Elevated [Ca 2+ ] i Levels Occur with Decreased Calpain Activity in Aged Fibroblasts and Their Reversal by Energy-Rich Compounds: New Paradigm for Alzheimer's Disease Prevention

Elevated intracellular Ca²⁺ levels in the aging brain are widely thought to hyperactivate Ca²⁺ signaling and Ca²⁺-dependent enzymes, leading to neuronal death through an excitatory mechanism in Alzheimer's disease (AD). This “Ca²⁺ overload” hypothesis has been questioned by our theoretical analyses. To better understand the relationship between the “level” and functionality of Ca²⁺ in aging, in this study we simultaneously measured intracellular Ca²⁺ transients and calpain activity in cultured human fibroblasts. We found that Ca²⁺ transitions elicited by bradykinin were indeed overstayed or elevated in levels in old cells but, remarkably, calpain activity was decreased compared to young cells. Also, treating young cells with the energy inhibitor rotenone or with H₂O₂ recapitulated the Ca²⁺ overstay and calpain inactivation found in old cells. More importantly, treating old cells with high-energy compounds such as phosphoenol pyruvate or phosphocreatine, which boosted cellular ATP content, reduced the Ca²⁺ overstay and re-activated calpain. Moreover, Ca²⁺ levels and calpain activity were dramatically raised in the dying cells killed by detergent. Finally, Ca²⁺ oscillations induced by low dose of bradykinin in old cells exhibited lower spike frequency, but higher overall levels. Collectively, these results suggest that (a) Ca²⁺ overload in old cells arises from an inefficient Ca²⁺ handling system compromised by age-related energy depletion and oxidative stress; and (b) despite elevated levels, the functionality of Ca²⁺ signaling has diminished in old cells. Thus, the study reinforces the concept that tonic promotion of bioenergetics and Ca²⁺ signaling function is a reasonable and new paradigm to protect the aging brain cells to prevent AD.