The Interplay between GSK3ß and Tau Ser262 Phosphorylation during the Progression of Tau Pathology.

Tau hyperphosphorylation has been linked directly to the formation of toxic neurofibrillary tangles (NFTs) in tauopathies, however, prior to NFT formation, the sequence of pathological events involving tau phosphorylation remains unclear. Here, the effect of glycogen synthase kinase 3ß (GSK3ß) on tau pathology was examined independently for each step of transcellular propagation; namely, tau intracellular aggregation, release, cellular uptake and seeding activity. We find that overexpression of GSK3ß-induced phosphorylated 0N4R tau led to a higher level of tau oligomerization in SH-SY5Y neuroblastoma cells than wild type 0N4R, as determined by several orthogonal assays. Interestingly, the presence of GSK3ß also enhanced tau release. Further, we demonstrated that cells endocytosed more monomeric tau protein when pre-phosphorylated by GSK3ß. Using an extracellular vesicle (EVs)-assisted tau neuronal delivery system, we show that exosomal GSK3ß-phosphorylated tau, when added to differentiated SH-SY5Y cells, induced more efficient tau transfer, showing much higher total tau levels and increased tau aggregate formation as compared to wild type exosomal tau. The role of a primary tau phosphorylation site targeted by microtubule-affinity regulating kinases (MARKs), Ser262, was tested by pseudo-phosphorylation using site-directed mutagenesis to aspartate (S262D). S262D tau overexpression significantly enhanced tau release and intracellular tau accumulation, which were concurrent with the increase of pathological states of tau, as determined by immunodetection. Importantly, phosphorylation-induced tau accumulation was augmented by co-transfecting S262D tau with GSK3ß, suggesting a possible interplay between Ser262 phosphorylation and GSK3ß activity in tau pathology. Lastly, we found that pre-treatment of cells with amyloid-ß (Aß) further tau phosphorylation and accumulation when Ser262 pre-phosphorylation was present, suggesting that S262 may be a primary mediator of Aß-induced tau toxicity. These findings provide a potential therapeutic target for treating tau-related disorders by targeting specific phospho-tau isoforms and further elucidate the GSK3ß-mediated pathological seeding mechanisms.

Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Heparan Sulfate Proteoglycans (HSPGs) Serve as the Mediator Between Monomeric Tau and Its Subsequent Intracellular ERK1/2 Pathway Activation.

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1-a key enzyme in HSPG synthesis-with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1ß. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1ß expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles.

Nuclear translocation of the unliganded glucocorticoid receptor is influenced by membrane fluidity, but not A2AR agonism.

Epidemiological evidence suggests that chronic consumption of caffeine, a non-selective antagonist of adenosine A2AR receptors (A2AR), can be neuroprotective in a number of age-related neurodegenerative disorders including Alzheimer's disease. A growing body of work shows that this neuroprotection may act via a synergistic interaction with the glucocorticoid receptor (GR) and its associated genetic response elements. Therefore, we hypothesized that A2AR signaling may directly stimulate glucocorticoid receptor translocation via downstream signaling elements within the cell. Surprisingly, we found no effect of A2AR agonism on GR translocation in the absence of steroid. As expected, membrane-bound dexamethasone was capable of stimulating full GR translocation, albeit at a slower rate. This non-liganded translocation was unaffected by A2AR ligands, providing strong evidence that GR translocation occurs independently of activation of A2ARs. To identify other potential mechanisms of translocation, membrane fluidity was increased significantly by benzyl alcohol, which also induced full nuclear translocation of the GR, but unlike the membrane-bound dexamethasone, benzyl alcohol did result in transcriptional upregulation of GR-dependent genes. Taken together, our data shows that the unliganded GR is sensitive to changes in membrane state and can be transcriptionally active.

Redox-sensitive residue in the actin-binding interface of myosin.

We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.