Study of the role of Rabconnectin-3a at the auditory hair cell ribbon synapse

• Autor(a) principal: Moreira, Mariana Brandão
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/29675
• topic: Rabconnectin-3a; IHC; Ribbon synapses; V-ATPase; Otoferlin

Inner hair cells (IHCs) are the sound sensing cells of the auditory organ. The synaptic activity capabilities that these cells have can be attributed to the presence of ribbons anchored in the active zone of the IHC synapse. The ribbons are an electron dense structures responsible for the tethering of synaptic vesicles. Synaptic vesicles are composed of lipids and many proteins, one of which is Rabconnectin-3a, a subunit of the Rabconnectin-3 complex. This complex is found abundantly in the brain, particularly at the synapses where it is important for neurotransmission. Rabconnectin-3a protein is a putative V-ATPase regulator with an important role in brain development. This protein is encoded by the DMXL2 gene. Mutations in the DMXL2 gene are linked to hearing impairment and neurodevelopmental disorders. In this work it was proposed to study the role of Rabconnectin-3a in IHC ribbon synapse. To achieve this goal a Rabconnectin-3a knock-out (KO) mouse model in IHCs was used, where apical turns of the organ of Corti were dissected, immunolabelled and imaged. Also, the protein samples were prepared from the whole cochlea and subjected to Western blotting (WB) technique. Together, these two techniques have enabled the quantification of protein levels, as well as their distribution in the ribbon synapse. A significant decrease in the expression of V-ATPase subunits needed for synaptic vesicle acidification and refilling, and otoferlin protein needed for calcium sensing were observed in the Rabconnectin-3a IHC KO mice in comparison with the Wild-Type (WT) controls, suggesting an impairment in IHC transmission. Also, the number of ribbons was higher in the Rabconnectin-3a KO IHC in comparison with the WT. This could be due to a developmental impairment. On the other hand, both Rabconnectin-3β (other component of Rabconnectin-3 complex) and vesicular glutamate transporter 3 (vGlut3) protein levels are increased in the Rabconnectin-3a KO mouse. Overall, the results obtained in this work provided new insights on the role of Rabconnectin-3a at the mammalian IHC ribbon synapse.