Oxidative stress and ROS metabolism via down-regulation of sirtuin 3 expression in Cmah-null mice affect hearing loss

Abstract

CMP-Neu5Ac hydroxylase (Cmah) disruption caused several abnormalities and diseases including hearing loss in old age. However, underling molecular mechanisms that give rise to age-related hearing loss (AHL) in Cmah-null mouse are still obscure. In this study, Cmah-null mice showed age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. To identify differential gene expression profiles and pathway associated with AHL, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip and pathway-focused PCR array in the cochlear tissues of Cmah-null mouse. Pathway and molecular mechanism analysis using differentially expressed genes provided evidences that altered biological pathway due to oxidative damage by low expressed antioxidants and dysregulated reactive oxygen species (ROS) metabolism. Especially, low sirtuin 3 (Sirt3) gene expressions in Cmah-null mice decreased both of downstream regulator (Foxo1 and MnSod) and regulatory transcription factor (Hif1αand Foxo3α) gene expression. Taken together, we suggest that down-regulation of Sirt3 expression leads to oxidative stress and mitochondrial dysfunction by regulation of ROS and that it could alter various signaling pathways in Cmah-null mice with AHL.

Keywords: CMP-N-acetylneuraminic acid hydroxylase; ROS metabolism; hearing loss; mitochondria dysfunction; oxidative stress.

Figure 1. Age-related inner ear abnormality and neuron cell loss in Cmah-null mice

(A) Analysis of Neu5Gc expression in cochlear tissues of WT- and Cmah-null mice by IHC using a chicken anti-Neu5Gc antibody. (B) Histological abnormality in inner ears of Cmah-null mice. Deposition of unusual and apparently a cellular material in the vestibular otoconial epithelia (upper). The area of the outer hair cells showed degeneration of the sensory cells throughout the cochlea in the cochlear sensory epithelium, (bottom). SV: scala vestibule, SM: scala media, ST: scala tympani, SG: spiral ganglion, BM: sasilar membrane, RM: reissner's membrane, Sv: stria vascularis, OT: otoconia, OM: otolithic membrane, NHC & SC: neuroepithelial hair cells and supporting cells, HC: hair cells, OHCs: outer hair cells, SL: Spiral lamina. (C) Apoptotic cell death in the cochlea of Cmah-null mice. TUNEL labeling performed on paraffin sections the cochlea from WT- and Cmah-null mice. (D) The expression of ganglion cells in cochlear tissues from WT- and Cmah-null mice by IHC. Rectangular box indicate a higher magnification images (5x) in left panel. GFAP: Glial fibrillary acidic protein, NSE: Neuron specific enolase, NF: Neurofilament, VIM: Vimentin. SL: spiral lamina, OC: organ of corti, SG: spiral ganglion. (E) mRNA expression pattern of neuronal cell markers in cochlear tissues from WT- and Cmah-null mice. RT-qPCR was used to measure the expression of Gfap, Nse, Nf, and Vim in the cochlea from WT- and Cmah-null mice.

Figure 2. Gene expression profile in cochlea of Cmah-null mice

(A) Scatterplot of normalized mRNA expression by microarray. Data are log2 normalized. Grey- and orange-spots indicate no significant genes and up- or down-regulated genes, with more than 1.5-fold change. (B) Venn diagram showing differential expression of genes in the cochlear tissues of Cmah-null mice. Numbers in red and blue Venn diagram present up- and down-regulated genes, respectively. (C) The differentially up- or down-regulated genes were clarified according to biological process, molecular function, and cellular component. GO classification significantly enriched with a Benjamini-Hochberg corrected P value < 0.05 among all differentially expressed genes with ≥ 1.5-fold expression between WT- and Cmah-null mice.

Figure 3. Functional categorization of neurological disease and network of canonical pathways objected by IPA using genes differentially expressed in the cochlea of Cmah-null mice

(A) Pie chart analysis of the disease or functions annotation for neurological disease. (B) The network of canonical pathways highlights the relationships between pathways and classifies the functional groups by IPA analysis. Group I and II represent oxidative stress and mitochondrial dysfunction-related signaling (Group I), and Wnt/β-catenin signaling (Group II), respectively.

Figure 4. Molecular mechanisms of sirtuins, oxidative stress regulation, and Wnt signaling involved in Cmah-null mice

(A) IHC in cochlea of WT- and Cmah-null mouse for detection of Sirt3. Bar: 200 um. SV: scala vestibule, RM: reissner's membrane, SM: scala media, ST: scala tympani, Sv: stria vascularis, SL: spiral lamina. (B) Expression levels of sirtuins were determined by RT-qPCR of RNA samples from cochlear tissues of WT- and Cmah-null mice. (C) Expression of AHL related genes on the microarray data from cochlear tissues of Cmah-null mice. (D) Expression levels of genes involved in the regulatory factor of sirtuin, Wnt signaling, and mitochondrial functional regulation were determined by RT-qPCR of RNA samples from cochlear tissues of WT- and Cmah-null mice. (E) Measurement of mitochondrial activity in the cochlea of WT- and Cmah-null mice. Immunofluorescence was performed using a mitochondrial marker antibody. The intensity of the fluorescent signal indicates mitochondria numbers or the level of integrity. (F) Summary model describing the molecular mechanisms for sirtuins and oxidative stress regulation, Wnt signaling, and mitochondrial dysfunction by identified RT-qPCR results and array date set. The down-regulated Sirt3 lead to oxidative damage and mitochondrial dysfunction and could be alter various signaling pathways in Cmah-null mice with AHL. Sirt3-5: NAD-dependent protein deacetylase sirtuin3-5, Hif1α: Hypoxia-inducible factor 1-alpha, Foxo3a: Forkhead box O3, Foxo1: Forkhead box protein O1, MnSod: superoxide dismutase 2, mitochondrial, Lrp5-6: Low-density lipoprotein receptor-related protein 5-6, Wnt: Wingless, the Drosophila melanogaster segment-polarity gene, and Integrase-1 the vertebrate homologue, Gsk3β: Glycogen synthase kinase 3 beta, Cbr1: carbonyl reductase 1, Imp1: inner mitochondrial membrane peptidase-like, Mtfp1: mitochondrial fission process 1, RhoT2: Mitochondrial Rho GTPase 2, CytoC: Cytochrome C, Soc1-2: soluble oxidase component 1-2.