Hippocampal Transcriptome-Wide Association Study Reveals Correlations Between Impaired Glutamatergic Synapse Pathway and Age-Related Hearing Loss in BXD-Recombinant Inbred Mice

Abstract

Age-related hearing loss (ARHL) is associated with cognitive dysfunction; however, the detailed underlying mechanisms remain unclear. The aim of this study is to investigate the potential underlying mechanism with a system genetics approach. A transcriptome-wide association study was performed on aged (12-32 months old) BXD mice strains. The hippocampus gene expression was obtained from 56 BXD strains, and the hearing acuity was assessed from 54 BXD strains. Further correlation analysis identified a total of 1,435 hearing-related genes in the hippocampus (p < 0.05). Pathway analysis of these genes indicated that the impaired glutamatergic synapse pathway is involved in ARHL (p = 0.0038). Further gene co-expression analysis showed that the expression level of glutamine synthetase (Gls), which is significantly correlated with ARHL (n = 26, r = -0.46, p = 0.0193), is a crucial regulator in glutamatergic synapse pathway and associated with learning and memory behavior. In this study, we present the first systematic evaluation of hippocampus gene expression pattern associated with ARHL, learning, and memory behavior. Our results provide novel potential molecular mechanisms involved in ARHL and cognitive dysfunction association.

Keywords: BXD mice strain; cognitive dysfunction; gene network; glutamine synthetase (Gls); hearing loss; systems genetics; transcriptome-wide association study (TWAS).

FIGURE 1

(A) The 26 strains with both 56 strains for auditory-evoked brainstem response (ABR) thresholds and 57 strains for hippocampus mRNA transcriptomic analysis are involved in the association analysis. (B) The average ABR thresholds of 26 BXD mouse strains at 2 years old age. The x-axis shows the BXD strains and the two parental strains. The y-axis shows the average ABR thresholds for 8K, 16K, and 32 kHz tone bursts were averaged as hearing acuity. (C,D) Bubble charts of the Gene Ontology analysis enriched for age-related hearing loss (ARHL) correlated genes. Gene over-representation analysis for Gene Ontology analysis of the ARHL correlated genes (p-value < 0.05 and r > 0.3) were performed with WebGestalt (http://www.webgestalt.org/). The x-axis represents an enriched ratio, and the y-axis represents enriched pathways/terms. The size of the dots represents the number of genes, and the color indicates the p-values. An enriched ratio is defined as the number of observed divided by the number of expected genes from the annotation category in the gene lists. (E) Glutamatergic synapse is involved in ARHL correlated pathway based on the mice hippocampus transcriptome data. The ARHL correlated genes were analyzed by KEGG. The top 20 pathways associated with the network genes were tested alongside the p-values calculated using a right-tailed Fisher’s exact test. (F) Chord-Chart of the Glutamatergic synapse pathway enriched for ARHL correlated gene. The outer color green represents negative correlation, red represents positive correlation.

FIGURE 2

Scatterplots of the correlations of ABR threshold [dB sound pressure level (SPL)] with all glutamate receptors expression (A), Gria3 expression (B), Grm7 expression (C), Grik1 expression (D), Gria4 expression (E), Grm5 expression (F), Grm8 expression (G), and Gria2 expression (H). The Pearson correlation coefficient was used to determine the relationship. Pearson correlation and p-value are indicated. Gene expression levels are log2 transformed and modified with Z score.

FIGURE 3

Scatterplots of the correlations of ABR threshold (dB SPL) with glutamine synthetase (Gls) expression (A), Slc38a2 expression (B), Dlg4 expression (C), Shank3 expression (D), 10 known glutamate receptor chaperone proteins expression (E), and Nos1 expression (F). The Pearson correlation coefficient was used to determine the relationship. Pearson correlation and p-value are indicated. Gene expression levels are log2 transformed and modified with Z score.

FIGURE 4

Scatterplots of the correlations of ABR threshold (dB SPL) with Adcy4 expression (A). Adenylate cyclase 4 (Adcy4) catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP) (B). Scatterplots of the correlations of ABR threshold (dB SPL) with all Adenylate Cyclase family expression (C). The Pearson correlation coefficient was used to determine the relationship. Pearson correlation and p-value are indicated. Gene expression levels are log2 transformed and modified with Z score.

FIGURE 5

(A) The network includes the genes enriched in the Glutamatergic pathway, as well as the glutamate receptors that were mapped. The orange dots are in postsynapse, the blue dots are the glutamate receptors with moderate correlation with ARHL, and the dark gray dots is in presynapse, the size of the point indicates its connection degree, the red line indicates a significant positive correlation, the green line indicates a significant negative correlation, and the thickness of the line indicates the value of the correlation. Scatterplots of the correlations of Gls expression with Gria3 expression (B), Adcy4 expression (C), Dlg4 expression (D), Shank3 expression (E). The Pearson correlation coefficient was used to determine the relationship. Pearson correlation and p-value are indicated. Gene expression levels are log2 transformed and modified with Z score.

FIGURE 6

Scatterplots of the correlations of Gls expression with the learning latency in a touch screen test (A), the percentage of time spent freezing in Contextual fear conditioning test (B), the percentage of successful alternations in Y-maze test (C), the number of new entries during the first 8-arm choices in an 8-arm radial maze test (D). The Pearson correlation coefficient was used to determine the relationship. Pearson correlation and p-value are indicated. Gene expression levels are log2 transformed and modified with Z score.

FIGURE 7

ARHL associated Glutamatergic synapse pathway mapping. Glutamate released from presynaptic terminals acts through the activation of glutamate receptors located at the postsynaptic terminal. The interaction between glutamate and glutamate receptor favors the activation of several metabolic pathways. Glutamine is converted into glutamate by glutaminase. GLN, glutamine; GLS, Glutaminase; GLU, glutamate; Grm2, glutamate metabotropic receptor 2; Grm3, glutamate metabotropic receptor 3; Grm5, glutamate metabotropic receptor 5; Grm7, glutamate metabotropic receptor 7; Grm8, glutamate metabotropic receptor 8; Slc38a2, solute carrier family 38 member 2; Adrbk2, adrenergic, beta, receptor kinase 2; Adcy4, adenylate cyclase 4; NMDAR, N-methyl-D-aspartate receptor; KA, kainate receptors; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; PSD-95, postsynaptic density protein 95; Shank3, SH3 and multiple ankyrin repeat domains 3; Grik1, glutamate ionotropic receptor kainate type subunit 1; Gria2, glutamate ionotropic receptor AMPA type subunit 2; Gria3, glutamate ionotropic receptor AMPA type subunit 3; Gria4, glutamate ionotropic receptor AMPA type subunit 4. The blue sphere indicates GLU, the purple sphere indicates GLN. Green indicates negative correlation with ABR, red indicates positive correlation with ABR. Gray indicates non-significant correlation with ABR.