Cell-type-specific dysregulation of mitochondrial calcium signaling in Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid plaques, tau tangles, and synaptic dysfunction. Despite decades of research, effective disease-modifying therapies remain elusive, highlighting the need for alternative therapeutic targets. While neurons have traditionally been the focus of AD studies, increasing evidence underscores critical roles for glial cells particularly microglia and astrocytes in disease progression. Mitochondrial calcium ( m Ca 2+ ) dysregulation has emerged as a key contributor to neurodegeneration, yet how m Ca 2 ⁺ signaling varies across brain cell types and contributes to AD pathology remains poorly understood. We developed stable human SH-SY5Y (neuroblastoma-derived cells), HMC3 (microglial-like cells), and SVGp12 (astrocytic-like cells) immortalized cell lines expressing APP mutations (Swedish, Florida, and London; APPswe/F/L). We assessed mitochondrial calcium uniporter (mtCU) expression, m Ca 2+ flux using ratiometric calcium (Ca 2+ ) indicators, and evaluated calcium retention capacity (mito-CRC) as a readout of mitochondrial permeability transition pore opening. Bioenergetic parameters including ATP, NADH, membrane potential, and oxygen consumption rate (OCR) were measured alongside structural mitochondrial changes, ROS levels, and cell death using imaging and biochemical assays. APPswe/F/L expression induced mitochondrial dysfunction across all brain immortalized cell types, with neuroblastoma-derived cells exhibiting the highest susceptibility to m Ca 2+ overload, energy failure, and cell death. Compared to neuroblastoma-derived cells, glial-like cells (astrocytic-like and microglial-like cells) showed higher expression of mtCU components, elevated m Ca 2+ uptake at high Ca²⁺ concentrations, and greater mito-CRC. Conversely, neuroblastoma-derived cells displayed faster m Ca 2+ uptake at low Ca 2+ levels, indicating distinct regulatory thresholds. Glial-like cells exhibited more elaborate mitochondrial networks and enhanced metabolic capacity, yet all cell types showed impaired mitochondrial structure, reduced membrane potential and respiration, and increased ROS under mutant APP expression. This study reveals cell-type-specific differences in m Ca 2+ signaling and mitochondrial function in AD, uncovering unique vulnerabilities in neuroblastoma-derived and glial-like cells. These findings highlight the need for cell-targeted strategies to restore m Ca 2+ homeostasis and mitochondrial function in AD.