AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.     

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ～3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.     

p62/SQSTM1 is required for the protection against endoplasmic reticulum stress-induced apoptotic cell death

Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.     

Oxidation state of binuclear iron site in azidosemimethemerythrin

Electron paramagnetic resonance spectra of azidosemimethemerythrin (from Phascolopsis gouldii) have been integrated to find the total number of spins per monomer unit. The value observed, 1.0 +/- 0.1 spins per Fe2 pair, confirms the assignment of a hybrid oxidation state, FeIIFeIII, to each site.     

Electron transport chain and energy transduction in Paracoccus denitrificans under autotrophic growth conditions

Cells of Paracoccus denitrificans grown autotrophically with H₂ as energy source contained a branched respiratory chain. The presence of two terminal oxidases was indicated by two cyanide sensitive sites (K _i =10^-5 M and K _i =10^-3 M). While oxidation of NADH and succinate apparently proceeded via both electron pathways as shown by the inhibition of respiration with cyanide and Antimycin A, oxidation of H₂ involved only the terminal oxidase which was less sensitive to KCN. Oxidation of H₂ was not inhibited by rotenone, and sensitive to only relatively high concentrations of Antimycin A (50 nmol/mg).Under our growth conditions, autotrophic cells contained only very small amounts of cytochrome a +a ₃ . A cytochrome b was able to bind CO (with a peak at 418 nm and a trough at 434 nm in the reduced plus CO minus reduced difference spectrum). This cytochrome b had the spectral characteristics of cytochrome o and could be the alternate oxidase. The respiratory chain contained two b cytochromes (b ₅₅₆ and b ₅₆₂ at 77°K); under steady state conditions only b ₅₅₆ was significantly reduced by NADH and succinate while both b ₅₅₆ and b ₅₆₂ were reduced by H₂.Measurement of respiration-driven proton translocation by spheroplasts showed that the oxidation of H₂ by O₂ was associated with a vectorial ejection of H⁺ (in the outward direction) with aH⁺/O value of 6 to 7.A similar result was obtained with succinate. Oxidation of endogenous substrates gave H⁺/O values corresponding to a H⁺/site ratio of 3 with 3 sites functioning in absence of inhibitors, two sites in the presence of rotenone and one site in the presence of antimycin. The H⁺/O values indicated that two energy transducing sites were involved in the oxidation of H₂ by O₂.Measurement of ATP synthesis in membrane vesicles confirmed that phosphorylation was coupled to H₂ oxidation. However, such determinations which necessitated the use of inverted vesicles, gave P/O values too low to allow any conclusions to be made on the number of coupling sites.     

Loss of p62 impairs bone turnover and inhibits PTH-induced osteogenesis

The p62 (also named sequestosome1/SQSTM1) is multidomain and multifunctional protein associated with several physiological and pathological conditions. A number of studies evidenced an involvement of p62 on the disruptive bone scenarios due to its participation in the inflammatory/osteoclastogenic pathways. However, so far, information regarding the function of p62 in the fine-tuned processes underpinning the bone physiology are not well-defined and are sometime discordant. We, previously, demonstrated that the intramuscular administration of a plasmid coding for p62 was able to contrast bone loss in a mouse model of osteopenia. Here, in vitro findings showed that the p62 overexpression in murine osteoblasts precursors enhanced their maturation while the p62 depletion by a specific siRNA, decreased osteoblasts differentiation. Consistently, the activity of osteoblasts from p62^−/− mice was reduced compared with wild-type. Also, morphometric analyses of bone from p62 knockout mice revealed a pathological phenotype characterized by a lower turnover that could be explained by the poor Runx2 protein synthesis in absence of p62. Furthermore, we demonstrated that the parathyroid hormone (PTH) regulates p62 expression and that the osteogenic effects of this hormone were totally abrogated in osteoblasts from p62-deficient mice. Therefore, these findings, for the first time, highlight the important role of p62 both for the basal and for PTH-stimulated bone remodeling.