Chronic lithium administration to FTDP-17 tau and GSK-3beta overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert

Glycogen synthase kinase-3 (GSK-3) has been proposed as the main kinase able to aberrantly phosphorylate tau in Alzheimer's disease (AD) and related tauopathies, raising the possibility of designing novel therapeutic interventions for AD based on GSK-3 inhibition. Lithium, a widely used drug for affective disorders, inhibits GSK-3 at therapeutically relevant concentrations. Therefore, it was of great interest to test the possible protective effects of lithium in an AD animal model based on GSK-3 overexpression. We had previously generated a double transgenic model, overexpressing GSK-3beta in a conditional manner, using the Tet-off system and tau protein carrying a triple FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation. This transgenic line shows tau hyperphosphorylation in hippocampal neurones accompanied by neurofibrillary tangles (NFTs). We used this transgenic model to address two issues: first, whether chronic lithium treatment is able to prevent the formation of aberrant tau aggregates that result from the overexpression of FTDP-17 tau and GSK-3beta; second, whether lithium is able to change back already formed NFTs in aged animals. Our data suggest that progression of the tauopathy can be prevented by administration of lithium when the first signs of neuropathology appear. Furthermore, it is still possible to partially reverse tau pathology in advanced stages of the disease, although NFT-like structures cannot be changed. The same results were obtained after shut-down of GSK-3beta overexpression, supporting the possibility that GSK-3 inhibition is not sufficient to reverse NFT-like aggregates.     

Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture

Alzheimer's disease is characterized by beta-amyloid (Abeta) overproduction and tau hyperphosphorylation. Recent studies have shown that synthetic Abeta promotes tau phosphorylation in vitro. However, whether endogenously overproduced Abeta promotes tau phosphorylation and the underlying mechanisms remain unknown. Here, we used mouse neuroblastoma N2a stably expressing wild-type amyloid precursor protein (APPwt) or the Swedish mutant APP (APPswe) to determine the alterations of phosphorylated tau and the related protein kinases. We found that phosphorylation of tau at paired helical filament (PHF)-1, pSer396 and pThr231 epitopes was significantly increased in cells transfected with APPwt and APPswe, which produced higher levels of Abeta than cells transfected with vector or amyloid precursor-like protein 1. The activity of glycogen synthase kinase-3 (GSK-3) was up-regulated with a concomitant reduction in the inhibitory phosphorylation of GSK-3 at its N-terminal Ser9 residue. In contrast, the activity of cyclin-dependent kinase-5 (CDK-5) and protein kinase C (PKC) was down-regulated. Inhibition of GSK-3 by LiCl, but not inhibition of CDK-5 by roscovitine, arrested Abeta secretion and tau phosphorylation. Inhibition of PKC by GF-109203X activated GSK-3, whereas activation of PKC by phorbol-12,13-dibutyrate inhibited GSK-3. These results suggest that endogenously overproduced Abeta induces increased tau phosphorylation through activation of GSK-3, and that inactivation of PKC is at least one of the mechanisms involved in GSK-3 activation.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.     

Hsp90α/β associates with the GSK3β/axin1/phospho-β-catenin complex in the human MCF-7 epithelial breast cancer model

Hsp90α/β, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/β with its client kinases form the framework of multiple signalling networks. GSK3β, a known Hsp90α/β client, mediates β-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-β-catenin to the 26S proteasome. The canonical Wnt/β-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/β involvement in Wnt/β-catenin signalling needs clarification. Here, we describe the association of Hsp90α/β with GSK3β, β-catenin, phospho-β-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/β, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/β modulates the phosphorylation of β-catenin by interaction in common complex with GSK3β/axin1/β-catenin.     

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 μg/mL corresponding to 50 μM, 24–48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3β), and with degradation of Mcl-1. With α-tocopherol (400 μM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3β dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3β/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.     

Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals

Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research.     

Why does the brain (not) have glycogen?

In the present paper we formulate the hypothesis that brain glycogen is a critical determinant in the modulation of carbohydrate supply at the cellular level. Specifically, we propose that mobilization of astrocytic glycogen after an increase in AMP levels during enhanced neuronal activity controls the concentration of glucose phosphates in astrocytes. This would result in modulation of glucose phosphorylation by hexokinase and upstream cell glucose uptake. This mechanism would favor glucose channeling to activated neurons, supplementing the already rich neuron-astrocyte metabolic and functional partnership with important implications for the energy compounds used to sustain neuronal activity. The hypothesis is based on recent modeling evidence suggesting that rapid glycogen breakdown can profoundly alter the short-term kinetics of glucose delivery to neurons and astrocytes. It is also based on review of the literature relevant to glycogen metabolism during physiological brain activity, with an emphasis on the metabolic pathways identifying both the origin and the fate of this glucose reserve.     

Critical scaffolding regions of the tumor suppressor Axin1 are natively unfolded

The Wnt pathway tumor-suppressor protein Axin coordinates the formation of a critical multiprotein destruction complex that serves to downregulate β-catenin protein levels, thereby preventing target gene activation. Given the lack of structural information on some of the major functional parts of Axin, it remains unresolved how the recruitment and positioning of Wnt pathway kinases, such as glycogen synthase kinase 3β, are coordinated to bring about β-catenin phosphorylation. Using various biochemical and biophysical methods, we demonstrate here that the central region of Axin that is implicated in binding glycogen synthase kinase 3β and β-catenin is natively unfolded. Our results support a model in which the unfolded nature of these critical scaffolding regions in Axin facilitates dynamic interactions with a kinase and its substrate, which in turn act upon each other.