the Amaxa 96-well nucleoporation plate was loaded with the mixture
of 75 000 neurons in 20 ml of Amaxa transfection solution, and 400 ng
of total DNA (including mCherry reporter with ratio plasmid to
reporter 6:1) in a volume of 2 ml. The rat neuron transfection, â??highefficiency'
program was used, and the neurons were recovered with
80 ml of ENBܾHEPES (20mM, Invitrogen 15630). Several control
plasmids were used including pSport mCherry (reporter alone) and
pSport CAT, a plasmid with no CMV promoter (chloramphenicol
acetyltransferase gene). Cells were then plated at two different
densitiesâ??8000 and 12 000 cells per well. Transfection efficiency
was validated by cotransfecting mCherry with plasmids from the
library containing myelin basic protein, vimentin, GFAP, and NCAM
cDNAs and subsequent identification by antibody staining
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase alpha (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.