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The Rho Kinase Pathway Regulates Mouse Adult Neural Precursor Cell Migration



Article Focus for this Month’s Edition of Stem Cells

Paper commentary by Carla B. Mellough

The subventricular zone (SVZ) is a multicellular structure that lines the lateral walls of the lateral ventricles of the brain. SVZ ependymal cells face the ventricular lumen and are involved in the production and circulation of cerebrospinal fluid. Further, the SVZ is an established site of adult neurogenesis, boasting the largest population of proliferative cells in the brain of mature rodents, monkeys and humans. The neural precursor cells (NPCs) of the SVZ produce neuroblasts which migrate to the olfactory bulb via the rostral migratory stream (RMS). These ordinarily act to replenish olfactory neurons, however following central nervous system (CNS) damage they become capable of migrating towards ectopic sites of injury. The mobilisation and guidance of NPCs towards a distinct neural destination involves numerous external signals which must be integrated and translated by neuroblasts to produce an appropriate response, allowing specific and directed migration. The Rho-GTPase family of molecules and their related regulatory members such as the Rac and PIk3 proteins have previously been demonstrated to influence cell migration by regulating the translation of external signals into cytoskeletal reorganisation, yet their role in the migration of neuroblasts through the adult RMS had not yet been established. In the February edition of Stem Cells, new results by Leong et al. from Ann Turnley’s laboratory at the Centre for Neuroscience at The University of Melbourne, begin to reveal the role of the Rho-GTPase pathway in the migration of adult mouse SVZ NPCs.

The authors started by looking at the effect of Rho Kinase (ROCK), Rac1 and PI3K inhibitors on the morphology of neurosphere cultures derived from the adult mouse SVZ. Under normal conditions, NPCs migrate as sheets of interconnected cells with lamellipodia, chain migration being the preferred mode of migration. Treatment with ROCK inhibitor Y27632, however, caused ‘thinning’ of NPC cell bodies, decreased lamellipodia formation and reduced cell-cell contact, promoting single cell migration over chain formation. This effect was reversed upon substitution with Y27632 free culture medium and chain migration of NPCs was resumed. In contrast, Rac1 inhibition resulted in the loss of cellular processes and promoted cell-cell contact, resulting in round, tightly packed cells that did not migrate away from the neurosphere. Inhibition of ROCK increased the migratory capacity of NPCs and increased NPC ‘spread’ (distance between migrating cells), an effect which was dose-dependent, whilst Rac1 inhibition led to the opposite effect. PI3K inhibition had no effect. Addition of these inhibitors did not influence proliferation, apoptosis or the number of cells per sphere. The authors also demonstrate that treatment with Y27632 decreases the phosphorylation of myosin light chain II, a major regulator of the cytoskeleton, a likely explanation for Y27632’s effects on NPC cell contact and migration.

Leong et al. then knocked down the expression of ROCK1 and ROCK2 RNA and protein in neurospheres using siRNA alongside a red fluorescent RNA. Whilst knockdown of ROCK1 alone had no apparent effect on NPCs, the downregulation of ROCK2 enhanced migration and promoted an elongated NPC morphology. Combined knockdown of both subtypes resulted in bipolar morphology and further enhanced migration compared to ROCK2 downregulation alone.

To further investigate the effects of ROCK inhibition on NPC migration, they studied migration patterns of NPCs in neonatal (P3) SVZ explants which under normal conditions show both chain and dissociation migration. They demonstrate again that treatment with Y27632 impairs chain migration and enhances dissociation migration of NPCs in SVZ explants. They went on to investigate these effects in vivo by infusing Bromodeoxyuridine (BrdU) plus Y27632 into the lateral ventricle of adult mice for 7 days or 1 month. They found that in treated mice there was a decrease in the number of newborn NPCs in the RMS and an overall reduction in cell density, suggesting reduced migration of NPCs to the olfactory bulb. Further, NPCs in the RMS were found to be more dispersed, with many NPCs found outside the RMS boundary indicating that the treatment also induced the ectopic migration of NPCs. This occurred alongside an increase in the number of BrdU+ cells in regions outside of the RMS, including the anterior cortex and the accessory olfactory nuclei (AON). The BrdU+ cells of the AON in treated mice were often immunopositive for the neuronal marker NeuN, although only a very small minority (<1%) of BrdU labelled cells in the anterior cortex were found to be NeuN positive.

In summary, this work has shown that inhibition of the Rho pathway greatly enhances NPC migration whilst migration is reduced by Rac1 pathway inhibition. Understanding the cues which govern the directed migration of endogenous resources of de novo neurons has great potential benefit for the development of methods which aim to enhance endogenous repair of the adult brain following injury. It should be noted here that although ROCK inhibition increased the migratory capacity of NPCs it was also associated with reduced navigational capacity and therefore the ectopic localisation of NPCs within adjacent brain regions. As noted by the authors, the detrimental effects of this disrupted migration must also be considered. The effect of ROCK inhibition on neuron formation in the AON is also very interesting and clearly deserves further study. The mobilisation of NPCs from the SVZ has already been implicated during the progression of neurodegenerative conditions and following brain injury and the use of ROCK inhibitors is already underway in a variety of human trials for various conditions including cardiovascular disease and cerebral infarction. Unlocking the mechanisms which control the directed migration of replacement cell types towards a site of CNS injury and that promote the appropriate and functional differentiation has great potential clinical value and may even remove the need for exogenous replacement cell types.

Original Research Article

Leong SY, Faux CH, Turbic A, Dixon KJ, Turnley AM. The Rho Kinase Pathway Regulates Mouse Adult Neural Precursor Cell Migration. Stem Cells. 2010 Dec 9.