Abstract
We have shown previously that intraocular elevation of cAMP using the cAMP analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) failed to promote axonal regeneration of axotomized adult retinal ganglion cells (RGCs) into peripheral nerve (PN) grafts but significantly potentiated ciliary neurotrophic factor (CNTF)-induced axonal regeneration. Using the PN graft model, we now examine the mechanisms underlying spontaneous and CNTF/CPT-cAMP-induced neuronal survival and axonal regrowth. We found that blockade of the cAMP pathway executor protein kinase A (PKA) using the cell-permeable inhibitor KT5720 did not affect spontaneous survival and axonal regeneration but essentially abolished the CNTF/CPT-cAMP-induced RGC survival and axonal regeneration. Blockade of CNTF signaling pathways such as phosphotidylinositol 3-kinase (PI3K)/akt by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) by 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059), or Janus kinase (JAK)/signal transducer and activators of transcription (STAT3) by tyrphostin AG490 also blocked the CNTF/CPT-cAMP-dependent survival and regeneration effects. PKA activity assay and Western blots showed that KT5720, LY294002, and PD98059 almost completely inhibited PKA, PI3K/akt, and MAPK/ERK signal transduction, respectively, whereas AG490 substantially decreased JAK/STAT3 signal transduction. Intraocular injection of CPT-cAMP resulted in a small PKA-dependent increase in CNTF receptor alpha mRNA expression in the retinas, an effect that may facilitate CNTF action on survival and axonal regeneration. Surprisingly, in the absence of CNTF/CPT-cAMP, LY294002, PD98059, and AG490, but not KT5720, significantly enhanced spontaneous RGC survival, suggesting differential roles of these pathways in RGC survival under different conditions. Our data suggest that CNTF/CPT-cAMP-induced RGC survival and axonal regeneration are a result of multiple pathway actions, with PKA as an essential component, but that these pathways can function in an antagonistic manner under different conditions.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Axons / drug effects
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Axons / physiology
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Carbazoles / pharmacology
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Cell Survival / drug effects
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Chromones / pharmacology
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Ciliary Neurotrophic Factor / antagonists & inhibitors
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Ciliary Neurotrophic Factor / pharmacology
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Ciliary Neurotrophic Factor / physiology*
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Cyclic AMP / analogs & derivatives*
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Cyclic AMP / pharmacology
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / physiology*
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DNA-Binding Proteins / antagonists & inhibitors
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Flavonoids / pharmacology
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Indoles / pharmacology
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MAP Kinase Signaling System / drug effects
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Morpholines / pharmacology
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Nerve Regeneration* / drug effects
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Optic Nerve Injuries / enzymology
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Optic Nerve Injuries / physiopathology
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Peroneal Nerve / transplantation
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Phosphoinositide-3 Kinase Inhibitors
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein-Tyrosine Kinases / antagonists & inhibitors
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Proto-Oncogene Proteins / antagonists & inhibitors
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Proto-Oncogene Proteins c-akt
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Pyrroles / pharmacology
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Rats
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Rats, Inbred F344
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Receptor, Ciliary Neurotrophic Factor / biosynthesis
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Receptor, Ciliary Neurotrophic Factor / drug effects
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Receptor, Ciliary Neurotrophic Factor / genetics
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Retinal Ganglion Cells / drug effects
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Retinal Ganglion Cells / physiology*
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STAT3 Transcription Factor
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Thionucleotides / pharmacology
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Trans-Activators / antagonists & inhibitors
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Tyrphostins / pharmacology
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Up-Regulation
Substances
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Carbazoles
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Chromones
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Ciliary Neurotrophic Factor
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DNA-Binding Proteins
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Flavonoids
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Indoles
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Morpholines
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Phosphoinositide-3 Kinase Inhibitors
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Proto-Oncogene Proteins
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Pyrroles
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Receptor, Ciliary Neurotrophic Factor
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STAT3 Transcription Factor
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Stat3 protein, rat
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Thionucleotides
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Trans-Activators
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Tyrphostins
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alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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8-((4-chlorophenyl)thio)cyclic-3',5'-AMP
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KT 5720
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Cyclic AMP
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Protein-Tyrosine Kinases
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Akt1 protein, rat
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Cyclic AMP-Dependent Protein Kinases
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2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one