STAT3Pathway Regulates Transdifferentiation Of Cochlear Hair Cell In Neonatal Mice And Virally Mediated Gene Therapy In Pmca2Mutant Mice

Fart I Stat3pathway regulates transdifferentiation of cochlear hair cell in neonatal miceObjective Irreversible damage and death of inner ear hair cells caused by aging, noise and ototoxic drugs is a major factor responsible for human hearing loss and balance dysfunction. Hearing loss is the third highest incidence of the disease in elderly population. Due to the inability to regenerate hair cells in mammals, stimulating the auditory hair cell regeneration to reconstruct the structure and function of auditory organ is an ideal choice for the treatment of hearing loss. Mouse cochlea in vitro model is effective to study hair cell regeneration. During the screens of potential drugs promoting hair cell regeneration, a small molecule compound SD19is identified to be effective in stimulating hair cell regeneration. It induces an increase of the number of hair cells in cultured p0-pl CD1mice cochlea, by promoting supporting cell to hair cell transdifferentiation. In this study, I demonstrate that SD19acts through activating STAT3signaling pathway, leading to upregulation of hair cell fate determinator Atohl and suppression of Notch pathway. Thus screening of small molecules is an effective way to identify those with properties in hair cell regeneration, and with a potential to be developed into drug-like compounds.Methods1) CD1neonatal mouse model of cochlea Basilar membrane and spiral ligament co-culture in vitro was established, hair cells and supporting cells survival situation were investigated, different concentrations STAT3pathway activator SD19impact on hair cells are studied.2) The differences between with neomycin and without neomycin before using SD19were investigated.3) The effects of SD19on cochlea hair cell in different develpment stage were investigated.4) Combined with STAT3pathway inhibitor, the hair cell number was counted.5) Using FM1-43for labeling the original hair cell then treatment with SD19in explanted cochlear, the new hair cells were investigated.6) Sox-2CreER+/-/tdTomato transgenic mice was used to identify the source of new hair cell. 7) Genes in STAT3and hair cell development were investigated after SD19treatment.Results1) There was a significantly increase in the number of hair cells in apex and middle turn of the cochlea in PO/1CD1mice after treatment with4μM and8μM of STAT3pathway activator SD19, but not in P3and P5cochleas. Lineage tracing and FM1-43uptake was used to demonstrate new hair cells are derived from supporting cell transdifferentiation. By EdU labeling, I did not detect proliferation during regeneration, an indication that cell cycle is not involved. In the presence of a STAT3inhibitor, SD19failed to induce an increase in the number of hair cells, a strong indication that STAT3is involved in SD19function.2) qRT-PCR showed upregulation of Stat3and Jakl genes after treatment with8μM SD19in PO stage CD1mouse cochlea culture system, Hesl and Mathl was also unregulated but Notch and Sox2downregulated.ConclusionI identified a small molecule SD19that activates STAT3pathway, with a role in neonatal mouse cochlear hair cell transdifferentiation. STAT3pathway activation promotes transdifferentiation from supporting cells to hair cell. STAT3maybe have some regulatory role in Notch signaling pathway. Part Ⅱ Virally mediated gene therapy in Pmca2mutant miceObjective17percent of American adults suffer from hearing loss, in which hundreds of mutations caused hereditary deafness for which there is no treatment available. Inner ear exogenous gene delivery and gene therapy is new approach could be potentially developed as a treatment for genetic deafness. Adenovirus and adeno-associated virus (AAV) are common inner ear gene delivery vectors, with different serotypes infecting different types of cells. Pmca2is plasma membrane Calcium-ATPase II located in the outer hair cell bundles, with a mutation leading to intracellular calcium accumulation, ultimately resulting in hearing loss. Both homozygous and heterozygous mutant Pmca2mice exhibit severe deafness. As a first step to develop gene therapy for genetic deafness, I screened AAV and adenovirus for their capacity to infect hair cell in vivo. I further used adenovirus carrying exogenous Pmca2gene into neonatal Pmca2mutant inner ear to explore the treatment of hereditary deafness gene level.Methods1) Using Nanoliter2010micromanipulator system, injected six different serotypes of AAV (AAV2.2-CMV-eGFP, AAV2.2-CASI-eGFP, AAV2.8-CMV-eGFP, AAV2.8-CASI-eGFP, AAV2.9-CMV-eGFP, AAV2.9-CASI-eGFP) carrying enhanced green fluorescent protein and one type of adenovirus (Ad5-CMV-GFP) into the inner ear from scale medium. After injection3d,5d,1w,2w, lm, cochlear were taken out and detected eGFP or GFP expression by bilateral cochlear basilar membrane immunohistochemistry.2) AAV2.8-CMV-eGFP and AAV2.9-CMV-eGFP, which can infect hair cells, were injected into cochlear of neonatal wild-type mice (P0/P1) to the right in order cochlea, ABR and DPOAE were evaluated of bilateral hearing after6weeks.3) Clone Pmca2plasmid and packaged into AAV2.8-CMV and Ad5-CMV-GFP virus, respectively.4) Transfect cos7cells with Pmca2plasmid using Lipofectamine2000, and infect cos7cell line with AAV2.8-CMV-Pmca2-HA and Ad5-CMV-Pmca2-GFP. The efficacy of the virus was evaluated by immunohistochemistry and Western Blot.5) Injected into right scale medium of neonatal Pmca2mutant mice (P0-P1) with0.3/0.6μL AAV2.8-CMV-Pmca2-HA and Ad5-CMV-Pmca2-GFP.1w,6w later, bilateral cochlear were taken out and investigated by basilar membrane immunohistochemistry. ABR and DPOAE were evaluated after6w.Results1) Scale medium injection1w later, AAV2.8-CMV-eGFP and AAV2.9-CMV-eGFP and Ad5-CMV-GFP were detected in supporting cells, hair cells, spiral ligament, spiral ganglion, vestibular sensory cells GFP, sustained expression of at least2months.2) In wild type mice which injected with AAV2.8-CMV-eGFP and AAV2.9-CMV-eGFP on P0/P1, compared to the ear, hearing loss0-15dB in different frequencies after6weeks.3) Pmca2plasmid can transfect Cos7cell lines by Lipofectamine2000and produce Pmca2protein, AAV2.8-CMV-Pmca2-HA can’t infect cos7cell lines, Ad5-CMV-Pmca2-GFP can infect cos7cell line and expression Pmca2.4) HA tag could not be detected in inner ear of Pmca2mutant mice after injection with AAV2.8-CMV-Pmca2-HA, likely due to limitation in the size of gene cloned.5)) Pmca2mutant mice injected with Ad5-CMV-Pmca2-GFP on P0/P1showed Pmca2 over-expression in hair cells one week after injection. Limited hearing recovery was detected three weeks after injection, a strong indication the validity of our approach.Conclusion1) Type5adenovirus gene could infect hair cell of neonatal mice by micro-injection into scala medium, and the target gene was successfully expressed in cochlear tissue.2) AAV2.8-CMV-eGFP and AAV2.9-CMV-eGFP could infect hair cell of neonatal mice by micro-injection into scala medium, and the target gene was successfully expressed in cochlear tissue. There is a slight hearing loss causing by injection.3) Adeno-associated virus was not toxic to cochlear hair cells and could effectively transfect least two months, but it was limited by packaging capacity. Adenovirus exhibits some ototoxicity in vivo.4) The capacity of AAV limited its application in Pmca2gene therapy.5) Exogenous Pmca2gene could be expressed by adenovirus mediated delivery in mice, with some hearing recovery. Future experiment needs to be focused on the identification of vectors for long-term effect with minimum ototoxicity. Gene therapy could be effective in the treatment of genetic deafness.