High-content cellular screening of genes dysregulated in Parkinson’s disease identifies regulators of cell viability, mitochondrial activity and axon growth

In this chapter high-content cellular screening (HCS) was used to functionally test 62 genes that are dysregulated in the SN of Parkinson’s disease (PD) patients. HCS was performed in SH-SY5Y cells, a well characterized dopaminergic cell line, and was designed to test the effect of siRNA mediated knockdown or lentivirus (LV)-mediated overexpression of target genes on mitochondrial activity, cell viability, and neurite growth. HCS of 62 genes revealed that RGMA and PTMA knockdown increased mitochondrial activity only in cells treated with MPP(+) (which induced mitochondrial inhibition), and FOXO4 knockdown increased neurite outgrowth independent of MPP(+). RGMA knockdown showed an interaction effect on mitochondrial activity and cell viability when coupled with MPP(+) treatment, indicating that its signaling is linked to mitochondrial activity. Overexpression of 12 out of 14 genes had an effect on at least one of the cellular parameters investigated. The most dramatic effects on cell viability were induced by FOXO4 and HIP1R, independent of MPP(+) treatment, and ALDH1A1 only after MPP(+) treatment. SOX2 most dramatically decreased neurite outgrowth of SH-SY5Y differentiated cells. CTDSP1, RGMA, PTMA, SLITRK5 and WWC1 induced effects on both cell survival and neurite outgrowth, suggesting that these genes contribute to both of these cellular parameters. Apart from gene-only overexpression effects, ALDH1A1, HIP1R and WWC1 overexpression showed clear interaction effects on cell survival and mitochondrial activation when treated with MPP(+), strongly suggesting a functional interaction between these genes and the level of mitochondrial activity and/or cellular oxidative stress. A total of 9 genes identified by this HCS may be causally or consequentially involved in the complex process of DAergic neuron degeneration in PD patients, and are likely candidates for testing their role in vivo in the initiation of PD-like neurodegeneration in a rodent model of PD. HIGH CON TEN T CELLU L A R SCR EEN OF SELECTED PD GENES