bafilomycin-a and Alzheimer-Disease

Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. We previously showed that MGO treatment targets the thioredoxin and the glyoxalase systems, leading to a decrease in Trx1 and Glo2 proteins in immortalized mouse hippocampal HT22 nerve cells. Here, we propose that autophagy is the underlying mechanism leading to Glo2 and Trx1 loss induced by MGO. The autophagic markers p62, and the lipidated and active form of LC3, were increased by MGO (0.5mM). Autophagy inhibition with bafilomycin or chloroquine prevented the decrease in Trx1 and Glo2 at 6 and 18h after MGO treatment. Proteasome inhibition by MG132 exacerbated the effect of MGO on Trx1 and Glo2 degradation (18h), further suggesting a role for autophagy. ATG5 small interfering RNA protected Trx1 and Glo2 from MGO-induced degradation, confirming Trx1 and Glo2 loss is mediated by autophagy. In the search for the signals that control autophagy, we found that AMPK activation, a known autophagy inducer, was markedly increased by MGO treatment. AMPK activation was confirmed by increased acetyl coenzyme A carboxylase phosphorylation, a direct AMPK substrate and by decreased mTOR phosphorylation, an indirect marker of AMPK activation. To confirm that MGO-mediated Trx1 and Glo2 degradation was AMPK-dependent, AMPK-deficient mouse embryonic fibroblasts (MEFs) were treated with MGO. Wildtype MEFs presented the expected decrease in Trx1 and Glo2, while MGO was ineffective in decreasing these proteins in AMPK-deficient cells. Overall, the data indicate that MGO activates autophagy in an AMPK-dependent manner, and that autophagy was responsible for Trx1 and Glo2 degradation, confirming that Trx1 and Glo2 are molecular targets of MGO.

Topics: Acetyl-CoA Carboxylase; Alzheimer Disease; AMP-Activated Protein Kinase Kinases; Animals; Autophagy; Cell Line, Transformed; Hippocampus; Humans; Inflammation; Macrolides; Mice; Neurons; Protein Kinases; Proteolysis; Pyruvaldehyde; Thiolester Hydrolases; Thioredoxins; TOR Serine-Threonine Kinases

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Autophagy; Avoidance Learning; Brain; Cells, Cultured; Cognition Disorders; Enzyme Inhibitors; Gene Expression Regulation; Humans; Macrolides; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Tissue Proteins; Neurons; Presenilin-1; Reaction Time; Selenomethionine; tau Proteins

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development.

Topics: ADAM Proteins; Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Animals; Brain; Cathepsin D; Cell Line, Tumor; Down-Regulation; HEK293 Cells; Humans; Lysosomes; Macrolides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Patch-Clamp Techniques; Pepstatins; RNA Interference; RNA, Small Interfering

Accumulation of amyloid β-peptide (Aβ) in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neurodegeneration. Recent studies suggested sestrin2 as a crucial mediator for reactive oxygen species (ROS) scavenging and autophagy regulation that both play a pivotal role in age-dependent neurodegenerative diseases. However, the potential link between sestrin2 and Aβ neurotoxicity has never been explored. The present study was therefore undertaken to test whether sestrin2 may be induced by Aβ and its possible role in modulating Aβ neurotoxicity. We showed that sestrin2 expression was elevated in primary rat cortical neurons upon Aβ exposure; a heightened extent of sestrin2 expression was also detected in the cortices of 12-month-old APPswe/PSEN1dE9 transgenic mice. Exposure of cortical neurons to Aβ led to formation of LC3B-II, an autophagic marker; an increased LC3B-II level was also observed in the cortices of 12-month-old AD transgenic mice. More importantly, downregulation of sestrin2 by siRNA abolished LC3B-II formation caused by Aβ that was accompanied by more severe neuronal death. Inhibition of autophagy by bafilomycin A1 also enhanced Aβ neurotoxicity. Together, these results indicate that sestrin2 induced by Aβ plays a protective role against Aβ neurotoxicity through, at least in part, regulation of autophagy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Humans; Macrolides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Mutation; Nuclear Proteins; Organ Culture Techniques; Peptide Fragments; Presenilin-1; Rats; Rats, Sprague-Dawley; Time Factors

The amyloid precursor protein (APP) metabolism is central to pathogenesis of Alzheimer's disease (AD). Parenchymal amyloid deposits, a neuropathological hallmark of AD, are composed of amyloid-beta peptides (Abeta). Abeta derives from the amyloid precursor protein (APP) by sequential cleavages by beta- and gamma-secretases. Gamma-secretase cleavage releases the APP intracellular domain (AICD), suggested to mediate a nuclear signaling. Physiologically, AICD is seldom detected and thus supposed to be rapidly degraded. The mechanisms responsible of its degradation remain unknown. We used a pharmacological approach and showed that several alkalizing drugs induce the accumulation of AICD in neuroblastoma SY5Y cell lines stably expressing APP constructs. Moreover, alkalizing drugs induce AICD accumulation in naive SY5Y, HEK and COS cells. This accumulation is not mediated by the proteasome or metallopeptidases and is not the result of an increased gamma-secretase activity since the gamma-secretase cleavage of Notch1 and N-Cadherin is not affected by alkalizing drug treatments. Altogether, our data demonstrate for the first time that alkalizing drugs induce the accumulation of AICD, a mechanism likely mediated by the endosome/lysosome pathway.

Topics: Alkalies; Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Cadherins; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Kidney; Lysosomes; Macrolides; Neuroblastoma; Protein Structure, Tertiary; Receptors, Notch; Solubility; Transfection