RCAN1 regulates mitochondrial function and increases susceptibility to oxidative stress in mammalian cells

Abstract

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1(ox)) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12(RCAN1)). Similar to RCAN1(ox) neurons, PC12(RCAN1) cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.

Figure 1

RCAN1^ox neurons display decreased viability upon exposure to H₂O₂. Primary neuronal cultures from E15 wild-type (light bars) and RCAN1^ox mice (dark bars) were exposed to varying concentrations (0–500 μm) of H₂O₂ for 18 hours and cell viability was measured via a standard MTT assay. n = 4 neuronal cultures from individual wild-type and RCAN1^ox mice. Data presented as a percentage of the number of starting viable cells, error bars represent SEM, and ***P < 0.001.

Figure 2

RCAN1 regulates mitochondrial morphology. Representative electron-micrographs from (a) wild-type and (b) RCAN1^ox chromaffin cells captured at 4000x magnification (scale bar = 300 nm). RCAN1^ox chromaffin cells (black bars) have (c) a greater density of mitochondria and (d) significantly smaller mitochondria when compared to wild-type controls (white bars). (e) RCAN1^ox cells (red line) have a higher frequency of smaller mitochondria when compared to wild-type controls (black line). n = 9 cells from 3 animals for each genotype, error bars represent the SEM, *P < 0.05, and **P < 0.01.

Figure 3

RCAN1^ox cells have altered mitochondrial membrane potential in response to ROS. Wild-type (light bars) and RCAN1^ox (dark bars) chromaffin cells were stained with the fluorescent mitochondrial membrane potential marker JC-1 under the following conditions: basal (30 cells), 56 mM K⁺ solution (30 cells), 500 nM Valinomycin (10 minutes/10 cells), and 100 μm H₂O₂ (60 minutes/9 cells). Error bars represent the SEM; *P < 0.05.

Figure 4

Generation of a stable PC12 cell line overexpressing RCAN1, PC12^RCAN1. (a) Diagrammatic representation of the generation of the FLAG-fused RCAN1 construct, which was cloned into pGEM-T easy vector and finally ligated to the EcoR1 restriction site of pcDNA3 expression vector containing a pCMV promoter. (b) Agarose gel electrophoresis of the PCR products from the 3 selected clones containing the 900 bp RCAN1-FLAG tag sequence, a water only (−) control, and (+) control using DNA encoding the pGEM-T/RCAN1-FLAG construct. (c) Immunoblot against the FLAG tag in the 3 selected clones and a negative control lacking FLAG normalized to β-tubulin loading control. (d) Immunocytochemistry of control PC12 and PC12^RCAN1 cells with an antibody against RCAN1 demonstrates the elevated RCAN1 expression in PC12^RCAN1 cells (scale bar = 50 μm).

Figure 5

RCAN1 overexpression increases mitochondrial ROS production. Representative images of (a) control and (b) PC12^RCAN1 cells stained with 100 μM MitoSOX Red at 20x magnification (scale bar = 50 μm). (c) Quantification of MitoSOX fluorescence intensity revealed that control cells (light bars) have significantly less mean fluorescence compared to PC12^RCAN1 cells (dark bars) under basal conditions. The addition of the antioxidant N-acetylcysteine (NAC) significantly reduces florescence in PC12^RCAN1 cells while having no effect on control cells. Data has been normalized to basal mean fluorescence in control cells and error bars represent SEM. n = 30 cells from each group; ***P < 0.001.

Figure 6

PC12^RCAN1 cells exhibit reduced viability upon exposure to H₂O₂. Control (lights bars) and PC12^RCAN1 cells (dark bars) were exposed to increasing concentrations of H₂O₂ from 0–250 μm for 18 hours. At 50 μM and 100 μM H₂O₂, fewer PC12^RCAN1 cells were viable when compared to control PC12 cells under the same conditions. Data has been normalized to percentage of starting viable cells for each group and error bars represent the SEM. n = 13 experiments in control and PC12^RCAN1 cells; *P < 0.05 and **P < 0.01.

Figure 7

Summative roles of RCAN1 in neurodegeneration and cell function. Elevated amyloid-β levels in neuronal cells can lead to elevated RCAN1 expression which inhibits calcineurin activity resulting in accumulation of hyperphosphorylated tau protein. This causes the formation of neurofibrillary tangles that lead to neurodegenerative disorders. We have further shown that elevated RCAN1 expression also leads to mitochondrial dysfunction, resulting in elevated intracellular oxidative stress, both of which are implicated in AD pathogenesis.