Next-Generation Sequencing Identifies Polyunsaturated Fatty Acid Responsive Genes in the Juvenile Rat Cerebellum

Abstract

Dietary n-3 polyunsaturated fatty acids (PUFA) influence postnatal brain growth and development. However, little data exist regarding the impacts of dietary n-3 PUFA in juvenile animals post weaning, which is a time of rapid growth. We tested the hypothesis that depleting dietary n-3 PUFA would result in modifications to the cerebellar transcriptome of juvenile rats. To test this hypothesis, three week old male rats (an age that roughly corresponds to an 11 month old child in brain development) were fed diets containing either soybean oil (SO) providing 1.1% energy from α-linolenic acid (ALA; 18:3n-3; ALA-sufficient) or corn oil (CO) providing 0.13% energy from ALA (ALA-deficient) for four weeks. Fatty acids (FAs) in the cerebellum were analyzed and revealed a 4-fold increase in n-6 docosapentaenoic acid (DPA; 22:5n-6), increases in arachidonic acid (AA; 20:4n-6) and docosatetraenoic acid (DTA; 22:4n-6), but no decrease in docosahexaenoic acid (DHA; 22:6n-3), in animals fed CO versus SO. Transcript abundance was then characterized to identify differentially expressed genes (DEGs) between the two diets. Upper quartile (UQ) scaling and transcripts per million (TPM) data normalization identified 100 and 107 DEGs, respectively. Comparison of DEGs from the two normalization methods identified 70 genes that overlapped, with 90% having abundance differences less than 2-fold. Nr4a3, a transcriptional activator that plays roles in neuroprotection and learning, was elevated over 2-fold from the CO diet. These data indicate that expression of Nr4a3 in the juvenile rat cerebellum is responsive to dietary n-3 PUFA, but additional studies are needed clarify the neurodevelopmental relationships between n-3 PUFA and Nr4a3 and the resulting impacts.

Keywords: brain development; cerebellum; gene expression; juvenile; n-3 PUFA; next-generation sequencing.

Figure A1

Correlation heat map using transcripts per kilobase million normalization. The heat map represents the graphical display resulting from unsupervised clustering of the correlations between samples. The x- and y-axis are the same and the red diagonal line represents a correlation of 1. The pink labeled IDs are associated with the soybean diet while the blue labeled IDs are from the corn oil diet.

Figure A2

Volcano plot for upper transcripts per kilobase million normalization. The x-axis displays the difference in log scale, and the y-axis plots the –log10(p-value) for each transcript, represented by a point. Significance threshold is graphed with a dotted red line (3.430). Points to the right of 0 and above the significance threshold are significantly increased transcripts, and transcripts that are significantly decreased are to the left of 0 and above the threshold line.

Figure 1

Correlation heat map of NGS-identified transcripts using upper quartile scaling normalization. The heat map represents the graphical display resulting from unsupervised clustering of the correlations between samples. The x- and y-axis are the same and the red diagonal line represents a correlation of 1. The pink labeled IDs are associated with the soybean diet while the blue labeled IDs are from the corn oil diet.

Figure 2

Volcano plot of NGS-identified transcripts using upper quartile scaling normalization. The x-axis displays the difference in log scale, and the y-axis plots the –log10(p-value) for each transcript, represented by a point. Significance threshold is graphed with a dotted red line (3.550). Points to the right of 0 and above the significance threshold are significantly increased transcripts and transcripts that are significantly decreased are to the left of 0 and above the threshold line.

Figure 3

Venn diagram comparison of differentially expressed genes identified with NGS using separate data normalizations. The light pink circle represents the number of genes that were exclusively identified using TPM normalization, while the blue circle represents the number of genes that were exclusively identified using UQ scaling normalization. The dark pink oval represents the genes that overlapped between the TPM and UQ scaling normalizations.

Figure 4

Cerebellar qPCR measurement of Nr4a3. Data are reported as mean ± SD, n = 10. * Statistical significance was assessed using Student’s t-test performed on the initial ΔCt values for the gene with p ≤ 0.05. CO: Corn oil; SO: Soybean oil; ΔCt: Change in cycle threshold normalized to β-actin.