CS is the key enzyme of the tricarboxylic acid cycle, catalyzing the condensation of acetyl-Coenzyme A and oxaloacetate to citric acid, controlling the entrance of the tricarboxylic acid cycle and playing a decisive role in regulating energy generation.
several lines of evidence had been provided that a missense mutation of Cs is responsible for the hearing loss of A/J strain mice[4]. A/J strain mice have been widely used in the study of the anatomical, physiological, pathological and molecular mechanisms of age-related hearing loss (AHL)[7]. The onset of hearing loss is much earlier, showing an elevated hearing threshold as early as 25 days of age[8]. Genetic studies of hearing loss in A/J mice revealed an additional AHL locus, named ahl 4, on the distal-most 5.5 Mb chromosome (Chr) 10 [4, 7]. The rs29358506 SNP in the third exon 3 of Cs gene (11 exons, cDNA 1395 nt, encoding 464 amino acids) is the root cause of ahl 4-related hearing impairment which resulted in a missense mutation (H55N) of Cs. Our previous studies have shown that the mRNA levels of apoptosis-related genes (caspase-3 and caspase-9, etc.) were increased in inner ears of A/J mice at postnatal day 1, and anti-apoptosis treatment can reduce the hearing threshold of A/J mice to some extent. Furthermore, downregulation of Cs expression HEI-OC1 cells significantly increased the expression of Caspase-3, indicating that the low expression of Cs could cause the enhancement of cell apoptosis signal.
In the present study, 225 proteins were confirmed as DEPs between shRNA-NC and shRNACs-1429 cells, including 99 proteins downregulated and 126 proteins upregulated. The following bioinformatics analysis showed that the first ten HUB proteins were enriched in the oxidative phosphorylation signaling pathway and all downregulated differentially. This suggested that low expression of CS lead to the dysregulation of oxidative phosphorylation which is one of the important causes of mitochondrial dysfunction. Mitochondrial dysfunction is a typical feature of aging diseases and aging-related degenerative diseases, such as Alzheimer’s disease and Parkinson’s disease.[9] In our study, the top 3 KEGG pathways were shown as DEPs were oxidative phosphorylation, Parkinson’s disease and Alzheimer’s disease. This indicated mitochondrial dysfunction occurred in shRNACs-1429 cells. Ongoing mitochondrial dysfunction resulted in cell dysfunction and death[10], which was reported in our previous work that the proliferation ability of shRNACs-1429 cells was decreased and apoptosis was increased.[5] Mitochondrial dysfunction also was a important cause of hearing loss because of the disorder or death of cochlear cells.[11]
The mitochondrial oxidative phosphorylation system (OXPHOS) generates ATP by phosphorylating ADP, which occurs in conjunction with the transit of electrons from reducing equivalents (i.e., NADH, FADH2) down the electron transport chain(ETC), a series of transmembrane protein complexes in the mitochondrial inner membrane.[12] The electron transport chain is composed with four enzyme multi-subunit complexes, Complex I (NADH-Coenzyme Q reductase), Complex II (succinate-Coenzyme Q reductase), Complex III (ubiquinol-cytochrome c reductase or cytochrome bc1 Complex) and Complex IV (cytochrome c oxidase).[13] Complex I is the largest component of respiratory chain, transporting two electrons from NADH to reduce ubiquinone. In our study, 9 subunits of Complex I were detected as DEFs with downregulated expression in shRNACs-1429 cells. Ndufv1 is one of the core subunits of the dehydrogenase domain of Complex I, catalyzing the NADH oxidation by a non-covalently bound flavin-mononucleotide (FMN).[14] NDUFS7 involves in the formation of a binding pocket in which electrons are transferred along iron-sulfur (FeS) to ubiquinone. Ndufs3, Ndufb5, Ndufa13, Ndufa2, Ndufb8, Ndufb3 and Ndufv5, as accessory subunits of complex I, involve in respiratory chain assembly. The downregulation of these proteins was detected in our study, which indicated the depress of electrons transfer in the mitochondrial respiratory chain of shRNACs-1429 cells. Complex III (Cytochrome b-c1 complex ) transfers electrons from QH2 to cytochrome c coupling with the translocation of protons, and is composed of three highly conserved core subunits and eight supernumerary subunits.[15] Uqcrb, also named as Cytochrome b-c1 complex subunit 7, was found to be significantly down-regulated in shRNACs-1429 cells. Uqcrb is one of supernumerary subunits, and required for assembly, stability, modulation and regulation of complex III. [16]Complex IV (Cytochrome c oxidase) transfers electrons from cytochrome c to oxygen, catalyzing the production of H2O. Cytochrome c oxidase subunit 4 is the largest of the accessory subunits. It is responsible for the allosteric inhibition of complex IV by binding ATP with increased ATP/ADP ratios. [17] Cytochrome c oxidase subunit 4 isoform 1(Cox4i1) was decreased in shRNACs-1429 cells. The generation of ATP was decreased in shRNACs-1429 cells which may be caused by the downregulation of multiple subunits of OXPHOS.
During the process of electrons transfer to O2 along ETC, some of the electrons leaking out noncovalently reduces O2 to hydroxyl radicals[2] and superoxide radicals, which can be immediately dismutated into hydrogen peroxide by superoxide dismutase.[18] The generation of mitochondrial ROS is closely related with complex I and complex III, [19] especially complex I.[20] The production of ROS was increased when complex I was inhibited by its specific inhibitor Rotenone.[21] In our study, 9 subunits were decreased in low-expressed CS cells, which resulted in the inhibition of complex I. Additionally, ROS was detected increasingly in our previous study.[5] It indicated that the inhibition of complex I induced by the low expression of CS in HEI-OC1 cells may be responsible for the enhancement of ROS production.