Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Abstract

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y-27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.

The Rho Kinase Pathway Regulates Mouse Adult Neural Precursor Cell Migration

“…HA1077 is in clinical use for managing cerebral vasospasm associated with subarachnoid hemorrhage and is under clinical trial for acute ischemic stroke [37], among other conditions. Application of Y27632 has also been investigated for promotion of neurite outgrowth and axonal regeneration [38][39][40][41][42][43][44][45] and inactivation of Rho or ROCK signaling promoted functional recovery and/or axonal regeneration after neural injuries [46][47][48].…”

Expression of a dominant-negative Rho-kinase promotes neurite outgrowth in a microenvironment mimicking injured central nervous system

“…Pharmacological inhibition of RhoA with C3 transferase or inhibition of ROCK with Y27632 has been shown to stimulate neurite growth on inhibitory substrates in vitro. Inhibition of RhoA or ROCK has also been found to promote regeneration of optic nerves and corticospinal axons in vivo [8,[12][13][14][15] . However, the clinical use of these antagonists may be limited by undesired side effects and pharmacokinetics.…”

Blocking Nogo Receptor 1 Promotes Functional Regeneration after Spinal Cord Injury

“…NTera2 (human embryonal carcinoma (EC) stem cells; German National Resource Center for Biologicals, DMSZ, Braunschweig, Germany) were thawed and plated in 175 cm 2 culture flasks (Greiner bio-one) with DMEM medium (Gibco), containing 10% fetal calf serum (FCS) and 5% Horse Serum, as described before [29]. For differentiation, retinoic acid (SIGMA) at a final concentration of 10 µM was added twice-weekly to the NTera2 cells over a period of three weeks.…”

“…Fixed cultures were stained with Alexa-phalloidin and analysis of neurite outgrowth was performed automatically with the software AxioVision LE Rel. 4.1 (Carl Zeiss, Jena, Germany), according to a published protocol [29]. Data were analyzed by ANOVA followed by Bonferroni's test using GraphPad Prism software (Graph Pad Software, La Jolla, USA).…”

“…Neurons differentiated from NTera2 cells are less well studied in this respect, but allow to expand studies to human postmitotic neurons. Recently, these neurons were shown to be sensitive to chondroitin sulfate proteoglycans [29], which are also known inhibitors for neurite outgrowth in primary rodent neurons. Interestingly, human NTera2 derived neurons had a 25-fold higher sensitivity for neutralization of neurite outgrowth inhibition through mAb50 compared to rat DRG neurons.…”