Automated Western Blot Analysis of Ototoxin-Induced Prestin Burst in the Blood after Cyclodextrin Exposure

Abstract

Hypothesis Ototoxin cyclodextrin (CDX) will induce a burst in serum prestin when quantified with automated Western blot analysis. Background 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. Previously, we demonstrated a burst in serum prestin after cisplatin exposure using enzyme-linked immunosorbent assayELISA. Methods Guinea pigs were treated with either 3,000 or 4,000 mg/kg CDX, and serum samples were obtained through 3 days after exposure. Serum prestin levels were quantified using automated blot analysis, western and hair cell counts were obtained. Results Both 3,000 and 4,000 mg/kg resulted in robust OHC loss, although more variability was seen at the lower dose. Automated Western blot analysis demonstrated that the prestin profile after CDX exposure is different than baseline. Specifically, a new ~134- kDa band accounted for the prestin burst after ototoxin ablation of OHCs at both doses. Conclusions We reproduced the prestin burst seen after cisplatin administration using CDX. Automated Western blot western analysis revealed that a ~a ~ 134- kDa species of prestin is responsible for the burst. We suggest that the induced band may be a prestin dimer, which could serve as a biomarker for early detection of ototoxicity in the clinical setting. These results add further promise to the potential of serum prestin to serve as an ototoxicity biomarker when using therapeutics with ototoxic properties.

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

“…1C). Although it remains unclear whether the ~58- and ~83-kDa bands represent nonspecific proteins or prestin fragments, the SCBT antibody detects the ~134-kDa band with better sensitivity than the INVT antibody under the conditions used; the band can be more easily quantified at baseline where the ~134-kDa levels are very low, and the magnitude of ~58- and ~134-kDa bands is more comparable to each other (see (6), Fig. 5A).…”

“…Serum samples diluted to 0.5 μg/μl total protein were analyzed using INVT anti-prestin at 1:25 and SCBT anti-prestin at 1:2.5, 1:5, 1:10, and 1:20. IHC and OHC counts for G19 are shown in (6), Figure 3. CDX indicates cyclodextrin.…”

“…The kinetics of the prestin burst could be informative in interpreting the mechanism by which prestin ends up in circulation. We previously reported that the prestin burst is maintained 1, 2, and 3 days after CDX administration (6), although previous studies using other models have described an eventual decline of prestin to baseline levels (2–4). Here, blood samples were collected as indicated (Fig.…”

“…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).…”