Down-regulation of cAMP-dependent protein kinase by over-activated calpain in Alzheimer disease brain

Abstract

Impaired cognition and memory may be associated with down-regulation of cAMP-response element-binding protein (CREB) in the brain in patients with Alzheimer disease, but the molecular mechanism leading to the down-regulation is not understood. In this study, we found a selective reduction in the levels of the regulatory subunits (RIIalpha and RIIbeta) and the catalytic subunit (Cbeta) as well as the enzymatic activity of cAMP-dependent protein kinase (PKA), which is the major positive regulator of CREB. We also observed that PKA subunits were proteolyzed by calpain and the levels of PKA subunits correlated negatively with calpain activation in the human brain. These findings led us to propose that in the brain in patients with Alzheimer disease, over-activation of calpain because of calcium dysregulation causes increased degradation and thus decreased activity of PKA, which, in turn, contributes to down-regulation of CREB and impaired cognition and memory.

Fig. 1

cAMP-dependent protein kinase (PKA) activity in the extracts of Alzheimer disease (AD) and control brains. (a) PKA activity of the 16 000 g extract of the medial temporal cortex from six AD and seven age-matched control brains was assayed in the absence or presence of 5 μmol/L cAMP. (b) Ratio of PKA activity in the absence or in the presence of cAMP. *p < 0.05 as compared with the control group.

Fig. 2

Levels of cAMP-dependent protein kinase (PKA) subunits/isoforms in Alzheimer disease (AD) and control brains. (a) Western blots of the medial temporal cortical homogenate from six AD and seven control brains, which were probed with antibodies against various subunits/isoforms of PKA, as indicated on the left of the blots. (b) Densitometric quantitation of the blots shown in panel (a). Mean ± SD of the relative immunoreactivities are shown. *p < 0.05 as compared with the control groups.

Fig. 3

In vitro proteolysis of cAMP-dependent protein kinase (PKA) subunits/isoforms. The cytosolic and nuclear fractions of mouse brain homogenate were incubated at 30° C for 10 or 30 min in the presence of calcium and selective protease inhibitors, as indicated above the blots. The levels and proteolysis of various PKA subunits/isoforms of the samples were then analyzed by western blots.

Fig. 4

cAMP-dependent protein kinase (PKA) degradation in mouse brain. Western blots of calcineurin and various isoforms of PKA subunits in the brain homogenate of mice after a single intraperitoneal injection of kainic acid at the indicated time points before killing the animals.

Fig. 5

Correlation analysis between the levels of cAMP-dependent protein kinase (PKA) subunits/isoforms and calpain activation in human brain. (a) Western blots of calpain I of medial temporal cortical homogenate from six Alzheimer disease (AD) and seven control cases. The apparent molecular weights of the full length and the truncated calpain are marked on the right of the blot. (b–h) Linear correlation analyses between the levels of each PKA subunit/isoform and calpain activation in 13 human brains (six AD and seven controls).

Fig. 6

A proposed scheme of calpain–cAMP-dependent protein kinase (PKA)–cAMP-response element-binding protein (CREB) dysregulation in Alzheimer disease (AD) brain.