Periodic Reporting for period 1 - LipiSyn (Study of membrane lipid composition and alpha-synuclein spreading)
Période du rapport: 2023-09-01 au 2025-08-31
Neurodegenerative diseases (NDs), including Parkinson’s disease (PD), represent an escalating health and societal challenge with growing prevalence due to population aging. These disorders, characterized by progressive neuronal loss and the accumulation of misfolded proteins, impose a substantial economic and emotional burden on patients, families, and healthcare systems. Despite decades of research, disease-modifying therapies remain elusive, largely due to an incomplete understanding of the molecular mechanisms underlying neuronal dysfunction and protein aggregation.
Lipids, as fundamental constituents of cellular membranes, are emerging as key modulators of protein aggregation, trafficking, and toxicity. Specific lipid species—such as phosphoinositides, sphingolipids, and oxysterols—regulate autophagy, lysosomal activity, and mitochondrial quality control, processes that are crucial for neuronal homeostasis. Alterations in lipid metabolism therefore profoundly affect the clearance of aggregation-prone proteins such as α-synuclein (aSyn), a central player in PD pathogenesis.
Within this framework, the present MSCA project aimed to elucidate the contribution of a specific class of lipids, the N-acylphosphatidylethanolamines (NAPEs), to α-synuclein processing and spreading. NAPEs, normally a minor component of brain membranes, accumulate under pathological conditions, suggesting a potential role in disease-associated lipid remodeling. The project’s main objectives were to: (i) investigate how NAPEs influence aSyn intracellular localization and trafficking in neuronal cultures; (ii) determine their involvement in aSyn intercellular transfer in vitro; and (iii) characterize their effects in vivo using relevant animal models.
By integrating advanced lipidomics, cell biology, and neurobiology approaches, this research provides novel mechanistic insights into the interplay between lipid metabolism and protein aggregation. Ultimately, the project contributes to the European priority of improving the understanding of brain function and dysfunction, laying the groundwork for future therapeutic strategies targeting lipid–protein interactions in PD and other neurodegenerative diseases.
Lipids, as fundamental constituents of cellular membranes, are emerging as key modulators of protein aggregation, trafficking, and toxicity. Specific lipid species—such as phosphoinositides, sphingolipids, and oxysterols—regulate autophagy, lysosomal activity, and mitochondrial quality control, processes that are crucial for neuronal homeostasis. Alterations in lipid metabolism therefore profoundly affect the clearance of aggregation-prone proteins such as α-synuclein (aSyn), a central player in PD pathogenesis.
Within this framework, the present MSCA project aimed to elucidate the contribution of a specific class of lipids, the N-acylphosphatidylethanolamines (NAPEs), to α-synuclein processing and spreading. NAPEs, normally a minor component of brain membranes, accumulate under pathological conditions, suggesting a potential role in disease-associated lipid remodeling. The project’s main objectives were to: (i) investigate how NAPEs influence aSyn intracellular localization and trafficking in neuronal cultures; (ii) determine their involvement in aSyn intercellular transfer in vitro; and (iii) characterize their effects in vivo using relevant animal models.
By integrating advanced lipidomics, cell biology, and neurobiology approaches, this research provides novel mechanistic insights into the interplay between lipid metabolism and protein aggregation. Ultimately, the project contributes to the European priority of improving the understanding of brain function and dysfunction, laying the groundwork for future therapeutic strategies targeting lipid–protein interactions in PD and other neurodegenerative diseases.
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.
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.
This project extends the state of the art by identifying a previously unknown NAPE–LRRK2–lysosome regulatory axis and its influence on αSyn homeostasis. Prior studies had described NAPEs as neuroprotective lipids but lacked a mechanistic explanation. The results of this work now provide that mechanistic foundation.
Key advances include:
1. Generation of novel neuronal models with stable manipulation of NAPE metabolism.
These SH-SY5Y lines (PLA2G4e OE, NAPE-PLD KO, NAPE-PLD OE) represent the first human neuronal systems allowing long-term, controlled modulation of NAPE levels. They will be valuable tools for future research into lipid-driven processes in neurodegeneration.
2. Discovery of a NAPE–LRRK2 interaction pathway.
Increased NAPE levels downregulate both LRRK2 expression and kinase activity, whereas NAPE depletion produces the opposite effect. As LRRK2 hyperactivity is linked to lysosomal dysfunction and aSyn accumulation in PD, this identifies NAPEs as endogenous negative regulators of LRRK2, offering a new entry point for therapeutic intervention.
3. Functional restoration of lysosomal activity and reduction of aSyn accumulation.
NAPE-enriched cells displayed enhanced lysosomal function and decreased αSyn burden even upon fibril exposure, suggesting that NAPE signaling promotes protein clearance. This provides a molecular rationale for the previously observed neuroprotective effects of NAPEs.
4. Identification of a link between NAPE metabolism and intercellular communication.
The increased formation of TNTs in NAPE-enriched cells introduces a new conceptual dimension, suggesting that lipid metabolism influences not only intracellular degradation pathways but also aSyn spreading between cells.
Together, these findings bridge lipid signaling and PD molecular pathology, revealing that NAPE enrichment simultaneously dampens LRRK2 activity, enhances lysosomal function, and may modulate intercellular communication. This integrated model advances the understanding of how lipid metabolism impacts neurodegenerative cascades at multiple levels.
Future impact and exploitation potential:
These results pave the way for translational studies aimed at pharmacologically modulating NAPE metabolism. Potential directions include:
• in vivo validation of the NAPE–LRRK2–lysosome axis in rodent PD models;
• identification of bioactive NAPE analogues with improved stability and brain permeability;
• exploration of NAPE-mimetic or NAAA-inhibiting compounds as novel therapeutic candidates for PD and related synucleinopathies;
• integration of lipidomics and proteomics data to uncover downstream effectors and biomarkers of NAPE signaling.
The combination of mechanistic insight and translational potential ensures that the outcomes of this project contribute significantly beyond the state of the art, establishing a foundation for future lipid-based neuroprotective strategies.
Key advances include:
1. Generation of novel neuronal models with stable manipulation of NAPE metabolism.
These SH-SY5Y lines (PLA2G4e OE, NAPE-PLD KO, NAPE-PLD OE) represent the first human neuronal systems allowing long-term, controlled modulation of NAPE levels. They will be valuable tools for future research into lipid-driven processes in neurodegeneration.
2. Discovery of a NAPE–LRRK2 interaction pathway.
Increased NAPE levels downregulate both LRRK2 expression and kinase activity, whereas NAPE depletion produces the opposite effect. As LRRK2 hyperactivity is linked to lysosomal dysfunction and aSyn accumulation in PD, this identifies NAPEs as endogenous negative regulators of LRRK2, offering a new entry point for therapeutic intervention.
3. Functional restoration of lysosomal activity and reduction of aSyn accumulation.
NAPE-enriched cells displayed enhanced lysosomal function and decreased αSyn burden even upon fibril exposure, suggesting that NAPE signaling promotes protein clearance. This provides a molecular rationale for the previously observed neuroprotective effects of NAPEs.
4. Identification of a link between NAPE metabolism and intercellular communication.
The increased formation of TNTs in NAPE-enriched cells introduces a new conceptual dimension, suggesting that lipid metabolism influences not only intracellular degradation pathways but also aSyn spreading between cells.
Together, these findings bridge lipid signaling and PD molecular pathology, revealing that NAPE enrichment simultaneously dampens LRRK2 activity, enhances lysosomal function, and may modulate intercellular communication. This integrated model advances the understanding of how lipid metabolism impacts neurodegenerative cascades at multiple levels.
Future impact and exploitation potential:
These results pave the way for translational studies aimed at pharmacologically modulating NAPE metabolism. Potential directions include:
• in vivo validation of the NAPE–LRRK2–lysosome axis in rodent PD models;
• identification of bioactive NAPE analogues with improved stability and brain permeability;
• exploration of NAPE-mimetic or NAAA-inhibiting compounds as novel therapeutic candidates for PD and related synucleinopathies;
• integration of lipidomics and proteomics data to uncover downstream effectors and biomarkers of NAPE signaling.
The combination of mechanistic insight and translational potential ensures that the outcomes of this project contribute significantly beyond the state of the art, establishing a foundation for future lipid-based neuroprotective strategies.