Elevated [Ca2+]i Levels Occur with Decreased Calpain Activity in Aged Fibroblasts and Their Reversal by Energy-Rich Compounds: New Paradigm for Alzheimer's Disease Prevention

Abstract

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.

Aging-related increase in store-operated Ca2+ influx in human ventricular fibroblasts

“…The age-related increase in the angiotensin II-induced Ca 2ϩ influx could be due to an increase in the function of SOCE, as evident from the increase in Ca 2ϩ -ATPase inhibition (thapsigargin)-induced store-operated Ca 2ϩ influx without a change in the thapsigargin-induced intracellular Ca 2ϩ release between the two age groups. Similar to our observation, elevated intracellular Ca 2ϩ levels induced by bradykinin have been reported in human skin fibroblasts from an elderly (96yr-old) individual (29). Similar results with enhanced sensitivity to Ca 2ϩ influx have also been demonstrated in human lung fibroblasts but not to a similar extent for human skin fibroblasts (30), suggesting a tissue origin-related difference in response to age.…”

“…The reason underlying enhanced SOCE with aging in (35), supporting the idea that SOCE may represent a therapeutic target to prevent excessive CF. Additionally, the energy status of the cells can also influence Ca 2ϩ homeostasis, as reported in a previous study (29), wherein Ca 2ϩ overload occurred in fibroblasts from elderly individuals and was determined to be due to age-related compromised bioenergetics leading to inefficient Ca 2ϩ handling. We also observed that when both groups of hVFs were kept in the presence of a high-energy compound, such as phosphoenolpyruvate, the significant difference in the AUC of SOCE between the young and elderly groups was negated.…”

“…Effect of age on angiotensin II-induced Ca 2ϩ release and Ca 2ϩ influx under enhanced cellular energy status. Intracellular elevated Ca 2ϩ levels in aging cells have been reported to be potentially due to age-related compromised bioenergetics and oxidative stress, leading to inefficient Ca 2ϩ homeostasis (29). Therefore, we determined if enhancement of the energy status of cells in vitro would negate the correlation of age with angiotensin II-induced intracellular Ca 2ϩ release and SOCE.…”

Our “energy-Ca2+ signaling deficits” hypothesis and its explanatory potential for key features of Alzheimer’s disease

“…Furthermore, we have also found that Ca 2+ /calpain is indeed sharply activated in the dying cells ( Nguyen et al, 2013 ), suggesting that Ca 2+ /calpain undergoes a “bi-phasic” change in the “sAD process”: first inactivated during aging, then dramatically activated at the cell-death stage, as observed in the AD brain ( Saito et al, 1993 ). The latter change, however, may not have any therapeutic values ( Figures 1A,B ).…”

“…This intriguing phenomenon may happen because the frequency and amplitude of the Ca 2+ waves in vivo may reduce by aging, but this change can manifest as a “Ca 2+ overstay” or elevated steady-state “levels” in vitro ( Figure 1B ). For this reason, the rational approach to reduce the observed “higher Ca 2+ levels” in the aging brain cells should make use of energy metabolism stimulators and Ca 2+ agonists, not antagonists as currently believed ( Nguyen et al, 2013 ).…”