Automated western blot analysis of ototoxin-induced prestin burst in the blood after cyclodextrin exposure

Abstract

In the clinical realm, we primarily rely on audiological measures for diagnosis and surveillance of sensorineural hearing loss (SNHL) and have limited therapeutic options. We have proposed a blood-based biomarker approach to overcome this challenge by measuring the outer hair cell’s (OHC) electromotile protein, prestin, in the blood. In a guinea pig model of cyclodextrin (CDX) ototoxicity, using western blots, we show that prestin in the blood may have several different forms and specifically the ~ 134 kDa form spikes after ototoxin ablation of OHCs. This form appears to be a glycosylated dimer likely secreted by the inner ear as exosomes reflecting increased expression after ototoxin exposure. These results suggest that the ~ 134 kDa dimer may serve as a biomarker for early detection of ototoxicity in the clinical setting. However, because prestin can still be measured in the blood after total ablation of OHCs, its ability to inform on OHC health is restricted to a narrow window after ototoxin-induced injury. Monitoring prestin, when using therapeutics with ototoxic properties, could guide dosage and administration schedule to minimize damage.

Serum Prestin After Ototoxin Exposure Is Not Dependent on Outer Hair Cell Loss

“…Western Blot Using Different Antibodies Raised to Two Different Targets Within the Prestin Molecule Confirm a 134-kDa CDX-Induced Band The apparent molecular weight of the CDX-responsive serum prestin peak is ~134 kDa (6), which is larger than the apparent molecular weight that is predicted for fulllength human prestin, which is ~81.4 kDa (8). Automated Western systems generally result in apparent molecular weights that are larger than what is predicted or found using standard Western blotting (9), which still does not account for the large difference between 81 and 134 kDa.…”

“…Most of the above reports used the enzyme-linked immunosorbent assay technique, which cannot distinguish between prestin fragments, monomers, or multimers. Recently, we used an automated Western blot approach to determine if the characteristics of prestin differed before and after exposure to an ototoxin (6).…”

“…We recently introduced a guinea pig CDX-ototoxicity model with targeted ablation of the outer hair cells (OHCs) (6). Using Western blot analysis, it was demonstrated that CDX ototoxicity induced a species of prestin (~134 kDa) in the serum that was different from pre-exposure species (~58 and ~83 kDa).…”

“…Prestin in the serum has been proposed as a biomarker for ototoxicity (1) with the potential to detect ototoxicity in advance of audiometric measures (2,3). Since the original proposal by our senior author, proof of concept has been offered in cisplatin (2–5), amikacin (5), and cyclodextrin (CDX) (6) experimental models and in cisplatin-treated human patients (3,7). These reports agree in that prestin rises shortly after exposure to ototoxins.…”

Outer Hair Cell Motor Protein, Prestin, Is Not a Reliable Serological Biomarker for Mouse Cochlear Damage

“…By using a sandwich enzyme-linked immunosorbent assay (ELISA), Prestin was detected in the bloodstream of humans [13][14][15], rats [16,17], guinea pigs [18,19], and mice [20]. Several reports suggested that Prestin in the bloodstream could be used as a biomarker for hearing loss such as idiopathic sudden sensorineural hearing loss [13-15, 21, 22], noise-induced hearing loss [16,17,[23][24][25], sensory hearing loss [26][27][28], age-related hearing loss [29], ototoxic regents induced hearing loss, such as HPβCD [30] and cisplatin [18][19][20]31], and also hearing loss observed in various diseases like Meniere's Disease and Vestibular Migraine [32], COVID-19 [33], lead poison [34], and even surgical related damage [21]. However, the data reported in several studies lack proper negative controls and show inconsistency [13,14].…”