Crocetin treatment differentially changed the expression of genes in the hippocampi of aged mice to bring about a change in memory behavior
To understand the molecular basis of changes observed in the memory behavior of treated mice, we isolated the hippocampi from the brain of mice and sequenced the whole transcriptome. To achieve greater accuracy in transcriptome sequencing, we used a depth of 40 million reads per sample. We conducted PCA analysis to compare the control and crocetin-treated groups. The results revealed significant insights into the variance and underlying patterns within the datasets. The initial nine principal components (PC1 to PC9) accounted for 84.06% of the total variance, suggesting potential treatment-related effects (Figure 2A & B). Further, differential gene expression analysis revealed that out of 22,096 protein-coding genes, 154 genes were differentially expressed (Figure 2C). A detailed differential gene expression list is provided as supplementary spreadsheet SS1.
We performed a hierarchical clustering to ensure to distinguish the upregulated and downregulated genes. We found a significant upregulation of Bdnf, Gabbr2, Gad2, and Drosha that correlated to the improved brain function and behavior of treated mice (Figure 2D and Supplementary Figure S3). Further, some of the genes downregulated after crocetin treatment could also be linked to improvement in brain function, these included Hck, Amigo3, Top1mt, and Lcp1 (Figure 2E).
Further analysis revealed that mitochondrial genes belonging to different complexes of ETC were significantly upregulated. The upregulated genes included ND1, ND2, ND4, ND4L, ND5, and ND6 related to NADH dehydrogenase from complex I, CYTB from complex III, Uqcrq from complex IV, and ATP6 and ATP8 from complex V (Figure 2F). Few more ETC genes, such as ND3, COX1, COX2, and COX3, were also found upregulated, though non-significantly (Supplementary Spreadsheet SS1).