Targeted neutralization of calcineurin, by expression of an inhibitor peptide under the control of a cholinergic specific promoter in PC12 cells, promotes neurite outgrowth in the presence of NGF.
We have characterized a region of the mouse vesicular acetylcholine transporter(VAChT)/choline acetyltransferase (ChAT) gene locus that serves as a cholinergic-specific promoter for the expression of both VAChT and ChAT genes, as well as a reporter gene (LacZ) in vivo. We have used this promoter to direct the expression of an inhibitor peptide, derived from the calcineurin (CalN) autoregulatory domain, to directly neutralize the function of CalN to define the role of this Ca2+/Calmodulin regulated phosphatase in neurite outgrowth. Targeted inhibition of CalN promotes neurite outgrowth in PC12 cells in the presence of NGF, as early as 24 h after transfection. Inhibition of CalN-mediated enhancement of neurite outgrowth in PC12 cells reaches a maximum effect within the first 4 to 6 days after transfection, and does not cause adverse effects when highly expressed for up to 12 days. Cyclosporin A, a nontargeted CalN inhibitor, increases the number of neurites in mock transfected cells by 1.5 fold, while in transfected PC12 cells, the expression of the CalN inhibitor peptide increases the neurite number by 1.8 fold. These data demonstrate that CalN is an important regulator of the neurotrophic response in cholinergic cells and may prove valuable in developing treatment strategies to promote recovery from neurological injury.
Naciff
J M
JM
Department of Molecular and Cellular Physiology, University of Cincinnati, OH 45267-0576, USA.
King
K L
KL
Dedman
J R
JR
D000818:Animals; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002795:Choline O-Acetyltransferase / Q000235:genetics*; D018390:Gene Targeting; D026901:Membrane Transport Proteins; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects* / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D016716:PC12 Cells / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D020932:Nerve Growth Factor; D002795:Choline O-Acetyltransferase; D019703:Calcineurin