Mapping the lipidome in mitochondria-associated membranes (MAMs) in an in vitro model of Alzheimer's disease.

The disruption of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) plays a relevant role in Alzheimer's disease (AD). MAMs have been implicated in neuronal dysfunction and death since it is associated with impairment of functions regulated in this subcellular domain, including lipid synthesis and trafficking, mitochondria dysfunction, ER stress-induced unfolded protein response (UPR), apoptosis, and inflammation. Since MAMs play an important role in lipid metabolism, in this study we characterized and investigated the lipidome alterations at MAMs in comparison with other subcellular fractions, namely microsomes and mitochondria, using an in vitro model of AD, namely the mouse neuroblastoma cell line (N2A) over-expressing the APP familial Swedish mutation (APPswe) and the respective control (WT) cells. Phospholipids (PLs) and fatty acids (FAs) were isolated from the different subcellular fractions and analyzed by HILIC-LC-MS/MS and GC-MS, respectively. In this in vitro AD model, we observed a down-regulation in relative abundance of some phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) species with PUFA and few PC with saturated and long-chain FA. We also found an up-regulation of CL, and antioxidant alkyl acyl PL. Moreover, multivariate analysis indicated that each organelle has a specific lipid profile adaptation in N2A APPswe cells. In the FAs profile, we found an up-regulation of C16:0 in all subcellular fractions, a decrease of C18:0 levels in total fraction (TF) and microsomes fraction, and a down-regulation of 9-C18:1 was also found in mitochondria fraction in the AD model. Together, these results suggest that the over-expression of the familial APP Swedish mutation affects lipid homeostasis in MAMs and other subcellular fractions and supports the important role of lipids in AD physiopathology.

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.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-ß (Aß) 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.

Amyloid-beta disrupts calcium and redox homeostasis in brain endothelial cells.

In Alzheimer's disease, the accumulation of amyloid-beta (Aß) in the brain occurs in the parenchyma and cerebrovasculature. Several evidences support that the neuronal demise is potentiated by vascular alterations in the early stages of the disease, but the mechanisms responsible for the dysfunction of brain endothelial cells that underlie these cerebrovascular changes are unknown. Using rat brain microvascular endothelial cells, we found that short-term treatment with a toxic dose of Aß1-40 inhibits the Ca(2+) refill and retention ability of the endoplasmic reticulum and enhances the mitochondrial and cytosolic response to adenosine triphosphate (ATP)-stimulated endoplasmic reticulum Ca(2+) release. Upon prolonged Aß1-40 exposure, Ca(2+) homeostasis was restored concomitantly with a decrease in the levels of proteins involved in its regulation operating at the plasma membrane, endoplasmic reticulum, and mitochondria. Along with perturbations in Ca(2+) regulation, an early increase in the levels of oxidants and a decrease in the ratio between reduced and oxidized glutathione were observed in Aß1-40-treated endothelial cells. Under these conditions, the nuclear levels of oxidative stress-related transcription factors, namely, hypoxia-inducible factor 1α and nuclear factor (erythroid-derived 2)-related factor 2, were enhanced as well as the protein levels of target genes. In conclusion, Aß1-40 affects several mechanisms involved in Ca(2+) homeostasis and impairs the redox homeostasis simultaneously with stimulation of protective stress responses in brain endothelial cells. However, the imbalance between cell death and survival pathways leads to endothelial dysfunction that in turn contributes to cerebrovascular impairment in Alzheimer's disease.