To investigate intracellular regulatory processes, we interrogated global gene expression by RNAseq. Results identified mRNAs differentially expressed between D398 and N398 (Fig. 3C and Supplemental Table 3). The most informative and significant functional enrichment was for KEGG pathways specific for neuroactive ligand receptor interaction, calcium signaling, and axon guidance (red bars in Fig. 3D). Whether the altered expression of these genes is an effect of the N398 variant or whether it is only a correlation due to differences in neuronal function, the RNAseq results demonstrate identifiable differences between cultures based on CHRNA5 genotype and they predict that N398 cells will have altered response to stimuli. Because some of the differentially regulated genes were components of the glutamatergic pathway, and because we detected as much as 20% excitatory glutamatergic neurons in the mDA cultures (Fig. 1O,P), we reasoned that the increased excitability in mDA cultures may be due to nAChR activity in the DA neurons or to pre-synaptic input from other cells in culture—possibly an excitatory input. It is also possible that some TH+ cells co-release glutamate. Recent reports identify GABA release by DA neurons4849. To test this prediction we differentiated iPSC into glutamatergic neurons.
