Calcineurin links Ca2+ dysregulation with brain aging - PubMed (original) (raw)

T C Foster et al. J Neurosci. 2001.

Abstract

Brain aging is associated with altered Ca(2+) regulation. However, many Ca(2+) signal transduction mechanisms have not been explored in the aged brain. Here, we report that cytosolic expression and activity of the Ca(2+)-dependent protein phosphatase calcineurin (CaN) increases in the hippocampus during aging. CaN changes were paralleled by increased activation, but not expression, of CaN-regulated protein phosphatase 1 and a reduction in the phosphorylation state of CaN substrates involved in cell survival (i.e., Bcl-2-associated death protein and cAMP response element-binding protein). The age-related increase in CaN activity was not attributable to the inability of CaN to translocate to the membrane and was reduced by blocking L-type Ca(2+) channels. Finally, increased CaN activity correlated with memory function as measured with the Morris water escape task. The results suggest that altered regulation of CaN is one of the processes that could link Ca(2+) dyshomeostasis to age-related changes in neural function and cognition.

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Figures

Fig. 1.

Fig. 1.

Cytosolic CaN activity is increased in the hippocampus of aged (filled bars) relative to young adult (open bars) rats. Measures of phosphate released by CaN activity in the cytosolic fraction (A) and in the whole-tissue homogenate (B) from young adult and aged rats under the three conditions of activation, basal, and inhibition. The + and − signs indicate the presence or absence, respectively, of added activators (CA2+/CaM) or the CaN inhibitor FK506. Protein levels of the catalytic subunit of CaN in the cytosolic fraction (C) and in the whole-tissue homogenate (D) from young adult and aged rats.Insets show representative immunoblots of the two groups (A, aged; Y, young adult). The results suggest that increased cytosolic CaN activity in the aged group is attributable to an age-related increase in CaN protein levels in the cytosol. In this and the following figures, _asterisks_indicate a significant (p < 0.05) age difference, and error bars illustrate group means ± SEM.

Fig. 2.

Fig. 2.

Similar to CaN activity, cytosolic PP1 activity is increased in the hippocampus of aged (filled bars) relative to young adult (open bars) rats. Measures of phosphate released by PP1 activity in the cytosolic fraction (A) and in the whole-tissue homogenate (B) from young adult and aged rats. The + and − signs indicate the presence or absence, respectively, of added activators (CA2+/CaM) or the PP1 inhibitor okadaic acid (OKA). Protein levels of the catalytic subunit of PP1 in the cytosolic fraction (C) and in the whole-tissue homogenate (D) from young adult and aged rats.Insets show representative immunoblots of the two groups (A, aged; Y, young adult). Unlike CaN, the age-related increase in cytosolic PP1 activity is not associated with an increase in cytosolic PP1 protein levels.

Fig. 3.

Fig. 3.

Increased CaN activity is a function of Ca2+ regulation and not an inability to translocate in response to Ca2+ levels. Measures of phosphate released by CaN activity (A) and protein levels of the catalytic subunit of CaN (B) in the cytosolic fraction and in the whole-tissue homogenate of hippocampus tissue of young adult (open bars) and aged (filled bars) rats after a 3 hr incubation in no Ca2+ medium. Note that when Ca2+is removed from the incubation buffer, age differences in CaN activity and expression are ameliorated. C, Measures of phosphate released by CaN activity in cytosolic fractions after the in vivo injection (0.5–1 μl/min) of nimodipine (20 μ

m

) or vehicle (ACSF) into the hippocampus of aged rats anesthetized with ketamine–xylazine. D, Measures of phosphate released by CaN activity in cytosolic fractions from hippocampal slices bathed with ACSF or 10 μm of nifedipine.Plus signs indicate a significant difference (p < 0.05) from ACSF condition.

Fig. 4.

Fig. 4.

The CaN substrates BAD and CREB exhibit decreased phosphorylation in the hippocampus of aged (filled bars) relative to young adult (open bars) rats.A, Measures of the cytosolic protein levels of phosphorylation state-independent BAD (BAD) and phospho-BAD (p-BAD). B, Measures of the cytosolic levels of phosphorylation state-independent CREB (CREB) and phospho-CREB (p-CREB). In contrast to their phosphorylation-independent counterparts, phospho-CREB and phospho-BAD are decreased in aged animals, consistent with an age-related increase in CaN activity. _Insets_illustrate representative immunoblots.

Fig. 5.

Fig. 5.

Behavioral measures for aged (filled circles) and young adults (open circles) during behavioral training. Mean latency (A) and mean path length (B) to escape during cue discrimination training. Mean latency (C) and mean path length (D) to escape during spatial discrimination training. Each block consisted of three training trials, and training on each task was massed into a single day with 3 d between tasks. The break in the _x_-axis between blocks 5 and 6 indicates the time point at which a probe trial was administered to measure acquisition (see Fig. 6).Asterisks indicate a significant difference between the two age groups. Error bars indicate SEM.

Fig. 6.

Fig. 6.

Searching behavior during testing of acquisition and retention of spatial discrimination for aged (filled bars) and young adult (open bars) rats.A, After block 5 of training on the spatial version of the task, a probe trial was administered to measure acquisition. The mean percentage of time spent searching the goal quadrant (Goal) and the quadrant opposite the goal (Opposite) is illustrated for both age groups.B, An age-related decrease in percentage of time searching the goal quadrant was observed during the retention probe trial administered 24 hr after the acquisition probe trial.C, Relative to young adult animals, the aged groups exhibited larger variability in goal quadrant search time during the retention probe trial. D, An age-related decrease in platform crossings was observed during both the acquisition and retention probe trials. Asterisks indicate significant differences across age groups. Plus signs indicate significant difference from chance (dashed lines). Error bars indicate SEM.

Fig. 7.

Fig. 7.

Increased cytosolic CaN activity is associated with retention deficits examined by the water escape task.A, Mean cytosolic CaN activity for young adults (open bars) and aged animals (filled bars) that were behaviorally characterized on the water escape task. B, Correlation between cytosolic CaN activity and percentage of time in the goal quadrant during the retention probe trial for aged rats. C, Correlation between cytosolic CaN activity and number of platform crossings during the retention probe trial for young adult rats. Asterisks indicate a significant difference between young adult and aged groups (p < 0.05).

Fig. 8.

Fig. 8.

Model illustrating how, during aging, an increase in intracellular Ca2+ activates CaN, causing it to move into the cytoplasm. In turn, CaN induces the translocation of BAD from the cytosol to the mitochondrial membrane, a step involved in Ca2+-mediated apoptosis. Furthermore, CaN-mediated dephosphorylation of the CREB inhibits the passage of CREB into the nucleus and is associated with decreased cell viability. Finally, by dephosphorylating inhibitor-1 (I-1), CaN increases the activity of PP1, leading to dephosphorylation of glutamate receptors (NMDAR, AMPAR), resulting in altered synaptic transmission and plasticity.

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