PKC isozymes in the enhanced regrowth of retinal neurites after optic nerve injury.
OBJECTIVE
To establish an in vitro model of axonal regeneration from mammalian retinal ganglion cells and to evaluate the role of PKC isozymes in promoting such retinal axon regeneration.


METHODS
Postnatal day-3 mice were subjected to optic nerve crush, and then retinal ganglion cells (RGCs) were used for culture 5 days later. RGCs were selected using anti-Thy 1.2-coated magnetic beads and plated onto a merosin substrate. Changes in axonal localization of PKC and axonal regeneration were examined in cultured RGCs by immunofluorescence. Changes in PKC isozyme mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The role of PKC in RGC neurite outgrowth was examined by treatment with activators or pharmacological inhibitors of PKC activity.


RESULTS
RGCs subjected to optic nerve crush injury demonstrated more rapid neurite outgrowth in vitro when compared with RGCs isolated from naïve retina. The neurites of these injury-conditioned RGCs showed both an increased rate of extension and enhanced PKC localization in culture. Injury-conditioned RGCs had elevated PKC isozyme mRNA levels, which probably contributed to the increased level of PKC protein in injury-conditioned RGC axons. Pharmacological activation of PKC enhanced neurite growth, whereas inhibition of PKC suppressed neurite growth in both the conditioned and naïve RGCs.


CONCLUSIONS
RGCs actively respond to axonal injury by regulating expression of genes that promote neurite outgrowth. PKC-alpha and -beta isozymes are among the growth-associated proteins that are upregulated after injury. Results of pharmacological manipulation of PKC activity support the argument that increased PKC levels enhance neurite regrowth after axonal injury.
Wu
Da-Yu
DY
Departments of Cell and Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA. dayuwu@hsc.usc.edu
Zheng
Jun-Qi
JQ
McDonald
Marisa A
MA
Chang
Bieshia
B
Twiss
Jeffery L
JL
D000818:Animals; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D007527:Isoenzymes / Q000502:physiology; D051379:Mice; D009409:Nerve Crush; D009416:Nerve Regeneration / Q000502:physiology*; D016501:Neurites / Q000502:physiology*; D020221:Optic Nerve Injuries / Q000503:physiopathology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D012333:RNA, Messenger; D011493:Protein Kinase C