Fig. 7: 2-AG and AA are reduced in AP-4 knockout brains and MGLL inhibition rescues impaired neurite outgrowth in AP-4-deficient neurons.

a Diagram of the 2-AG biosynthesis pathway. Hydrolysis of diacylglycerol (DAG; blue) by DAG lipase (DAGL; blue) generates 2-arachidonoylglycerol (2-AG; green), which is hydrolysed by monoacylglycerol lipase (MGLL; green) to generate arachidonic acid (AA; purple). MGLL inhibition by the specific inhibitor ABX-1431 (red) blocks 2-AG hydrolysis and thereby increases the level of 2-AG³³. b–d Mass spectrometry-based quantification of (b) 2-AG, (c) AA and (d) 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), from wild-type (WT) and Ap4e1 knockout (KO) mouse brains (n = 5 animals per group; horizontal bar indicates median; mice were aged 6 months). Data were subjected to a two-tailed Mann–Whitney U-test. e Neurite outgrowth was assayed in iPSC-derived cortical neurons from a patient with AP4B1-associated AP-4 deficiency syndrome (SPG47; patient 1) and their unaffected same sex heterozygous parent (control), using automated live cell imaging. Neurons were cultured in the presence of DMSO (vehicle control) or the MGLL inhibitor ABX-1431 at 10, 50, 100 or 500 nM (the highest two doses were administered only to the patient neurons). Neurons were monitored from 4 h post-plating, with images captured every 3 h until 25 h post-plating. Representative images are shown at 4 h (t0) and 25 h post-plating, with cell bodies outlined in green and neurites traced in red. Scale bar: 100 μm. f–h Automated image analysis from neurite outgrowth assay of two separate neuronal differentiations per cell line shown in (e). Graphs show neurite length per image over time, normalised to cell body cluster area. Data are shown relative to normalised neurite length at t0. The same data are shown for the control plus DMSO condition in (f–h) and for the patient plus DMSO condition in (f) and (g). f Average neurite length of patient neurons was significantly reduced compared to control neurons at all time points. Per group, n = 108 images from two biological replicates were analysed. Data were subjected to a two-way repeated measures ANOVA with Šídák’s multiple comparisons test for comparisons at each time point between control and patient: ***p  ≤  0.001. g Average neurite length of patient neurons was rescued by treatment with 10 nM ABX-1431. 50 and 100 nM ABX-1431 doses also increased neurite length, whereas neurite length was not improved by the highest dose (500 nM). Per treatment group, n = 18 images from two biological replicates were analysed. Data were subjected to a two-way repeated measures ANOVA with Dunnett’s multiple comparisons test for comparisons at each time point between each dose of ABX-1431 and the patient plus DMSO control: **p  ≤  0.01; *p  ≤  0.05. The control plus DMSO condition is shown for reference, but was not included in the statistical analysis. h Average neurite length of control neurons was not affected by treatment with 10 or 50 nM ABX-1431. Per treatment group, n = 18 images from two biological replicates were analysed. Data were subjected to a two-way repeated measures ANOVA with Dunnett’s multiple comparisons test for comparisons at each time point between each dose of ABX-1431 and the DMSO control: *p  ≤  0.05. In (f–h) only significant changes are annotated; all other comparisons resulted in non-significant p values (p > 0.05). Source data are provided as a Source Data file.