Autism-Related Cc2d1a Heterozygous Mice: Increased Levels of miRNAs Retained in DNA/RNA Hybrid Profiles (R-Loop)

Abstract

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a highly variable expression of phenotypes (restricted interest or activity and repetitive behavior in communication and social interactions), genes (mutation), markers (alteration of transcription) and pathways. Loss of function of the CC2D1A gene appears to primarily affect the brain, leading to a range of behavioral problems in humans. In our study published in 2020, we found that the expressions of miR-19a-3p, miR-361-5p, miR-150-5p, miR-3613-3p, miR-126-3p and miR-499a-5p were downregulated in the serum samples of autistic patients, their families and mouse models (Cc2d1a +/- and valproic acid treated males). Here, acquired non-Mendelian hereditary character in a genetically defined mouse model of autism (Cc2d1a +/-) correlates with the transcriptional alteration of five miRNAs. We seek to test the hypothesis that miRNA levels vary by changes in RNA/DNA structure during development, thereby creating transcription alteration and cell memory. Behavioral tests were conducted on the offspring of Cc2d1a (+/-) mutant and control mice, such as novel object, social interaction, marble burying and tail suspension behavior. Two RNA fractions were isolated from mouse hippocampal tissues and sperm cells via standard TRIzol extraction: free RNA and the fraction of RNA bound to DNA in the form of a DNA/RNA hybrid (R-loop). The expression levels of miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p and miR-499a-5p were investigated by quantitative real-time RT-PCR. We report differences in the distribution of five miRNAs in the hippocampus between male and female mice, particularly in colonies of Cc2d1a (+/-) mice. Furthermore, the number of miRNAs engaged in the DNA/RNA hybrid fraction is generally higher in the mutant pedigree than in the control group. On the other hand, in sperm, both fractions are at lower levels than in controls. R-loops contribute to the physiology and pathology of organisms including human disease. Here, we report a variation in five miRNA levels between gender and tissue. Our results suggest that the transcription levels of these five miRNAs are directly regulated by their RNA.

Keywords: Cc2d1a; DNA/RNA hybrid; autism; microRNA; non-Mendelian heredity; transcripts.

Figure 1

Tail suspension test for parent and F1 generation. (A). Test results for parents (n = 10, each sex). (B). Test results for F1 generation (n = 10, each sex). (C). Marble burying test for parents (n = 10, each sex). (D). Test results for F1 generation (n = 10, each sex). (E). Social interaction test for parents and F1 generation (n = 10, each sex). Total time was measured as sec. (F). Novel object recognition test for parents (n = 10, each sex). Distance was measured as cm. (G). Test results for F1 generation (n = 10, each sex). (H). Novel object recognition test for female mice (n = 10, each sex). Duration was measured as sec. (I). Novel object recognition test for male mice (n = 10, each sex). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 1

Tail suspension test for parent and F1 generation. (A). Test results for parents (n = 10, each sex). (B). Test results for F1 generation (n = 10, each sex). (C). Marble burying test for parents (n = 10, each sex). (D). Test results for F1 generation (n = 10, each sex). (E). Social interaction test for parents and F1 generation (n = 10, each sex). Total time was measured as sec. (F). Novel object recognition test for parents (n = 10, each sex). Distance was measured as cm. (G). Test results for F1 generation (n = 10, each sex). (H). Novel object recognition test for female mice (n = 10, each sex). Duration was measured as sec. (I). Novel object recognition test for male mice (n = 10, each sex). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 1

Tail suspension test for parent and F1 generation. (A). Test results for parents (n = 10, each sex). (B). Test results for F1 generation (n = 10, each sex). (C). Marble burying test for parents (n = 10, each sex). (D). Test results for F1 generation (n = 10, each sex). (E). Social interaction test for parents and F1 generation (n = 10, each sex). Total time was measured as sec. (F). Novel object recognition test for parents (n = 10, each sex). Distance was measured as cm. (G). Test results for F1 generation (n = 10, each sex). (H). Novel object recognition test for female mice (n = 10, each sex). Duration was measured as sec. (I). Novel object recognition test for male mice (n = 10, each sex). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

(A). miR-126-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (B). miR-150-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (C). miR-361-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (D). miR-499a-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (E). miR-19a-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

(A). miR-126-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (B). miR-150-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (C). miR-361-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (D). miR-499a-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (E). miR-19a-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

(A). miR-126-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (B). miR-150-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (C). miR-361-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (D). miR-499a-5p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). (E). miR-19a-3p expression in hybrid and total groups. Graphs were shown for the female hippocampus, male hippocampus and male sperm. Data were expressed in mean ± standard deviation (n = 5, each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.