Martin Gamerdinger

Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3

The Hsc/Hsp70 co‐chaperones of the BAG (Bcl‐2‐associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin‐binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3‐II as well as a higher cathepsin activity. We conclude that the BAG3‐mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro‐oxidant and aggregation‐prone milieu characteristic of aging.

BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat‐shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome‐targeting and autophagic degradation. This pathway is regulated by the stress‐induced co‐chaperone Bcl‐2‐associated athanogene 3 (BAG3), which interacts with the microtubule‐motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome‐targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.

Emerging roles of molecular chaperones and co-chaperones in selective autophagy: focus on BAG proteins

Macroautophagy is a catabolic process by which the cell degrades cytoplasmic components through the lysosomal machinery. While initially acknowledged as a rather unspecific bulk degradation process, growing lines of evidence indicate the selectivity of macroautophagy pathways in the removal of misfolded or aggregated proteins. How such substrates are recognized and specifically targeted to the macroautophagy machinery has become a hotspot of investigation, and recent evidence suggests that here molecular chaperones and co-chaperones play a central role. One emerging pathway is mediated by the co-chaperone protein Bcl-2-associated athanogene 3 (BAG 3) which seems to utilize the specificity of molecular chaperones (heat-shock proteins) towards non-native proteins as basis for targeted macroautophagic degradation. In this short review, we focus on the molecular interplay between the macroautophagy system and molecular chaperones and highlight the relevance of the pathway mediated by BAG3 to aging and age-associated protein-misfolding diseases.

Inhibition of the myosin light chain kinase prevents hypoxia-induced blood–brain barrier disruption

Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia‐induced blood–brain barrier (BBB) opening. Measurements of trans‐endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co‐culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two‐vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using immunohistochemistry and immunoblot analysis. Hypoxia caused a decrease of TEER values by more than 40%, which was prevented by inhibition of the MLC‐kinase (ML‐7, 10 μmol/L). In addition, ML‐7 significantly reduced the brain fluid content in vivo after stroke. The NAD(P)H‐oxidase inhibitor apocynin (500 μmol/L) prevented the hypoxia‐induced TEER decrease. Hypoxia‐dependent ROS generation was completely abolished by apocynin. Furthermore, ML‐7 and apocynin blocked hypoxia‐dependent phosphorylation of MLC. Our data demonstrate that hypoxia causes a breakdown of the BBB in vitro and in vivo involving ROS and the contractile machinery.

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress‐resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress‐resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress‐resistant cells with neutral SMase reversed the stress‐resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress‐resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress‐sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type‐specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.

Oestrogen receptor subtype‐specific repression of calpain expression and calpain enzymatic activity in neuronal cells – implications for neuroprotection against Ca2+‐mediated excitotoxicity

Calpains represent a superfamily of Ca2+‐activated cysteine‐proteases, which are important mediators of apoptosis and necrosis. In the brain, m‐calpain and µ‐calpain, the two ubiquitous calpain‐isoforms, are strongly activated in neurones after an excitotoxic Ca2+ influx occurring, for example, during cerebral ischemia. Because oestrogen and its receptors (ERα/ERβ) can exert neuroprotective activity, we investigated their influence on expression of calpains and their endogenous inhibitor, calpastatin. We found that ectopic expression of ERα in human neuroblastoma SK‐N‐MC cells led to a ligand‐independent constitutive down‐regulation of m‐calpain accompanied by an up‐regulation of µ‐calpain expression. Up‐regulation of µ‐calpain was reversed in the presence of oestrogen, which, in turn, could be blocked by co‐treatment with the oestrogen‐receptor antagonist ICI 182 780. Expression of calpastatin was not altered, either in the absence or in the presence of oestrogen. Additional studies revealed that ERα‐expressing cells exhibited decreased calpain enzymatic activity and increased survival when cells were exposed to the Ca2+ ionophore, ionomycin. Since all investigated effects could be observed exclusively in the presence of ERα, but not ERβ, and since the effects are reduced when ERα and ERβ are co‐expressed, our data suggest a novel ER subtype‐specific neuroprotective action by repressing calpain expression and calpain activity under conditions of a massive Ca2+ influx.

Cholesterol-Like Effects of Selective Cyclooxygenase Inhibitors and Fibrates on Cellular Membranes and Amyloid-β Production

Strong evidence suggests a mechanistic link between cholesterol metabolism and the formation of amyloid-β peptides, the principal constituents of senile plaques found in the brains of patients with Alzheimers disease. Here, we show that several fibrates and diaryl heterocycle cyclooxygenase inhibitors, among them the commonly used drugs fenofibrate and celecoxib, exhibit effects similar to those of cholesterol on cellular membranes and amyloid precursor protein (APP) processing. These drugs have the same effects on membrane rigidity as cholesterol, monitored here by an increase in fluorescence anisotropy. The effect of the drugs on cellular membranes was also reflected in the inhibitory action on the sarco(endo)plasmic reticulum Ca2+-ATPase, which is known to be inhibited by excess ordering of membrane lipids. The drug-induced decrease of membrane fluidity correlated with an increased association of APP and its β-site cleaving enzyme BACE1 with detergent-resistant membranes (DRMs), which represent membrane clusters of substantial rigidity. DRMs are hypothesized to serve as platforms for the amyloidogenic processing of APP. According to this hypothesis, both cholesterol and the examined compounds stimulated the β-secretase cleavage of APP, resulting in a massive increase of secreted amyloid-β peptides. The membrane-ordering potential of the drugs was observed in a cell-free assay, suggesting that the amyloid-β promoting effect was analog to cholesterol due to primary effect on membrane rigidity. Because fenofibrate and celecoxib are widely used in humans as hypolipidemic drugs for prevention of atherosclerosis and as anti-inflammatory drugs against arthritis, possible side effects should be considered upon long-term clinical application.

Effects of sulindac sulfide on the membrane architecture and the activity of γ-secretase

gamma-Secretase is a membrane-embedded multi-protein complex that catalyzes the final cut of the Alzheimers disease-related amyloid precursor protein (APP) to amyloid-beta peptides of variable length (37-43 amino acids) via an unusual intramembrane cleavage. Recent findings propose that some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) have the ability to modulate specifically gamma-secretase activity without inhibiting the enzyme as a whole. These drugs may shift the processing of APP from the longer amyloid-beta 42 peptide towards shorter, less fibrillogenic and less toxic amyloid-beta species. We hypothesize that gamma-secretase activity, as an enzyme that is strictly associated with cellular membranes, is sensitive to alterations of the hydrophobic membrane environment. Here, we show that the gamma-secretase modulator and amyloid-beta 42-lowering drug sulindac sulfide alters the physical state of the membrane and strongly decreases fluidity of cellular membranes. Furthermore, sulindac sulfide changed the protein composition of membrane microdomains, the so-called lipid rafts. Most significantly, APP C-terminal fragments (CTFs) were redistributed from rafts towards non-raft membrane domains. This could be demonstrated also in cell-free assays, where in addition presenilin-1, the catalytic subunit of the gamma-secretase complex, was shifted out of lipid rafts. Together, these findings suggest that sulindac sulfide directly alters the membrane architecture and shifts the gamma-secretase-mediated cleavage of APP towards a hydrophobic environment where the enzyme-substrate complex is in a conformation for processing preferentially shorter amyloid-beta peptides.

The selective β1‐adrenoceptor antagonist nebivolol is a potential oestrogen receptor agonist with neuroprotective abilities

Background and purpose:  Nebivolol, a selective β1‐adrenoceptor antagonist mediating rapid vasodilating effects, is used clinically to treat hypertension. Recently, it was reported that nebivolol also acts as an oestrogen receptor (ER) agonist. To investigate the neuroprotective potential of oestrogens, we assessed the oestrogenic effects of nebivolol in several in vitro neuronal models.

BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.