In adult mammalian central nervous system (CNS) neurons fail to regenerate after injury and most of them die by apoptosis within few days. The neuronal pathways underlying such events are far from being fully understood. Rac1 is a rho-related small GTPase that regulates cytoskeletal dynamics critically involved in neuronal development, axon growth and cell survival. Its constitutively active (CA) mutant is able to promote the axonal growth through an inhibitory environment both in vitro and in vivo and also to promote cell survival in vitro. However Rac1 CA mutant simultaneously activates many downstream concurrent signaling pathways, because Rac1 can potentially bind several downstream effectors. Thus, in order to investigate the effect of Rac1 activation on neuronal survival and axonal regeneration after injury, we used two Rac1 double mutants in which a first L61 mutation leads to constitutive activation state whereas a second mutation (F37A or Y40C) prevents the specific interaction with some downstream effectors, conferring selectivity for the delivery of downstream signaling. A TAT Trojan nanovector sequence allows overstepping of the cell plasma membrane leading to accumulation into the cell. We injected these two mutants intravitreally and we investigated, by means of immunofluorescence and imaging techniques, the retina ganglion cell (RGCs) survival and the axonal regeneration at 15 and 30 days after optic nerve crush. These mutants have been previously characterized, but to our knowledge they have never been used in vivo in the CNS. Our results show that both Rac1-mutants were able to improve cell survival, however with different mechanisms: the F37A increased p21 activated kinase (PAK) and extracellular signal regulated kinases (ERK1/2) activation directly in RGCs, displaying a dose-dependent effect on survival, whereas the Y40C mutant increased the ERK1/2 phosphorylation selectively in Muller glial cells, giving an indirect effect on RGCs. Between the two mutants only the F37A was able to improve axonal regeneration until 15 days post-injury, suggesting that the manipulation of a single pathway is insufficient to obtain a massive regeneration. However, our data suggest also that long-term repetitive treatments could counteract the normal axonal degeneration occurring after crush. Our study clarifies the role of Rac1 as a pro-survival signaling molecule in neurons in vivo and supports the possibility to use Trojan nanovector-based approaches in the treatment of neuronal damages.

Selective Rac1 mutants promote retina ganglion cells survival and regeneration by ERK1/2 and PAK activation after optic nerve injury.

LORENZETTO, Erika;ETTORRE, Michele;Pontelli, Valeria;BOLOMINI VITTORI, Matteo;Bolognin, Silvia;ZORZAN, Simone;LAUDANNA, Carlo;BUFFELLI, Mario Rosario
2012

Abstract

In adult mammalian central nervous system (CNS) neurons fail to regenerate after injury and most of them die by apoptosis within few days. The neuronal pathways underlying such events are far from being fully understood. Rac1 is a rho-related small GTPase that regulates cytoskeletal dynamics critically involved in neuronal development, axon growth and cell survival. Its constitutively active (CA) mutant is able to promote the axonal growth through an inhibitory environment both in vitro and in vivo and also to promote cell survival in vitro. However Rac1 CA mutant simultaneously activates many downstream concurrent signaling pathways, because Rac1 can potentially bind several downstream effectors. Thus, in order to investigate the effect of Rac1 activation on neuronal survival and axonal regeneration after injury, we used two Rac1 double mutants in which a first L61 mutation leads to constitutive activation state whereas a second mutation (F37A or Y40C) prevents the specific interaction with some downstream effectors, conferring selectivity for the delivery of downstream signaling. A TAT Trojan nanovector sequence allows overstepping of the cell plasma membrane leading to accumulation into the cell. We injected these two mutants intravitreally and we investigated, by means of immunofluorescence and imaging techniques, the retina ganglion cell (RGCs) survival and the axonal regeneration at 15 and 30 days after optic nerve crush. These mutants have been previously characterized, but to our knowledge they have never been used in vivo in the CNS. Our results show that both Rac1-mutants were able to improve cell survival, however with different mechanisms: the F37A increased p21 activated kinase (PAK) and extracellular signal regulated kinases (ERK1/2) activation directly in RGCs, displaying a dose-dependent effect on survival, whereas the Y40C mutant increased the ERK1/2 phosphorylation selectively in Muller glial cells, giving an indirect effect on RGCs. Between the two mutants only the F37A was able to improve axonal regeneration until 15 days post-injury, suggesting that the manipulation of a single pathway is insufficient to obtain a massive regeneration. However, our data suggest also that long-term repetitive treatments could counteract the normal axonal degeneration occurring after crush. Our study clarifies the role of Rac1 as a pro-survival signaling molecule in neurons in vivo and supports the possibility to use Trojan nanovector-based approaches in the treatment of neuronal damages.
retinal ganglion cell; regeneration; apoptosis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/476958
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