ABSTRACT: The pathology of PD is caused by accumulation of oligomeric forms of alpha-synuclein (gene SNCA, alphaSyn). The Formation of alphaSyn fibrils is precipitated by glucosylceramides or their glucosylsphingosine metabolites. It has been shown that PD patients have increased plasma concentrations of glucosylceramides even if do not carry a pathogenic mutation in GBA1, which is the gene encoding the lysosomal enzyme glucocerebrosidase 1 necessary for degradation of hexosylceramides (doi: 10.1002/mds.28186.). The results suggested that accumulation of glucosylceramides may be common phenomenon in PD, not restricted to the about 10-15% PD patients carrying a pathogenic GBA1 mutation. To obtain further insight into the pattern of glycosphingolipids and their impact on other lipids we assessed lipidomic patterns in brain tissue of PD mice carrying a double PD-causative Pink1 deletion plus human mutant synuclein, SNCA A53T. The double mutant Pink1-/-SNCA-A53T mice are referred to a Pink1SNCA. The mice are available from Jackson lab as cryopreserved sperm (FVB;129-Pink1tm1Aub X Tg(Prnp-SNCA*A53T)AAub/J; Strain #:017678), and described in doi: 10.1111/nan.12734.
Targeted and untargeted lipidomic studies revealed increased ceramides in the brain (cortex, subcortex, midbrain) of double mutant Pink1-/-SNCAA53T mice compared with wildtype Sv129-FVB control mice. Importantly, Pink1-/-SNCAA53T mice had no clinical symptoms of PD motor disease at the time of tissue sampling at approximately 12 months of age. Ceramides, hexosylceramides (GlcCer or GalCer) and sulfatides (SHexCer) were increased in Pink1-/-SNCAA53T brains whereas acetyl-HexCer (AHexCer) were decreased. Lipidomic analyses further revealed increased diacylglycerols (DG) and lysophosphatidylethanolamines (LPE) in Pink1-/-SNCAA53T brains. DGs are normally low in the brain, and high brain DGs have been suggested to indicate CNS disease. LPEs are contained in Lewy bodies and likely precipitate alphaSyn aggregation, depending on C-chain length and saturation. The results further strengthen the notion that PD is caused or aggravated by pathologic hexosylceramide metabolism.
Lipid analyses
Mice were euthanized with carbon dioxide and blood withdrawal. The brain was dissected for lipidomic and proteomic analyses. Cerebellum and olfactory bulb were removed, and the brain was cut sagittal. Left and right halves were weighed with precision scales and snap frozen on dry ice. Samples were stored at -80 °C until analysis.
Tissue samples were homogenized prior to extraction and 20 µl of the resulting homogenate containing 1 mg of brain tissue were used for sample preparation. A methyl-tert-butyl-ether (MTBE) and methanol-based liquid-liquid extraction was used to allow for the simultaneous analysis of polar metabolites and lipids from the same sample. For chromatographic separation of lipids, a Zorbax RRHD Eclipse Plus C8 1.8 µm 50 x 2.1 mm ID column (Agilent, Waldbronn, Germany) with a pre-column of the same type was used. The mobile phases were (A) 0.1% formic acid and 10 mM ammonium formate and (B) 0.1% formic acid in acetonitrile:isopropanol (2:3, v/v).
Analyses of lipids and polar metabolites was performed on an Orbitrap Exploris 480 with a Vanquish horizon UHPLC system (both Thermo Fisher Scientific, Dreieich, Germany). Data was acquired using Thermo Scientific XCalibur v4.4 (RRID:SCR_014593) and relative quantification was performed in Thermo Scientific TraceFinder 5.1 (RRID:SCR_023045). Full scan spectra were acquired from 180-1500 m/z (lipidomics), 70-700 m/z (metabolomics positive ion mode) or 59-590 m/z (metabolomics negative ion mode) at 120,000 mass resolution each for 0.6 sec, and data dependent MS/MS spectra at 15,000 mass resolution in between. Relative quantification was performed in MultiQuant 3.02 software and peak identification in MasterView v1.1 software (both Sciex, Darmstadt, Germany).
For all lipid analyses, the area under the curve (AUC) was used for quantification. The AUCs were divided by the AUC of the internal standard referred to as AUC/IS. AUCs were transformed to square root of the AUCs (sqrt AUC) for data analysis. For multivariate analyses sqrt AUCs were scaled to have a common mean and SD of 1 (autoscaling in MetaboAnalyst).
Targeted sphingolipid analyses were done as described previously using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), according to procedures described in detail in doi: 10.1111/nan.12734 (further details in S-BSST389). Sphingolipids were separated using an Agilent 1260 Infinity II UHPLC System (RRID:SCR_019365) equipped with a Zorbax C18 Eclipse Plus UHPLC column (50 Å~ 2.1 mm, 1.8 μm, Agilent, Waldbronn, Germany). For mass spectrometry, we used a hybrid triple quadrupole-ion trap mass spectrometer QTRAP 5500 (Sciex, Darmstadt, Germany), equipped with a Turbo-V-source operating in positive ESI mode. Quality control samples of three different concentration levels (low, middle, high) were run as initial and final samples of each run. For all analytes, the concentrations of the calibration standards, quality controls and samples were evaluated by Analyst®TF Software (RRID:SCR_015785) v.1.6 and MultiQuant Software 3.02 (Sciex) using the internal standard method (isotope-dilution mass spectrometry). Calibration curves were calculated by linear or quadratic regression with 1/x weighting or 1/x2 weighting. Variations in accuracy of the calibration standards were less than 15 % over the range of calibration, except for the lower limit of quantification (LLOQ), where a variation in accuracy of 20 % was accepted. Lipid concentrations in tissue are expressed as pg/mg or ng/mg tissue.
