Repeated Clozapine Administration Causes Extensive Changes to the Expression of Coding and Non-coding RNAs, Including miR-124, in the Mouse Frontal Cortex

Abstract

Clozapine is arguably the most effective antipsychotic drug for the treatment of schizophrenia, but the mechanisms underlying its efficacy are poorly understood. Therefore, we perform deep RNA sequencing to test for differential transcription and exon use resulting from clozapine's effects in the mouse frontal cortex, and integrate our findings with known schizophrenia risk genes. We used a dose (4 mg/kg/day, i.p.) and duration (21 days) to approximate clinical exposure, followed by a 24-h washout to determine persistent changes resulting from biological remodeling. We observed significant (FDR < 0.05) differential expression of both mRNAs and long noncoding RNAs (lncRNAs), which were enriched in RNA processing and splicing pathways. Among the most significant lncRNAs, showing 2.3-fold upregulation, was the microRNA 124 host gene (Mir124a-1hg), a major source of miR-124, one of the most abundant microRNAs in the brain. Quantitative PCR analysis of the mature microRNAs miR-124-3p and miR-124-5p revealed a significant dose-dependent upregulation of miR-124-3p following 21-day repeated clozapine administration. RNA splicing was also profoundly impacted by clozapine, as revealed by differential exon use analysis, with mouse orthologs of 50 schizophrenia risk genes from the Psychiatric Genomics Consortium among the genes affected. These genes were enriched in "apical dendrite" and "distal axon" ontologies, supporting prior evidence that clozapine may target cortical pyramidal neuron deficits implicated in schizophrenia. Overall, this study demonstrates the profound effect of clozapine on cortical gene expression, affecting abundance of splicing of coding and non-coding transcripts. Future studies are needed to fully characterize our findings as potential preclinical markers of clozapine response.

Keywords: Antipsychotic drugs; Brain; Gene expression; Non-coding RNA; RNA sequencing; Schizophrenia.

Fig. 1

Experimental timeline. Male C57BL/6 mice were subjected to repeated 21 day i.p. clozapine injections. One day after the final injection, mice were sacrificed and the frontal cortex collected for subsequent gene expression analysis. C57BL/6 cartoon from reproduced from DataBase Center for Life Science (DBCLS) submission to Wikimedia Commons under a Creative Commons Attribution 4.0 International license

Fig. 2

Results from RNA-seq gene expression analysis. (A) Volcano plot showing differentially expressed genes. The x-axis is the log₂ fold change, while the y-axis is the –log₁₀ of the Benjamini–Hochberg adjusted p-value of differential expression. The horizontal dotted line shows the FDR < 0.05 threshold for genome-wide significance. Transcripts with p-values below this threshold, or those with less than twofold change in expression (up or down) are in gray, while significant transcripts with either > twofold increase in expression (orange) or > twofold reduction in expression (blue) are highlighted. (B) Dot plot of Gene Ontologies (GOs) found to be significantly enriched with genes differentially expressed following repeated clozapine administration (panel A, FDR < 0.05). The size of each dot is proportional to the number of genes in the pathway, while the color is of each dot corresponds to the p-value of enrichment (see legend)

Fig. 3

Dose-dependent response of cortical miR-124-3p to repeated clozapine administration for 21 days. Panel (A) shows the microRNA 124 host gene (Mir124a-1hg) that was significantly up-regulated in our RNA-seq analysis of the cortex following clozapine administration at 4 mg/kg for 21 days. Mir124a-1hg is spliced to yield the Pri-mir-124 hairpin, which is the further processed to yield the mature miR-124-3p and miR-124-5p microRNAs. Data from miRbase [48]. Panel (B) shows the results of qPCR analysis of the two mature microRNAs in the cortex of subjects exposed to three different clozapine doses of 1, 4, and 10 mg/kg/day respectively for 21 days. The upper set of three barplots (1, II and III) show the results for miR-124-3p while the lower set of three (IV, V and VI) show the results for miR-124-5p. For each plot, the x-axis shows the clozapine dose and vehicle groups, while the y-axis shows the normalized fold change (2^−(ΔΔCq)) relative to the control (vehicle, VEH) group. * = p < 0.05, ** = p < 0.01

Fig. 4

Plots showing differential exon use in the cortex following 4 mg/kg/day clozapine for 21 days. The exon structure of each of the four most significant genes are shown, with the relative abundance of each exon transcript in clozapine (CLOZ4, red) and vehicle (SAL, blue) plotted. Exons showing significant differences in abundance are colored in magenta. Genomic positions are for mouse genome build mm10. Plots were made using the DEXSeq package