Abstract
Purpose:
Central nervous system neurons including retinal ganglion cells (RGCs) die after axon injury. Many molecular signaling pathways, including cAMP, Ca2+ and mitogen-activated protein kinase (MAPK)-dependent pathways, have been implicated in the regulation of neuronal survival and apoptosis. Scaffold protein muscle A-kinase anchoring protein-beta (mAKAP-β) orchestrates localized cAMP, Ca2+, and MAPK-regulated stress signaling in non-neuronal cells. In contrast, the α-isoform of mAKAP expressed in the CNS has not been extensively investigated. The purpose of this study is to investigate the expression of mAKAP and its role in RGC survival and neurite growth.
Methods:
The expression of mAKAP in RGCs was investigated by western blot and immunostaining. In vitro, purified RGCs were electroporated either with control or anti-mAKAP siRNA and then cultured in growth media for 3 days. RGC survival was investigated using calcein/sytox assay while neurite length was analyzed by high-content microscopy (Cellomics). In vivo, mAKAP was knock out of mature RGCs by injection of AAV2-cre into adult mAKAPfl/fl mice. RGC survival was quantified by counting the number of fluorogold retrograde-labeled RGCs in retina flat mounts 1 week after optic nerve crush.
Results:
mAKAPα is expressed in RGCs where it facilitates RGC survival in cell culture and after optic nerve axon injury. mAKAPα was detected in the retinal ganglion cell layer and weakly in the inner plexiform and inner nuclear layers where amacrine, horizontal and bipolar neurons are located. When mAKAPα was deleted early in RGC development in a Math5-cre conditional mAKAP knock-out mouse, RGC density in the adult retina was similar to littermate control mice, indicating that mAKAPα was not necessary for RGC survival during development. In contrast, mAKAPα was necessary for RGC survival after optic nerve injury, revealing a new neuroprotective function for this scaffold.
Conclusions:
We have identified mAKAPα expression in RGCs and demonstrated a requirement for mAKAPα in neuronal survival and neurite growth in cell culture and after optic nerve injury in vivo. Future studies will identify the mAKAPα signalosome signaling modules relevant to these processes and may suggest new targets for neuroprotection after injury or in neurodegenerative diseases.
Keywords: 615 neuroprotection •
449 cell survival •
629 optic nerve