Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in usher syndrome type 1F

Abstract

Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model.

Cochlin expression in the rat perilymph during postnatal development

“…Previous 2D gel investigation identified a full-length isoform, as well as smaller isoforms with truncations of the N terminus. The molecular weight values of the predominant isoforms were 44 and 40 kDa [10,12]. Isoform-specific antibodies further identified the short truncated isoform CTP in the perilymph [10], and this isoform contains the N-terminal LCCL domain devoid of the C-terminal vWFA domain.…”

“…The potential effects that are conveyed by this molecular phenotype are currently unknown, but the observed change is counter to what has been reported in normal development. Our previous data indicated that the abundance of the full-length isoform p63s decreases relative to the abundance of the N-terminally truncated form p44s and p40s as a function of age in the rat, with the fulllength isoform predominant on DAB24 but decreased relative to the truncated form on DAB70 [11,12].…”

“…The molecular weight of CTP is different in different species, e.g. rat (11)(12)(13)(14)(15)(16)(17), human (16 kDa), and bovine (18-23 kDa) [13]. The importance of cochlin has been shown in a variety of pathological conditions, such as DFNA9, autoimmune hearing loss, glaucoma, and others.…”

“…One of the keys to understanding the pathogenetic mechanisms in these conditions is the analysis of the isoform expression patterns. Chance et al [12] identified certain proteins and protein networks associated with cochlear pathogenesis in the Ames Waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F). Sequence analysis by mass spectrometry showed that, in the av cochlea, a set of full-length cochlin isoforms was up-regulated, while the isoforms lacking the N-terminal FCH/LCCL domain were down-regulated.…”

“…In contrast, on DAB70, the expression of p63s had decreased and p40s had become the predominant isoform. The importance of isoform analysis was reported in a study of Usher syndrome type 1F (USH1F) [12], where altered levels of cochlin isoforms were suggested to be pathogenic.…”