Global Analysis of Protein Expression of Inner Ear Hair Cells

Abstract

The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

Deletion of Tmtc4 activates the unfolded protein response and causes postnatal hearing loss

“…Tmtc4 is a gene encoding a predicted 741 amino acid 83 kDa protein. Tmtc4 has been found by both proteomic and transcriptomic analyses to be expressed in the developing and young postnatal murine cochlea along with the other 3 isoforms, which is consistent with our data (17,18,34). Moreover, our finding that Tmtc1, Tmtc2, and Tmtc3 all increase substantially in transcript abundance in cochleae from Tmtc4-KO mice appears to underscore the likely interrelatedness of these genes.…”

“…Immunohistochemistry on cochlear sections from P21 Tmtc4 Het mice demonstrated broad expression of β-gal (as an indirect measure of Tmtc4 expression) driven by the Tmtc4 promoter in IHCs, OHCs, and cochlear supporting cells ( Figure 3). This corroborates recently published data from RNASeq and mass spectrometry experiments of flow-sorted outer and inner hair cells confirming that Tmtc4 protein is highly enriched in these cells (17,18). These published data also demonstrate that the level of Tmtc4 (both mRNA and protein) in the cochlea is higher than the other 3 isoforms (Tmtc1,2,3), although all 4 are expressed in the cochlea (18).…”

“…Thus, these examples provide both indirect and direct evidence linking ER stress and the UPR to hearing loss. Moreover, there is also data linking age-related hearing loss to the UPR (17,48). Our work with TMTC4 further strengthens the connection between the UPR and hearing loss, but also implicates the even more upstream pathway of Ca 2+ regulation, which may be a potentially more advantageous point for therapeutic intervention.…”

CNTNAP2 ectodomain, detected in neuronal and CSF sheddomes, modulates Ca2+ dynamics and network synchrony

“…Sample preparation for LC-MS/MS analysis. We followed the protocol described elsewhere (Hickox et al, 2017). The precipitated protein pellets were solubilized in 100 μL of 8 M urea for 30 min; 100 μL of 0.2% ProteaseMAX (Promega) was then added, and the mixture was incubated for an additional 2 h. The protein extracts were reduced and alkylated as described previously (Chen et al, 2008), followed by the addition of 300 μL of 50 mM ammonium bicarbonate, 5 μL of 1% ProteaseMAX, and 2 μg of sequence-grade trypsin (Promega).…”

Altered protein quality control contributes to noise-induced hearing loss

“…Mass spectrometry (MS)-based proteomics provides an opportunity to investigate complex biological phenomena by identifying and quantitating thousands of proteins. Previous proteomic studies of the auditory system have provided draft HC and organ of Corti proteomes, and identified protein substrates of chemically induced hearing loss (20-24). Transcriptomic analysis has also been informative and revealed a panel of genes acutely regulated in response to very high noise levels (25).…”