The project aimed to elucidate the role of N-acylphosphatidylethanolamines (NAPEs) in modulating alpha-synuclein (aSyn) membrane binding and spreading, with the goal of identifying novel lipid-mediated mechanisms involved in Parkinson’s disease (PD).
To achieve this, human neuronal cell lines (SH-SY5Y) with permanent alterations in NAPE metabolism were generated:
(i) PLA2G4e-overexpressing (OE) cells, in which NAPE synthesis is increased via stable transfection of a PLA2G4e–GFP plasmid;
(ii) NAPE-PLD knockout (KO) cells, generated using CRISPR–Cas9, lacking the enzyme responsible for NAPE degradation.
Both models exhibited increased basal NAPE levels compared with wild-type (WT) cells, although to different extents (PLA2G4e OE: +1000%; NAPE-PLD KO: +30%). In parallel, NAPE-PLD–overexpressing cells (NAPE-depleted) were provided by a collaborator, enabling full comparison across the NAPE metabolic spectrum.
Initial analyses showed no significant differences in αSyn endogenous expression or localization across the models, prompting a redirection toward LRRK2 biology, given previous evidence linking NAPE-PLD perturbation to LRRK2 regulation. In NAPE-enriched cells (PLA2G4e OE, NAPE-PLD KO), LRRK2 protein levels and kinase activity (pT72-Rab8a/total Rab8a ratio) were markedly reduced, whereas NAPE-PLD OE cells exhibited the opposite phenotype. Upon treatment with exogenous aSyn fibrils (500 nM, 16 h), all cell lines upregulated LRRK2, but this induction was blunted in NAPE-enriched cells, suggesting that NAPEs stabilize LRRK2 at lower expression/activity levels under both basal and αSyn-challenged conditions.
Because LRRK2 regulates vesicular trafficking and lysosomal homeostasis, lysosomal activity assays were performed. NAPE-enriched cells displayed higher lysosomal activity than WT or NAPE-depleted cells, both in basal conditions and after αSyn exposure. This was accompanied by reduced aSyn accumulation, indicating improved clearance rather than reduced uptake. Indeed, dextran endocytosis assays confirmed that NAPE-enriched cells have enhanced endocytic capacity.
Collectively, these findings identify a protective role of NAPEs, likely mediated through LRRK2 inhibition and restoration of lysosomal function. While a neuroprotective effect of NAPEs had been observed previously, this study provides the first mechanistic explanation linking NAPE signaling to the LRRK2–lysosome axis.
Furthermore, quantitative confocal imaging revealed an increased number of tunneling nanotubes (TNTs) in NAPE-enriched cells compared to WT, suggesting that NAPEs may also regulate intercellular communication and potentially influence αSyn propagation. No difference was observed in NAPE-PLD OE (NAPE-depleted) cells.
Ongoing experiments are assessing aSyn transfer between neuronal and microglial cells (HMC3) across all models to determine whether NAPE metabolism modulates aSyn spreading at the neuron–glia interface.
Overall, the fellowship achieved all major technical and scientific objectives, leading to the establishment of novel cell systems, functional assays, and mechanistic insights that substantially advance understanding of lipid signaling in PD.
