As a complement to the anti-miR-34a studies that showed an elevation of putative miR-34a target genes, to examine the effect of increasing miR-34a levels on target gene expression and neurodevelopment we stably overexpressed miR-34a in hNPCs from a healthy control subject and differentiated them for up to eight weeks (Figure 3A). miR-34a overexpression decreased levels of ANK3 and CACNB3 as observed in differentiated neurons relative to a negative control miRNA construct (miR-Ctrl) especially in the latest stages of differentiation (Figures 3B-E). Changes in target expression were not seen at the hNPC stage (Figures 3D-E), again pointing to a temporal dependency of the neurodevelopmental regulation of miR-34a targets. To further investigate how miR-34a upregulation observed in BD patient samples could affect pathophysiology, we assessed neuronal differentiation in miR-Ctrl and miR-34a overexpressing cells with a panel of biochemical markers (Figures 3D-E). Consistent with a blockade of neuronal differentiation, miR-34a overexpression delayed the expression of the presynaptic and postsynaptic neuronal markers SYP, SYN1 and PSD-95 over the neurodevelopmental time course (Figures 3D-E). In agreement with these findings, a similar phenotype was observed in
