Abstract
Apoptosis is closely associated with cochlear sensory hair cell injury caused by several kinds of ototoxic insults. The proteins involved in apoptosis have been identified; the application of antiapoptotic proteins could prevent the development of diseases resulting in hearing loss. However, this strategy, known as protein therapy, has a limitation; the barrier in the cochlea obstructs the entry of high molecular weight proteins into the cochlear, irrespective of whether the proteins are administered systemically or topically in the tympanic cavity.
To slove this problem, we developed the protein transduction domain (PTD) fusing technique. PTD is a peptide originally derived from the HIV Tat protein. When fused with this peptide, high molecular proteins can effectively penetrate the cell membrane. In this study, we constructed an antiapoptotic protein FNK, originally derived from Bcl-xL, and fused it with PTD. An in vitro study showed that PTD-FNK significantly attenuated cochlear hair cell (HC) death and reduced kanamycin sulfate (KM)-induced caspase-9 expression. Next, we examined if PTD-FNK could reach the cochlea in vivo. We tagged PTD-FNK with myc to form PTD-myc-FNK and examined its immunoreactivity to the myc antibody. PTD-myc-FNK was diffusely distributed throughout the cochlea on both systemic and topical administration of the protein. The distribution was most prominent in the organ of corti and spiral ganglion cells. Immunoreactivity was observed at 1-3 h after systemic administration and at 1-24 h after topical administration. Thereafter, we examined whether PTD-FNK could attenuate the cochlear damage induced by a combination of KM and ethacrynic acid (EA) in vivo. The PTD-FNK protein, irrespective of systemic or topical administration, significantly attenuated the ototoxic drug-induced ABR threshold shifts and reduced the extent of HC death. Further, systemic PTD-FNK administration significantly attenuated the effect of the ototoxic drug-induced cleavage of PARP.
These findings indicate that PTD-FNK can be successfully delivered to the cochlea, where it can effectively prevent the apoptotic cell death of the cochlear HCs induced by KM, and that protein therapy using the PTD fusing technique is a feasible approach for treating diseases resulting in hearing loss.
