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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release

doi: 10.1007/s00018-010-0494-4

Figure Lengend Snippet: Endocannabinoid system in gliosomes (grey bars) and synaptosomes (black bars). Endogenous levels of AEA (a) and 2-AG (b). Metabolism of AEA and 2-AG, which includes synthesis by NAPE-PLD activity for AEA (c) and by DAGL activity for 2-AG (d); hydrolysis by FAAH activity for AEA (e) and by MAGL activity for 2-AG (f). Results are expressed as pmol/mg for endogenous levels and pmol/min/mg protein for the enzymatic activities. Reported data are mean ± SEM of at least three independent experiments, each performed in duplicate or triplicate. In panel d, *p < 0.05 versus gliosomes

Article Snippet: Rabbit anti-NAPE-PLD polyclonal antibody was from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release

doi: 10.1007/s00018-010-0494-4

Figure Lengend Snippet: Western-blot analysis of the elements of the endocannabinoid system in gliosomes and synaptosomes. Gliosome and synaptosome homogenates were reacted with anti-NAPE-PLD, anti-FAAH, anti-DAGL, anti-MAGL, anti-CB1R, anti-CB2R, and anti-TRPV1 polyclonal antibodies. Expected molecular mass values are reported on the left-hand side

Article Snippet: Rabbit anti-NAPE-PLD polyclonal antibody was from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Western Blot

Journal: The FEBS journal

Article Title: In vitro and in vivo models of Huntington's disease show alterations in the endocannabinoid system.

doi: 10.1111/febs.12329

Figure Lengend Snippet: Fig. 5. The ECS in ST14A and HD43 cells. The activity values are expressed as percentage relative to ST14A cells, set to 100%: 40.4 1.3 pmolmin1mg of protein1 (FAAH); 21.0 2.2 pmolmin1mg of protein1 (NAPE-PLD); 77.6 3.5 pmolmin1mg of protein1 (MAGL); 740 50 pmolmin1mg of protein1 (DAGL). Data are means SDs. *P < 0.05 versus ST14A cells. ***P < 0.001 versus ST14A cells.

Article Snippet: Rabbit polyclonal antibodies against NAPE-PLD were from Novus Biologicals (Littleton, CO, USA).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Dysfunction in Fatty Acid Amide Hydrolase Is Associated with Depressive-Like Behavior in Wistar Kyoto Rats

doi: 10.1371/journal.pone.0036743

Figure Lengend Snippet: The CB1 receptor agonist-stimulated [ 35 S]GTPγS binding was significantly higher in frontal cortex (24%, p<0.05) and hippocampus (44%, p<0.01) of WKY rats compared to WIS rats (A). Data is presented as percentage of stimulation over basal binding. Western blot analysis revealed significantly higher levels of CB1 receptors in hippocampus (45%, p<0.05), while they were found to be slightly higher in frontal cortex of WKY rats (18%, B). Hippo; Hippocampus.

Article Snippet: Membrane was then incubated with antibodies for FAAH, NAPE-PLD and CB1 receptor, (Abnova, Taipei City, Taiwan) overnight at 4°C.

Techniques: Binding Assay, Western Blot

Journal: PLoS ONE

Article Title: Dysfunction in Fatty Acid Amide Hydrolase Is Associated with Depressive-Like Behavior in Wistar Kyoto Rats

doi: 10.1371/journal.pone.0036743

Figure Lengend Snippet: Basal BDNF levels were found to be significantly lower in frontal cortex (27%) and hippocampus (26%) of WKY compared to WIS rats (p<0.05; A). Subchronic treatment with URB597 (0.3 mg/kg, i.p. for 7 days) significantly elevated BDNF levels in frontal cortex (64%) and hippocampus (45%) of WKY rats compared to vehicle treated WKY rats (p<0.05; B). Inhibition of FAAH was accompanied by significant increase in AEA levels in frontal cortex (31%, p<0.01; C) and hippocampus (42%, p<0.001; C), and a subsequent decrease in CB1 receptor-mediated G-protein activation in frontal cortex of WKY rats (21%, p<0.05; D). Hippo; Hippocampus.

Article Snippet: Membrane was then incubated with antibodies for FAAH, NAPE-PLD and CB1 receptor, (Abnova, Taipei City, Taiwan) overnight at 4°C.

Techniques: Inhibition, Activation Assay