RESUMO
Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.
Assuntos
Animais Recém-Nascidos , Reprogramação Celular , Cóclea , Animais , Camundongos , Cóclea/metabolismo , Cóclea/citologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/citologia , Dependovirus/genética , Ciclo Celular , Camundongos Endogâmicos C57BL , Regeneração , Células Labirínticas de Suporte/metabolismo , Neomicina/farmacologiaRESUMO
Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Reprogramação Celular , Dependovirus , Células Ciliadas Auditivas , Regeneração , Animais , Camundongos , Dependovirus/genética , Reprogramação Celular/genética , Regeneração/genética , Regeneração/fisiologia , Células Ciliadas Auditivas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismo , Diferenciação Celular/genética , Vetores Genéticos/genética , Proteínas de Ligação a DNA , Fatores de Transcrição , Proteínas de HomeodomínioRESUMO
Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.