ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS.
The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.
Department of Neurology, University Medicine Göttingen, Göttingen, Germany.
J C
JC
Koch
Department of Neurology, University Medicine Göttingen, Göttingen, Germany.
L
L
Tönges
Department of Neurology, University Medicine Göttingen, Göttingen, Germany.
E
E
Barski
Department of Neurology, University Medicine Göttingen, Göttingen, Germany.
U
U
Michel
1] Department of Neurology, University Medicine Göttingen, Göttingen, Germany [2] Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.
M
M
Bähr
1] Department of Neurology, University Medicine Göttingen, Göttingen, Germany [2] Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.
P
P
Lingor
D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D001369:Axons / Q000201:enzymology / Q000473:pathology; D002154:Calpain / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D054461:Lim Kinases / Q000235:genetics / Q000378:metabolism; D009409:Nerve Crush; D009410:Nerve Degeneration; D009416:Nerve Regeneration; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000201:enzymology / Q000473:pathology; D009900:Optic Nerve / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D020221:Optic Nerve Injuries / Q000201:enzymology* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000201:enzymology* / Q000473:pathology; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C095114:collapsin response mediator protein-2; D054461:Lim Kinases; C515894:Limk1 protein, rat; C490644:Limk2 protein, rat; D051057:Proto-Oncogene Proteins c-akt; C580818:ROCK2 protein, rat; D054460:rho-Associated Kinases; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3