Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Schematic representation of the experimental strategy. Stable SH-SY5Y cell lines were generated either by knocking down NAPE-PLD using CRISP-Cas9 approach or by overexpressing PLA2G4E, both manipulations led to NAPE accumulation at the cell membranes. ( B ) NAPE-PLD mRNA levels in NAPE-PLD-knockdown (KD; violet bars) and wild-type (WT; blue bars) cells. ( C ) Western blot analysis of NAPE-PLD protein levels in KD and WT cells. Top, representative blot; bottom, densitometric quantification normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( D ) Immunofluorescence analysis of NAPE-PLD (gray) with representative images ( D1, D2 ) and quantification. Nuclei were counterstained with DAPI. ( E ) Total NAPE levels in KD and WT cells. ( F ) PLA2G4E mRNA levels in PLA2G4E-overexpressing (OE; pink bars) and WT (blue bars) cells. ( G ) Western blot analysis of PLA2G4E protein levels in OE and WT cells. Top, representative blot; bottom, densitometric quantification normalized to GAPDH. ( H ) Immunofluorescence analysis of PLA2G4E (gray) with representative images ( H1, H2 ) and quantification. Nuclei were counterstained with DAPI. ( I ) Total NAPE levels in OE and WT cells. Error bars represent SEM. *P < 0.05, **P < 0.01 ***P < 0.001, ****P < 0.0001 Student’s t -test with Welch’s correction. Scale bars, 10 μm.

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Generated, Knockdown, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of LRRK2 (gray) with representative images ( A1–A3 ) and quantification in wild-type (WT; A1 , blue), NAPE-PLD knockdown (KD; A2 , violet), and PLA2G4E-overexpressing (OE; A3 , pink) SH-SY5Y cells. Nuclei were counterstained with DAPI. ( B ) Quantification of the colocalization between LAMP1-positive structures and LRRK2, expressed as Manders’ overlap coefficient for the fraction of LAMP1 signal overlapping with LRRK2. ( C ) Western blot analysis of Rab8a and phosphorylated Rab8a (pT72-Rab8a) in KD and OE compared with WT cells. Top, representative blot; bottom, densitometric quantification normalized to actin. ( D ) Representative super-resolution immunofluorescence images of LAMP1 (magenta) and LRRK2 (green) in WT ( D1 ), NAPE-PLD KD ( D2 ), and PLA2G4E OE ( D3 ) cells. Nuclei were stained with DAPI (gray). Scale bar, 5 μm. ( E ) Fluorescence intensity quantification and representative images of the Magic Red–cathepsin B assay, indicating lysosomal degradative capacity per cell, in WT ( E1 ), NAPE-PLD KD ( E2 ), and PLA2G4E OE ( E3 ) cells. Representative images are displayed using the FIRE lookup table (LUT) to indicate fluorescence intensity. Scale bars, 10 μm. Mean values are indicated in the graphs; error bars represent SEM. *P < 0.05, ****P < 0.0001; one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Immunofluorescence, Knockdown, Western Blot, Staining, Fluorescence, Comparison

Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of LRRK2 (gray) with representative images ( A1-A4 ) and quantification in WT ( A1, A2 ), NAPE-PLD-knockdown (KD; A3 ), and PLA2G4E-overexpressing (OE; A4 ) cells treated or not with α-syn ( A2-A4 ). ( B ) Quantification of the colocalization between LAMP1-positive structures and LRRK2, expressed as Mander’s overlap coefficient for the fraction of LAMP1 signal overlapping with LRRK2. ( C ) Quantification and representative images of α-syn aggregate number per cell in WT ( C1 ), NAPE-PLD KD ( C2 ) and PLA2G4E OE ( C3 ) cells. LAMP1 is shown in yellow, α-syn in pink, and nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Mean values are indicated in the graphs; error bars represent SEM. ***P < 0.001, ****P < 0.0001; one-way ANOVA with Tuckey’s multiple-comparison test.

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Immunofluorescence, Knockdown, Comparison

Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Schematic representation of the generation of stable SH-SY5Y cell lines overexpressing NAPE-PLD, leading to reduced NAPE levels at cellular membranes. ( B ) NAPE-PLD mRNA levels in NAPE-PLD-overexpressing (OE; green) and wild-type (WT; blue) cells. ( C ) Western blot analysis of NAPE-PLD protein levels in OE and WT cells. Top, representative blot; bottom, densitometric quantification normalized to GAPDH. ( D ) Immunofluorescence analysis of NAPE-PLD (gray) with representative images ( D1, D2 ) and quantification. ( E ) Immunofluorescence analysis of LRRK2 (gray) with representative images ( E1, E2 ) and quantification in WT ( E1 ) and NAPE-PLD OE ( E2 ) cells. Nuclei were counterstained with DAPI (blue). ( F ) Western blot analysis of Rab8a and phosphorylated Rab8a (pT72-Rab8a) in NAPE-PLD OE and WT cells. Top: representative blot; bottom, densitometric quantification normalized to actin. ( G ) Schematic representation of the experimental strategy. Ionomycin treatment induces intracellular Ca 2+ release, promoting NAPE production and degradation in NAPE-PLD OE cells. NAPE-PLD activity was inhibited by overnight treatment with the NAPE-PLD inhibitor LEI-401. Ionomycin was administered together with Magic Red reagent for 30 min before image acquisition. ( H ) Fluorescence intensity quantification and representative images of the Magic Red – cathepsin B assay, indicating lysosomal degradative capacity per cell, in untreated WT ( H1, white bar and blue dots) and NAPE-PLD OE cells ( H4, white bar and green dots), ionomycin-treated WT ( H2, blue bar and blue dots) and NAPE-PLD OE cells ( H5, green bar and green dots) or WT ( H3, blue bar white dots) and NAPE-PLD OE cells ( H6 , green bar and white dots) treated with ionomycin and LEI-401. Representative images are displayed using the FIRE lookup table (LUT) to indicate fluorescence intensity. Scale bars, 10 μm. Mean values are indicated in the graphs; error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s t -test with Welch’s correction ( B-F ) and two-way ANOVA with Tuckey’s multiple-comparisons test ( H ).

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of LRRK2 (gray) with representative images ( A1-A3 ) and quantification in WT ( A1, A2 ), and NAPE-PLD OE ( A3 ) SH-SY5Y cells treated or not with α-synuclein (α-syn; A2, A3 ). ( B ) Quantification and representative images of α-syn aggregate number per cell in WT ( B1 ) and NAPE-PLD OE ( B2 ) cells. α-Syn is shown in pink, and nuclei were counterstained with DAPI. Scale bar, 10 μm. Mean values are indicated in the graphs; error bars represent SEM. *P<0.05, ***P < 0.001, ****P < 0.0001; one-way ANOVA with Tuckey’s multiple-comparison test ( A ) and Student’s t -test with Welch’s correction ( B ).

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: A NAPE–LRRK2 metabolic axis controls lysosomal homeostasis in Parkinson’s disease

doi: 10.64898/2026.03.19.712902

Figure Lengend Snippet: ( A ) Representative images of the Magic Red – cathepsin B assay, indicating lysosomal degradative capacity per cell, in healthy dopaminergic neurons (DANs; A1, A2 ) and G2019S-LRRK2 mutant DANs ( A3, A4 ) treated ( A2, A4 ) or not ( A1, A3 ) with the NAPE-PLD inhibitor LEI-401. ( B ) Fluorescence intensity quantification of the Magic Red assay in healthy DANs (green) and G2019S-LRRK2 mutant DANs (gray). ( C, D ) Quantification and representative images of α-synuclein (α-syn) aggregate number per cell in healthy DANs ( D1, D2 ) and G2019-LRRK2 mutant DANs ( D3, D4 ) treated ( D2, D4 ) or not ( D1, D3 ) with LEI-401. α-Syn is shown in pink, LAMP1 yellow, and nuclei were counterstained with DAPI. Scale bars, 10 μm. *P < 0.05, ****P < 0.0001; two-way ANOVA with Tuckey’s multiple-comparison test.

Article Snippet: The NAPE-PLD Human Gene Knockout Kit (CRISPR, Origene, KN409877) was used following manufacturer’s instructions.

Techniques: Mutagenesis, Fluorescence, Comparison