Remodeling of Intracellular Ca2+ Homeostasis in Rat Hippocampal Neurons Aged In Vitro

Abstract

Aging is often associated with a cognitive decline and a susceptibility to neuronal damage. It is also the most important risk factor for neurodegenerative disorders, particularly Alzheimer's disease (AD). AD is related to an excess of neurotoxic oligomers of amyloid β peptide (Aβo); however, the molecular mechanisms are still highly controversial. Intracellular Ca²⁺ homeostasis plays an important role in the control of neuronal activity, including neurotransmitter release, synaptic plasticity, and memory storage, as well as neuron cell death. Recent evidence indicates that long-term cultures of rat hippocampal neurons, resembling aged neurons, undergo cell death after treatment with Aβo, whereas short-term cultures, resembling young neurons, do not. These in vitro changes are associated with the remodeling of intracellular Ca²⁺ homeostasis with aging, thus providing a simplistic model for investigating Ca²⁺ remodeling in aging. In vitro aged neurons show increased resting cytosolic Ca²⁺ concentration, enhanced Ca²⁺ store content, and Ca²⁺ release from the endoplasmic reticulum (ER). Ca²⁺ transfer from the endoplasmic reticulum (ER) to mitochondria is also enhanced. Aged neurons also show decreased store-operated Ca²⁺ entry (SOCE), a Ca²⁺ entry pathway related to memory storage. At the molecular level, in vitro remodeling is associated with changes in the expression of Ca²⁺ channels resembling in vivo aging, including changes in N-methyl-D-aspartate NMDA receptor and inositol 1,4,5-trisphosphate (IP₃) receptor isoforms, increased expression of the mitochondrial calcium uniporter (MCU), and decreased expression of Orai1/Stim1, the molecular players involved in SOCE. Additionally, Aβo treatment exacerbates most of the changes observed in aged neurons and enhances susceptibility to cell death. Conversely, the solely effect of Aβo in young neurons is to increase ER-mitochondria colocalization and enhance Ca²⁺ transfer from ER to mitochondria without inducing neuronal damage. We propose that cultured rat hippocampal neurons may be a useful model to investigate Ca²⁺ remodeling in aging and in age-related neurodegenerative disorders.

Keywords: Alzheimer’s disease; aging; amyloid beta oligomers; calcium; endoplasmic reticulum; hippocampal neurons; mitochondria.

Figure 1

Intracellular Ca²⁺ remodeling in in vitro aged rat hippocampal neurons and effects of amyloid oligomers. Small arrows depict Ca²⁺ fluxes from the endoplasmic reticulum (ER) to mitochondria, increases or decreases in intracellular Ca²⁺ concentration and IP₃ signaling. Large arrows outside neurons represent phenotypic remodeling from young neurons induced by aging, amyloid oligomers (Aβ) or both. In vitro aging enhances Ca²⁺ store content, Ca²⁺ release, and Ca²⁺ transfer from ER to mitochondria associated in cultured rat hippocampal neurons. The remodeling is associated with changes in expression of metabotropic acetylcholine receptors (mAChR), inositol triphosphate (IP₃) receptors, and mitochondrial Ca²⁺ uniporter (MCU), thus improving energy production at the expense of an increased risk of mitochondrial Ca²⁺ overload. Treatment of young neurons with amyloid β oligomers (Aβo) enhances ER–mitochondria colocalization and Ca²⁺ transfer from ER to mitochondria without deleterious effects. However, Aβ treatment in aged neurons showing Ca²⁺ remodeling disrupts mitochondrial Ca²⁺ homeostasis leading to apoptosis.