Synapsin-RFP Lentivirus

Copy number variations (CNVs) in genes involved in nervous system development or function are often associated with neuropsychiatric disorders. A CNV located on chromosome 15q13.3 affects the alpha-7 nicotinic acetylcholine receptor subunit (CHRNA7) gene and causes multiple neuropsychiatric disorders with highly variable penetrance. Here, researchers generated induced pluripotent stem cell (iPSC) models from first-degree relatives with 15q13.3 duplications and analyzed their cellular phenotypes to reveal the basis for the different phenotypic manifestations. Potential factors contributing to neuropsychiatric disorders were modeled in iPSC-derived cortical excitatory and inhibitory neurons. AP-derived models uniquely exhibited disruptions in cellular physiology and neurodevelopment that were not observed in unaffected mothers (UM) or unrelated controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, as well as increased endoplasmic reticulum (ER) stress. Both defective neuronal migration and elevated ER stress could be selectively rescued by different drugs. Neuronal gene expression was also dysregulated in the AP, including decreased expression of genes associated with behavior, psychological impairments, neurite outgrowth, neuronal migration, and Wnt, axon guidance, and GABA receptor signaling. The UM model instead exhibited upregulated gene expression in many of the same pathways, suggesting that molecular compensation may contribute to the lack of a neurodevelopmental phenotype in this model. However, both AP- and UM-derived neurons exhibited common alterations in neuronal function, including increased action potential firing and enhanced cholinergic activity, consistent with increased homomeric CHRNA7 channel activity.

AP-derived neurons (compared to UM) exhibited reduced expression of genes regulating neuronal migration (Figure 1a), suggesting that they may have impaired migration. In vivo, cortical interneurons migrate tangentially from the ventral telencephalon to the cortex. To study the migration of cExN and cIN neurons, the researchers developed a method utilizing the fusion co-culture of two 3D spheroids composed of cExN and cIN. These 3D spheroids were generated by transducing cExNPCs and cINPCs, respectively, with lentiviral synapsin-eGFP or lentiviral synapsin-RFP expression constructs. cExN neurons expressing synapsin-GFP and cIN neurons expressing synapsin-RFP were juxtaposed and migration from one neuron to the other was assessed (Figure 1b). cINs from AP neuroids exhibited reduced migration compared to the other models (Figure 1c-d). In contrast, the migration of AP-derived cExNs was only slightly impaired, whereas UM-derived cExNs exhibited reduced migration compared with the control model, suggesting that migration of both CHRNA7 duplication carriers is somewhat impaired (Figure 1c, e).

Figure 1. Neuronal migration is compromised in AP-derived cINs. (Meganathan K, et al., 2021)