Fig 1: Loss of PLD3 impairs endosomal tubule formation or stability. a HeLa cells were treated with siRNA to abolish PLD3 expression and then fixed and labelled with anti-Snx1. Arrow heads indicate tubules that were rarely observed in PLD3 knockdown cells. Scale bar = 50 µm. b As in a, but cells were labelled with antibodies against the MICALL1 protein. Scale bar = 50 µm. c Snx1-decorated endosomal tubules were imaged in control HeLa cells, PLD3 siRNA-treated HeLa cells and HeLa cells stably expressing PLD3–GFP. Tubules were quantified. The results of two independent experiments are shown (average ± SD) with more than 50 cells counted in each condition in each experiment. Statistical significance was determined by Student’s t test in Microsoft Excel (*P < 0.05). d MICALL1 tubules were counted in three independent experiments, scoring more than 75 cells each time for each condition. Tubules that were PACSIN2- or SNAP29 positive were also counted in more than 100 cells and the data presented graphically. e Cell lysates were analysed by Western blotting. Loss of PLD3 expression does not affect SNX1 levels but does result in reduced levels of MICALL1 and the associated proteins (i.e. EHD1, PACSIN2 and SNAP29)
Fig 2: PLD3 immunoreactivity in Alzheimer’s disease brains (I). PLD3 immunoreactivity was studied in Alzheimer’s disease brains by immunohistochemistry. (a) to (d) Numerous senile plaques containing dystrophic neurites expressing intense PLD3 immunoreactivity in the frontal cortex. Arrowheads in (d) represent swollen neurites in senile plaques. Arrow in (b) indicates pericyte cytoplasmic labeling in the white matter. PLD, phospholipase D.
Fig 3: Reduced expression of PLD3 mRNA and protein in Alzheimer’s disease brains. (a), (b) PLD3 mRNA expression. mRNA expression was studied by quantitative reverse transcription-polymerase chain reaction (qPCR) in human brain tissues derived from a reference of the human frontal cortex (REF), four non-neurological controls (NC), six amyotrophic lateral sclerosis (ALS) patients, four Parkinson’s disease (PD) patients, and seven Alzheimer’s disease (AD) cases. PLD3 expression levels were standardized against those of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and the statistical significance in difference between AD and non-AD groups was evaluated by Student’s t test. *P =0.0499 (PLD3 vs. G3PDH). (c), (d) PLD3 protein expression. Protein expression was studied by western blot in human brain tissues derived from four NC, six ALS patients, four PD patients, and seven AD cases. PLD3 expression levels were standardized against those of HSP60 or G3PDH, and the statistical significance in difference between AD and non-AD groups was evaluated by Student’s t test. *P =0.0416 (PLD3 vs. HSP60). PLD, phospholipase D.
Fig 4: Modification of PLD3 correlates with its activity in neurons(A) PLD3 5' exonuclease activity assay correlates the amount of the soluble PLD3 form with its activity and shows the modification to inhibit the catalytic activity (n = 3, line indicates the mean, error bars show the standard deviation).(B) Directed differentiation protocol of human iPSCs into dopaminergic neurons. NDM, neural differentiation medium.(C) SDS-PAGE and Phos-tag ligand SDS-PAGE separations followed by western blot using the anti-PLD3 antibody show pronounced and nearly quantitative modification, likely AMPylation of PLD3 in dopaminergic neurons compared with the iNGN forward reprogramming.(D) ACP2 SDS-PAGE and Phos-tag ligand SDS-PAGE separations followed by western blot using anti-ACP2 antibody.(E) SDS-PAGE followed by western blot using the anti-PLD3 antibody detects solely the full-length PLD3 in iPSCs, GPCs, and astrocytes.
Fig 5: PLD3–GFP co-localises with APP in the endo-lysosomal system. HeLa cells (a) or SH-SY5Y (b) cells stably expressing PLD3–GFP were fixed and stained for GFP and APP and imaged using full-field (a) or confocal (b) microscopy. Colocalisation was observed and is highlighted in the enlarged inset areas. c HeLa cells stably expressing PLD3–GFP were fixed and stained for GFP and VPS35 and imaged using full-field microscopy. Colocalisation was observed and is highlighted in the enlarged inset areas. d The colocalisation of PLD3–GFP with markers of the Golgi and post-Golgi endo/lysosomal system was quantified using the M1 coefficient of colocalisation. e The localisation of PLD3–GFP was analysed in detail by structured illumination microscopy. A single 110-nm-thick section is shown. GFP signal is observed on the lumenal side of VPS35 endosomes, consistent with the predicted type II membrane topology of PLD3–GFP. Scale bars a–c 10 µm, insets 2 µm, e 5 µm, inset 1 µm
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