Chronic pharmacologic manipulation of dopamine transmission ameliorates metabolic disturbance in trappc9-linked brain developmental syndrome

Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (trappc9) cause autosomal recessive intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links trappc9 to non-alcoholic fatty liver disease (NAFLD). Trappc9-deficient mice have been shown to appear overweight shortly after weaning. Here, we analyzed serum biochemistry and histology of adipose and liver tissues to determine the incidence of obesity and NAFLD in trappc9-deficient mice and combined transcriptomic and proteomic analyses, pharmacological studies, and biochemical and histological examinations of postmortem mouse brains to unveil mechanisms involved. We found that trappc9-deficient mice presented with systemic glucose homeostatic disturbance, obesity and NAFLD, which were relieved upon chronic treatment combining dopamine receptor D2 (DRD2) agonist quinpirole and DRD1 antagonist SCH23390. Blood glucose homeostasis in trappc9-deficient mice was restored upon administrating quinpirole alone. RNA-sequencing analysis of DRD2- containing neurons and proteomic study of brain synaptosomes revealed signs of impaired neurotransmitter secretion in trappc9-deficient mice. Biochemical and histological studies of mouse brains showed that trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that trappc9 loss- of-function causes obesity and NAFLD by constraining dopamine synapse formation. Overall design: DRD2-containing neurons were immunomagnetically isolated from both WT and trappc9 KO mice at age 3-4 weeks. In brief, brain tissue was minced into small pieces and treated with 30 U/mL papain at 37 °C for 20 minutes in neurobasal medium supplemented with 1% penicillin-streptomycin, 1% L-glutamine, 2% B27, and 2500 U DNase I. The digestion was terminated by adding 1 mL fetal bovine serum (FBS). The digested tissues were gently dissociated by pipetting through a blue tip and subsequently passed through 70-µm and 40-µm cell strainers (ThermoFisher). Cells in the flowthroughs were collected by a centrifugation at 200 x g for 10 minutes and washed once with neurobasal medium with 10% FBS and all supplements as above. Cells were resuspended in PBS (pH 7.2) containing 0.5% bovine serum albumin, 0.5 mM EDTA, 5 µg/mL insulin, and 1 g/L glucose and incubated with DRD2-specific antibodies precoupled on magnetic beads at 4 °C for 15 minutes. Cells on beads were washed once in above PBS buffers, separated on a magnetic grate, and resuspended in 1 mL TRIzol reagents for RNAseq analysis.