Abstract
Purpose:
Krüppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and axon regeneration in vivo in retinal ganglion cells (RGCs) and other CNS neurons. Here we propose to discover new KLF gene targets that regulate axon growth and regeneration in central nervous system (CNS) neurons.
Methods:
To determine through what gene targets KLFs regulate axon growth, we examined genes regulated by KLFs in rat RGCs based on microarray data after KLF7, -9, -11 and -16 expression in RGCs by viral transduction. KLF candidate gene targets were screened by quantification of neurite growth in vitro. qRT-PCR, protein extraction and western blot analysis after gene expression using primary RGCs were performed to characterize candidate genes. RGC axon regeneration in a rat optic nerve crush model using shRNA knock down strategy against a candidate gene was used to examine physiology in vivo.
Results:
We identified Dual specificity phosphatase 14 (Dusp14) as a KLF gene target in RGCs, induced four-fold after transduction with KLF9, a KLF family member that significantly suppresses neurite outgrowth. Dusp14 strongly suppressed neurite outgrowth of RGCs in vitro, with no effect on neurite number. In P0 RGCs, which do not express detectable levels of Dusp14 and express very low levels of KLF9 mRNA, two different Dusp14-specific siRNAs and a pharmacological inhibitor, PTP inhibitor IV, fully rescued P0 RGC neurite growth after KLF9 overexpression. In P8 RGCs, which endogenously express KLF9 and Dusp14, the siRNAs and PTP inhibitor IV not only rescued neurite growth from KLF9-induced suppression, but also significantly promoted neurite elongation in control-transfected neurons. Dusp14 and KLF9 inhibited the activation of mitogen-activated protein kinases (MAPK) including ERK1/2, JNK and P38. Finally, knocking down Dusp14 expression in RGCs in vivo enhanced regenerative axon growth after optic nerve injury.
Conclusions:
KLF gene target Dusp14 is a key player limiting axon growth and regenerative ability downstream of KLFs’ ability to suppress axon growth in RCGs. Developing strategies to modulate Dusp14 for enhancement of MAPK signaling may lead to efficient promotion of axon regeneration after CNS injury.
Keywords: 531 ganglion cells •
687 regeneration •
629 optic nerve