RESUMO
Abortive cell cycle (ACC) re-entry of apoptotic neurons is a recently characterized phenomenon that occurs after central nervous system (CNS) injury or over the course of CNS disease. Consequently, inhibiting cell cycle progression is neuroprotective in numerous CNS pathology models. Primary cilia are ubiquitous, centriole-based cellular organelles that prevent cell cycling, but their ability to modulate abortive cell cycle has not been described. Here, we show that neuronal cilia are ablated in-vitro and in-vivo following injury by hypoxia or optic nerve transection (ONT), respectively. Furthermore, forced cilia resorption sensitized neurons to these injuries and enhanced cell death. In contrast, pharmacological inhibition or shRNA knockdown of the proteins that disassemble the cilia increased neuron survival and decreased the phosphorylation of retinoblastoma (Rb), a master switch for cell cycle re-entry. Our findings show that the stabilization of neuronal primary cilia inhibits, at least transiently, apoptotic cell cycling, which has implications for future therapeutic strategies that halt or slow the progression of neurodegenerative diseases and acute CNS injuries.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular , Sistema Nervoso Central/patologia , Cílios/fisiologia , Neurônios/patologia , Traumatismos do Nervo Óptico/patologia , Substâncias Protetoras/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/lesões , Cílios/efeitos dos fármacos , Etoposídeo/farmacologia , Feminino , Hipóxia , Neurônios/efeitos dos fármacos , Traumatismos do Nervo Óptico/tratamento farmacológico , Fosforilação , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologiaRESUMO
Purpose: Integrin adherence to the extracellular matrix (ECM) is essential for retinal ganglion cell (RGC) survival: damage causes production and release of ECM degrading matrix metalloproteinases (MMPs) that disrupt integrin ligation, leading to RGC death. The interplay of MMPs, integrins, and focal adhesion kinase (FAK) was studied in RGCs after optic nerve injury. Methods: Optic nerve transection and optic nerve crush were used to study RGC survival and regeneration, respectively. Treatments were administered intravitreally or into the cut end of the optic nerve. RGC survival was assessed by fluorescence or confocal microscopy; cell counting, peptide levels, and localization were assessed by Western blot and immunohistochemistry. Results: MMP-9 was most strongly increased and localized to RGCs after injury. Pan-MMP, MMP-2/-9, and MMP-3 inhibition all significantly enhanced RGC survival and increased RGC axon regeneration. FAK activation was decreased at 4 days postaxotomy, when apoptosis begins. FAK inhibition reduced RGC survival and abrogated the neuroprotective effects of MMP inhibition, whereas FAK activation increased RGC survival despite MMP activation. Integrin ligation with CD29 antibody or glycine-arginine-glycine-aspatate-serine (GRGDS) peptide increased RGC survival after axotomy. Conclusions: ECM-integrin ligation promotes RGC survival and axon regeneration via FAK activation.