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Role of N-kinase in regulating central nervous system regeneration

Final Activity Report Summary - AXON GROWTH KINASE (Role of N-kinase in regulating central nervous system regeneration)

Normally, connections that are injured in the mature mammalian Central nervous system (CNS), i.e. brain, optic nerve and spinal cord, do not regrow. As a result of this, the consequences of conditions such as stroke, spinal cord injury and various chronic progressive neurodegenerative diseases, including glaucoma, are devastating and permanent. Several factors, both intrinsic and extrinsic to neurons, regulate the extent of neuronal survival and CNS axonal regeneration in such conditions. Previous work has identified some components of the molecular program underlying axonal growth, even though current knowledge of the intracellular signalling pathways is very incomplete. The outgoing host institution at Harvard Medical School, United States of America, had isolated a specific cell signalling molecule, a N-kinase called mammalian sterile 20-like kinase3b (Mst3b), and experiments by the outgoing host institution and others pointed to N-kinase as being a potential 'master switch' for activating the axon growth programme in the CNS.

The main objective of this project was therefore to investigate a potential role for Mst3b in regulating axon regeneration in the adult CNS. In order to achieve this, an optic nerve regeneration model in the adult rat was employed in which axons arising from the ganglion cells in the retina could be stimulated to grow past a lesion site in the optic nerve. Using Adeno-associated virus (AAV) to transfect Retinal ganglion cells (RGC) with small interfering ribonucleic acids (shRNAs) to downregulate the expression of Mst3b, it could be shown that inhibition of Mst3b expression resulted in an up to 90 % reduction in the number of axons regenerating past the lesion site. This effect was mirrored when placing RGC in culture, as RGC in which Mst3b was downregulated showed a strongly reduced growth response. Importantly, this reduced growth potential could be rescued via transfection with plasmids expressing human Mst3b, proving the specificity of the shRNA.

In order to investigate a more general role in regeneration, the effect of downregulating Mst3b expression on axon regeneration in the Peripheral nervous system (PNS) was assessed. It was found that adult Dorsal root ganglia (DRG), in which Mst3b expression was downregulated, showed significantly reduced axon growth past a lesion site in the radial nerve. Furthermore, this effect was mirrored by a reduced growth potential in culture.

In summary, the present project established that Mst3b is one of the key regulators of axon regeneration in the adult CNS and PNS.