Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-β (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays. In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.

Hippocampal GFAP-positive astrocyte responses to amyloid and tau pathologies

“…Additionally, as APP is synthesized in the ER, alterations in MAMs and ER stress are emerging as important pathological mechanisms of AD and interesting therapeutic targets to modify protein misfolding and synaptic failure (27)(28)(29). Furthermore, the upregulation of ubiquitin-related genes (Uba52, Ubc) may indicate compensatory attempts to mitigate the results of ER stress.…”

Dysregulated Ca2+ Homeostasis as a Central Theme in Neurodegeneration: Lessons from Alzheimer’s Disease and Wolfram Syndrome

“…MAM homeostasis is key for cellular health [ 28 ] and is well-established to be affected in neurodegenerative diseases, including AD. Moreover, altered MAM function is even suggested as a main pathogenic cause in AD [ 125 ]. The evidence to support the role of MAMs in AD pathophysiology comes from the observation that APP, PSENS and the complete γ-secretase complex predominantly reside in the MAMs [ 126 ].…”

Disentangling Mitochondria in Alzheimer’s Disease

“…It is possible that Aβ directly interferes with the mitochondrial function and causes the metabolic deficiencies and neurological dysfunction observed in the brains of patients with AD [ 66 ]. For example, Aβ alters the physical and biochemical connections between the endoplasmic reticulum and mitochondria, as evidenced in AD brain and neuronal cultures, making the connections abnormally tight and interfering with mitochondrial morphology, motility, bioenergetics, autophagy, Ca 2+ signaling, and apoptosis [ 67 , 68 , 69 , 70 ]. A direct membrane-binding of Aβ peptides was recently shown to block the mitochondrial large-conductance, Ca 2+ -activated potassium channels [ 71 ].…”