Abstract
The decline in cognitive robustness with aging can be attributed to complex genetic pathways involving many cellular dysfunctions, cumulative over time, precipitating in frailty and loss of wellness in the elderly brain. The size and health of the neuronal cell population determines cognitive robustness in mammals. A transgenic mouse model over-expressing Bcl-2 has been shown to rescue neurons from naturally occurring cell death (NOCD). Here we show that in the brain of calorie-restricted (CR) mice, there is an age-dependent decreased expression of microRNAs mmu-miR-181a-1*, mmu-miR-30e and mmu-miR-34a, with a corresponding gain in Bcl-2 expression, and decreases in pro-apoptosis genes such as Bax and cleavage of Caspases. Functional characterization shows that these miRNAs repress Bcl-2 expression by the 3'UTR reporter assays, accompanied by loss of this gene's endogenous expression, and a gain in pro-apoptosome-specific proteins. Over-expression of these miRNAs increases the rate of apoptosis, accompanied by a decline in Bcl-2 expression in miRNA-transfected mouse and human cell lines. We report here that down-regulation of miR-34a, -30e, and -181a permits their shared target gene expression (Bcl-2) to remain at a high level without post-transcriptional repression, accompanied by concomitant low levels of Bax expression and Caspase cleaving; this chain event may be a part of the underlying mechanism contributing to the gain in neuronal survival in long-lived CR-fed mice.
Highlights
Most old-age perils, such as neurodegenerative, cardiovascular, and metabolic diseases, are accompanied by physiologic decline determined by the derailment of underlying molecular signaling networks at the cellular level
In calorie restriction (CR)-fed mice the Bcl-2/Bax ratio showed an increased trend in an age-dependent manner, compared to ad lib-fed mice (Figure 4A). To validate this antagonistic pattern between Bcl2 and Bax protein levels, we examined their ratios in microRNAtransfected Human embryonic kidney (HEK)-293 and NIH/3T3 cultures
This shows that these miRNAs suppress the target protein through the 3’‐UTR of Bcl‐2. This effect is absent when a plasmid carrying a scrambled sequence is used. (B) In line with the increase of apoptosome‐ specific proteins in the over‐expression assay for these three miRNAs, over‐expression in HEK‐293 and NIH/3T3 cells contributes to a decline of the LD50 dose of Staurosporine, more cell death at lesser concentration; this effect on the LD50 dose is not present in scrambled and vector alone controls. (**p
Summary
Most old-age perils, such as neurodegenerative, cardiovascular, and metabolic diseases, are accompanied by physiologic decline determined by the derailment of underlying molecular signaling networks at the cellular level. In CR-fed mice the Bcl-2/Bax ratio showed an increased trend in an age-dependent manner, compared to ad lib-fed mice (Figure 4A) To validate this antagonistic pattern between Bcl and Bax protein levels, we examined their ratios in microRNAtransfected HEK-293 and NIH/3T3 cultures. In line with Bcl-2 expression, the expression of cleaved Caspase-9 and Caspase-3 is significantly reduced in age-dependent manner from young to old age groups in CR-fed, compared to ad lib-fed, mouse brain samples (Figure 5A). Our work has shown that: during aging, miR-34a, -30e, and -181* are not up-regulated in CR brain, while pro-survival gene Bcl-2 is maintained at the same level in older mice as their younger counterparts, accompanied by a decrease of pro-apoptosome proteins such as Bax and Caspase 9 cleaved products. During aging, down-regulation of the three microRNAs may reflect a gain of survival signaling in neurons of older CR mice
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