Fig 2: DAGLB-mediated 2-AG biosynthesis in the nigral DANs is involved in locomotor skill learning.a Schematic diagram of eCB2.0 fiber photometry recoding while mice undergo rotarod locomotor skill training. Inset shows the average eCB2.0 (green), tdT (red), and normalized eCB2.0 (blue) signals during each trial from one representative mouse on day 2 of the 6-day training paradigm (n = 1). Data were presented as mean ± SD. b Normalized eCB2.0 signal intensity during the locomotor learning. n = 8 (4M/4F). Data were presented as mean ± SD. c Box and whiskers plot (the minimum, the 25th percentile, the median, the 75th percentile, and the maximum) of maximal daily increase of eCB2.0 signal intensity. n = 8 (4M/4F). Tukey’s multiple comparisons adjusted, *p = 0.02, **p = 0.001, ***p = 0.0003, ****p < 0.0001. d Performance of rotarod motor skill training. n = 8 (4M/4F). Data were presented as mean ± SEM. e Box and whiskers plot (the minimum, the 25th percentile, the median, the 75th percentile, and the maximum) of correlation coefficient of eCB2.0 signal intensity and rotarod performance. n = 8 (4M/4F). Tukey’s multiple comparisons adjusted, ***p = 0.0002 to 0.0006, ****p < 0.0001.

Fig 3: JZL184 treatment promotes DAN activity and dopamine release.a Schematic illustrates fiber photometry recording of GCaMP6f and tdT signals in the SNc of DAN-control and DAN-Daglb KD DatIRESCre/GCaMP6f/tdT trigenic mice. b Representative images of GCaMP6f (green), tDT (red), and TH (blue) staining. Scale bar: 200 µm. Six independent experiments were performed. c Alterations of calcium transients in the SNc of DAN-control and DAN-Daglb KD DatIRESCre/GCaMP6f/tdT trigenic mice (n = 3M per genotype) treated with vehicle or JZL184 (20 mg/kg). Data were presented as mean ± SEM. Multiple unpaired t test, two-tailed, *p < 0.05, **p < 0.01, ***p < 0.001. d Schematic illustrates fiber photometry recording of DA2m and tdT signals in the SN of DAN-control and DAN-Daglb KD mice. e Representative images of DA2m (green), tDT (red) and TH (magenta) staining. Scale bar: 200 µm. Nine independent experiments were performed. f Changes of dopamine release in the SN of DAN-control (n = 5 M) and DAN-Daglb KD (n = 4 M) mice treated with vehicle, JZL184 (20 mg/kg). Data were presented as mean ± SEM. Multiple unpaired t test, two-tailed, *p < 0.05, **p < 0.01.

Fig 4: Identification of homozygous DAGLB mutations in affected families.a Pedigrees are shown for the four affected families. A double bar represents parental consanguinity. Slash indicates deceased individuals. Males are represented by squares, females by circles, and affected individuals by shading. b Schematic view of DAGLB gene structure and encoded protein domains. The four transmembrane segments are shown in blue, and the catalytic domain in maroon. Within the catalytic domain, a regulatory loop is colored in green. Variant sites are indicated by arrows. c–e Representative western blot (c) and quantification of DAGLB protein (d) and mRNA (e) levels in fibroblasts derived from Family 1 II-3, Family 2 II-4 and three age-matched HCs. N = 3-4 independent fibroblast cultures. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein or mRNA levels as appropriate and represent mean ± SEM. One-way ANOVA, F = 33.09, ****p < 0.0001.

Fig 5: JZL184 treatment rescues the locomotor skill learning impairments of DAN-Daglb KD mice.a Rotarod locomotor skill learning of DAN-control (nvehicle = 7, 4M/3F; nJZL184 = 10, 5M/5F) and DAN-Daglb KD (nvehicle = 11, 6M/5F; nJZL184 = 12, 6M/6F) mice treated with vehicle or JZL184 (20 mg/kg). Data were presented as mean ± SEM. b A working model of nigral DAN-derived 2-AG signaling in regulating DAN neuron activity and dopamine release in SN region, where 2-AG modulates DAN calcium influx and somatodendritic dopamine release through retrograde inhibition of presynaptic GABA release from dSPNs via CB1-mediated intracellular signaling pathway. Genetic deletion of Daglb in DANs compromises the 2-AG-mediated feedback regulation, while pharmacological inhibition of 2-AG degradation by JZL184 restores the signaling. Our study suggests that this nigral DAN-derived 2-AG signaling in the SN is involved in regulating the across-session locomotor skill learning. DAG diacylglycerol, DA dopamine, AA arachidonic acid.