Gene Expression Profile of Olfactory Transduction Signaling in an Animal Model of Human Multiple Sclerosis

Abstract

Olfactory dysfunction occurs in multiple sclerosis in humans, as well as in an animal model of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyze differentially expressed genes (DEGs) in olfactory bulb of EAE-affected mice by next generation sequencing, with a particular focus on changes in olfaction-related signals. EAE was induced in C57BL/6 mice following immunization with myelin oligodendrocyte glycoprotein and adjuvant. Inflammatory lesions were identified in the olfactory bulbs as well as in the spinal cord of immunized mice. Analysis of DEGs in the olfactory bulb of EAE-affected mice revealed that 44 genes were upregulated (and which were primarily related to inflammatory mediators), while 519 genes were downregulated; among the latter, olfactory marker protein and stomatin-like 3, which have been linked to olfactory signal transduction, were significantly downregulated (log2 [fold change] >1 and p-value <0.05). These findings suggest that inflammation in the olfactory bulb of EAE-affected mice is associated with the downregulation of some olfactory signal transduction genes, particularly olfactory marker protein and stomatin-like 3, which may lead to olfactory dysfunction in an animal model of human multiple sclerosis.

Keywords: Differentially expressed gene; Experimental autoimmune encephalomyelitis; Olfactory bulb; Olfactory marker protein.

Fig. 1. Clinical scores in mice immunized with myelin oligodendrocyte glycoprotein (MOG). Immunized animals were observed daily for clinical signs of MOG-experimental autoimmune encephalomyelitis (EAE). The scores are presented as means±standard error of the mean (SEM).

Fig. 2. Histological examination of the spinal cord and olfactory bulb of control and EAE-affected mice (G.3, paralytic stage). (A) No inflammatory cells were present in the spinal cord of control mice. (B) In EAE-affected mice, inflammatory cells (arrow) and perivascular cuffing (arrowhead) were detected in the spinal cord. (C) In control mice, there was no inflammatory response in the olfactory bulb. (D) Inflammatory cells were identified in the olfactory bulb of EAE-affected mice. Some Iba-1-positive microglia (E) and glial fibrillary acidic protein (GFAP)-positive astrocytes (G) were detected in the olfactory bulb of the control. Iba-1-positive microglia (F) and GFAP-positive astrocytes (H) were increased in the olfactory bulb of EAE-affected mice. (A~D) Hematoxylin and eosin staining. (E and F) Iba-1 immunostaining. (G and H) GFAP immunostaining. Counterstained with hematoxylin. ePL, external plexiform layer; GL, glomerulus layer; GrL, granular cell layer; ML, mitral cell layer; ONL, olfactory nerve layer. Arrowheads, perivascular cuffing; arrows, inflammatory cells. Scale bars=50 µm.

Fig. 3. Heatmap of differentially expressed genes (DEGs) in the olfactory bulbs of control versus EAE mice at the paralytic stage. Green and red indicate low and high expression, respectively.

Fig. 4. Double immunofluorescence staining of GFAP (red) with interneuron markers, GAD 65/67 (green). Few GAD 65/67 positive cells (green in B) showed GFAP-positive immunoreaction (red in A) in the glomerulus layer (arrowheads in C). ePL, external plexiform layer; GL, glomerulus layer. Scale bars=20 µm.

Fig. 5. Heatmap of olfactory transduction signaling (Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway: mmu04740) and quantitative real-time reverse transcription-PCR (RT-PCR) of Omp and Stoml3 in the olfactory bulbs of control and EAE-affected mice. (A) Omp and Stoml3 were significantly downregulated. (B) Omp mRNA expression was significantly decreased at the paralytic stage (G.3; severe paraparesis, D23PI) and subsequently restored in the recovery stage (R.1; recovery with floppy tail, D83PI). (C) The expression level of Stoml3 mRNA was significantly decreased at the paralytic and recovery stages following immunization. PI, post-immunization. Data are presented as means±SEM. ^**p<0.01, vs. control group.

Fig. 6. Heatmap of olfactory transduction signaling (KEGG pathway: mmu04740) in the olfactory mucosa of control versus EAE mice at the paralytic stage. In total, the expression of 119 olfactory receptor genes was increased, while the expression of Gng13 was decreased in the olfactory mucosa of EAE-affected mice.

Fig. 7. Schematic illustration of the DEGs of olfactory transduction signaling and inflammatory responses in the olfactory bulb and olfactory mucosa of EAE-affected mice. At the paralytic stage, EAE-affected mice showed upregulation of inflammation-related genes and downregulation of olfactory transduction signaling in the olfactory bulb and olfactory mucosa. This schematic of neuron types in the olfactory bulb was modified from the rat nervous system [47].