RESUMO
Indirect evidence suggests that endogenous ciliary neurotrophic factor (CNTF) receptor signaling can promote motor neuron (MN) survival in the adult. If so, proper targeting of this signaling may selectively counteract the effects of adult MN diseases. However, direct evidence for CNTF receptor involvement in adult MN survival is lacking, presumably because the unconditional blockade of the mouse CNTF receptor in vivo [through genetic disruption of the essential CNTF receptor alpha (CNTFRalpha) gene] leads to uniform perinatal death of the mice. To overcome this limitation, we have developed a method to selectively disrupt CNTF receptor function in a targeted subset of adult MNs that are not required for survival. A 'floxed CNTFRalpha' mouse line was generated and characterized. In addition, an adeno-associated virus (AAV) vector that drives Cre recombinase (Cre) expression was constructed and shown, with reporter mouse lines, to selectively excise floxed genes in facial MNs following its stereotaxic injection into the facial motor nucleus. Adult floxed CNTFRalpha mice were then injected with the AAV-Cre vector to excise the CNTFRalpha gene in the targeted MNs. The resulting data indicate that adult CNTF receptor signaling, likely by the MNs themselves, can play an essential role in MN survival. The data further indicate that this role is independent of any developmental contributions CNTF receptor signaling makes to MN survival or function.
Assuntos
Sistema Nervoso Central/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Marcação de Genes/métodos , Neurônios Motores/metabolismo , Receptor do Fator Neutrófico Ciliar/genética , Fatores Etários , Animais , Sobrevivência Celular/genética , Dependovirus/genética , Regulação para Baixo/genética , Nervo Facial/citologia , Nervo Facial/metabolismo , Vetores Genéticos/genética , Integrases/genética , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , TransfecçãoRESUMO
Sphingosine 1-phosphate (S1P) is an endogenous growth factor with potent effects on many different cell types. Most of these effects are produced by activation of one or more of a family of G-protein coupled receptors. The S1P2 receptor can mediate S1P-induced proliferation, differentiation and survival in a wide variety of cells in culture. However, identifying essential in vivo functions for S1P2 has been hampered by its ubiquitous expression and the failure to detect any anatomical abnormalities in initial analyses of S1P2 knockout mice. We report here that all S1P2 knockout mice are profoundly deaf from postnatal day 22 and approximately half display a progressive loss of vestibular function with aging. Anatomically, both the auditory and vestibular systems appear to develop normally but then degrade. Morphological defects associated with hearing are first detected at 3 weeks postnatal as deformations of the organ of Corti/Nuel's space. By one year of age structures within the scala media are dramatically altered. The S1P2 knockout mice also display a loss of otoconia consistent with the vestibular impairment. The present data are the first to indicate that S1P signaling plays critical roles, in vivo, in auditory and vestibular functions. The data further establish that the S1P signaling occurs through the S1P2 receptor and makes an essential contribution to the structural maintenance of these systems, raising the possibility that properly targeted enhancement of this signaling may prove to be clinically beneficial.