried extracts to the metabolomics facility at Children’s Medical Center Research Institute at UT Southwestern data acquisition was performed by reverse-phase chromatography on a 1290 UHPLC liquid chromatography (LC) system interfaced to a high-resolution mass spectrometry (HRMS) 6550 iFunnel Q-TOF mass spectrometer (MS) (Agilent Technologies, CA). The MS was operated in both positive and negative (ESI+ and ESI-) modes. Analytes were separated on an Acquity UPLC® HSS T3 column (1.8 μm, 2.1 x 150 mm, Waters, MA). The column was kept at room temperature. Mobile phase A composition was 0.1% formic acid in water and mobile phase B composition was 0.1% formic acid in 100% ACN. The LC gradient was 0 min: 1% B; 5 min: 5% B; 15 min: 99%; 23 min: 99%; 24 min: 1%; 25 min: 1%. The flow rate was 250 μL min-1. The sample injection volume was 5 μL. ESI source conditions were set as follows: dry gas temperature 225 °C and flow 18 L min-1, fragmentor voltage 175 V, sheath gas temperature 350 °C and flow 12 L min-1, nozzle voltage 500 V, and capillary voltage +3500 V in positive mode and −3500 V in negative. The instrument was set to acquire over the full m/z range of 40–1700 in both modes, with the MS acquisition rate of 1 spectrum s-1 in profile format. Raw data files (.d) were processed using Profinder B.08.00 SP3 software (Agilent Technologies, CA) with an in-house database containing retention time and accurate mass information on 600 standards from Mass Spectrometry Metabolite Library (IROA Technologies, MA) which was created under the same analysis conditions. The in-house database matching parameters were: mass tolerance 10 ppm; retention time tolerance 0.5 min. Peak integration result was manually curated in Profinder for improved consistency and exported as a spreadsheet (.csv). Peak intensities were protein-normalized across samples.

Institutions

University of Texas Southwestern Medical School

Categories

Neurodegenerative Disorder, Epilepsy, Glycogen, Neuro-Inflammation

Funding

National Institute of Neurological Disorders and Stroke

R01NS128437

National Institute of Neurological Disorders and Stroke

P01NS097197

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