iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic/lysosomal dysfunction and impaired calcium homeostasis
Mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher’s disease (GD), are the strongest genetic risk factor for Parkinson’s disease (PD) known to date. To elucidate the mechanisms underlying neurodegeneration in these patients, we generated induced pluripotent stem cells from subjects with GD and PD harboring GBA1 mutations and differentiated them to midbrain dopaminergic neurons. Highly enriched neurons showed a reduction of glucocerebrosidase activity and protein levels, increased glucosylceramide and α-synuclein levels and autophagic/lysosomal defects. Quantitative proteomics profiling revealed an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. We found dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium in mutant neurons. Importantly, correction of the mutations rescued such pathological phenotypes. Our findings provide evidence for a link between GBA1 mutations and complex changes in autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.
Sample Processing Protocol
Please refer to paper: iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic/lysosomal dysfunction and impaired calcium homeostasis David C. Schöndorf, Nature Communications, 2014.
Data Processing Protocol
Tandem mass tag labeling Desalted peptides were resuspended in 35L 100 mM HEPES pH 8.5. TMT reagents (0.8 mg) were dissolved in anhydrous acetonitrile (40 L) of which 4 L was added to the peptides. Acetonitrile (10 L) was added to achieve a final concentration of ca. 30% (v/v). The mixture was incubated at room temperature for 1 hour. The labelling reaction was then quenched by the addition of hydroxylamine to a final concentration of 0.3% (v/v). Each of the TMT-labeled samples were combined at a 1:1:1:1:1:1:1 ratio (equal protein/peptide concentrations). The combined labelled sample was then dried using vacuum centrifugation and desalted using C18 solid-phase extraction (SPE) (Sep-Pak, Waters). Peptide fractionation using high pH reversed-phase (HPRP) The desalted peptides labelled with TMT were separated by HPRP into 96 fractions which were subsequently combined into just 12 fractions for 12 runs on the mass spectrometer. HPRP was performed on an Agilent 1100 quaternary pump equipped with a photodiode array detector (220 and 280-nm wavelength) and an Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID and 220 mm in length). A 50 minute linear gradient from 5% to 30% acetonitrile in 10 mM ammonium bicarbonate pH 8 (flow rate 0.8 mL/min) was used. Following fractionating the samples were acidified to 1% formic acid (v/v), dried, desalted using StageTips, dried and then reconstituted in 5% acetonitrile and 5% formic acid ready for LC-MS/MS analysis. LC-MS (Liquid chromatography – Mass spectrometry) analysis Liquid Chromatography (LC) and Mass Spectrometry (MS) analysis was performed on an LTQ Orbitrap Elite mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) linked to an Accela 600 quaternary LC pump (Thermo) and a Famos autosampler (LC Packings). Flow rates of ~300 nL/minute over the column were achieved using a flow-split method. Peptides were separated on a hand-pulled fused silica microcapillary column (100 µm x 20 cm) that was first packed with ~ 0.5 cm of Magic C4 resin (5 µm, 100 Å, Michrom Bioresources) followed by ~ 20 cm of Maccel C18 AQ resin (3 µm, 200 Å, Nest Group). The total LC-MS run length for each sample was 180 minutes and this consisted of a 150 minute gradient from 6% to 33% acetonitrile in 0.125% formic acid. A recently developed MS3 method was used to overcome the interference problem in acquisition of TMT data 9,10. Briefly, a high resolution MS1 scan in the Orbitrap (300-1500 m/z, 60k resolution, AGC target 1 x 106, maximum injection time 100 ms ) was collected from which the top ten precursors were selected for MS2/MS3 analysis followed by MS3 analysis. The MS2 scan was performed in the quadrupole ion trap (CID, AGC 2 x 103, normalized collision energy 35, max injection time 100 ms) and the MS3 scan was analyzed in the Orbitrap (HCD, 30k resolution, max AGC 1.5 x 105, max injection time 250 ms, normalized collision energy 50). Multiple fragment ions from each MS2 spectrum were selected for MS3 analysis using isolation waveforms with multiple frequency notches 10 11
Schöndorf DC, Aureli M, McAllister FE, Hindley CJ, Mayer F, Schmid B, Sardi SP, Valsecchi M, Hoffmann S, Schwarz LK, Hedrich U, Berg D, Shihabuddin LS, Hu J, Pruszak J, Gygi SP, Sonnino S, Gasser T, Deleidi M. iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis. Nat Commun. 2014 Jun 6;5:4028 PubMed: 24905578