The psychiatric risk gene NT5C2 regulates AMPK signalling and protein translation in human neural progenitor cells

Background The cytosolic 5’-nucleotidase II gene (NT5C2, cN-II) is associated with disorders characterised by psychiatric and psychomotor disturbances. Common psychiatric risk alleles at the NT5C2 locus reduce expression of this gene in the foetal and adult brain, but downstream biological risk mechanisms remain elusive. Methods Distribution of the NT5C2 protein in the human DLPFC and cortical human neural progenitor cells (hNPCs) was determined using immunostaining, publicly available expression data, and reverse transcription quantitative PCR (RT-qPCR). Phosphorylation quantification of AMPK-alpha (Thr172) and ribosomal protein S6 (RPS6) (Ser235/Ser236) were performed using western blotting, to infer the degree of activation of AMPK signalling and the rate of protein translation. Knockdowns were induced in hNPCs and Drosophila melanogaster using RNA interference (RNAi). Transcriptomic profiling of hNPCs was performed using microarrays, and motility behaviour was assessed in flies using the climbing assay. Results Expression of NT5C2 was higher during neurodevelopment, and was neuronally enriched in the adult human cortex. Knockdown in hNPCs affected AMPK signalling, a major nutrient sensing mechanism involved in energy homeostasis, and protein translation. Transcriptional changes implicated in protein translation were observed in knockdown hNPCs, and expression changes to genes related to AMPK signalling and protein translation were confirmed using RT-qPCR. The knockdown in Drosophila was associated with drastic climbing impairment. Conclusions We provide an extensive neurobiological characterisation of the psychiatric risk gene NT5C2, describing its previously unknown role on the regulation of AMPK signalling and protein translation in neural stem cells, and its association with Drosophila melanogaster motility behaviour. The transcriptomic effect of the knockdown of the psychiatric disease susceptibility gene NT5C2 was assessed in the human neural stem cell line CTX0E16. The Trilencer 27-mer NT5C2 siRNA kit (SR307908, Origene) was used to knockdown NT5C2 using the siRNA sequences A (n = 3) and B (n = 4), in parallel with a negative scramble control sequence (SR30004, n = 4). Cells were incubated with the siRNA sequences for 72 hours, followed by RNA/protein extraction. RT-qPCR analysis revealed a significant knockdown of NT5C2 in the samples analysed by microarray. For additional information, see Methods.