aldehyde phosphatase dehydrogenase; GSK-3, glycogen synthase kinase 3; GFP, green fluorescent protein; Hsc, heat shock cognate; Hsp, heat shock protein; IGF, insulin-like growth factor; IRS, insulin receptor substrate; MB, methylene blue; LC, liquid chromatography; MS, mass spectroscopy; mTOR, mammalian target of rapamycin; PI3, phosphoinositide 3-kinase; PBS, phosphate buffered saline; p70, p70S6 kinase; pp70, phosphorylated p70S6 kinase; RFP, red fluorescent protein; shRNA, short hairpin RNA More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubulebinding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

BackgroundIt has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.ResultsHere, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.ConclusionsNeuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D 1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the ␣ isoform of GRK4; the ␤, ␥, and ␦ isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4␣ is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D 1 receptor by GRK4␣ results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number-all of which are abolished in a D 1 receptor mutant containing T428V and S431A. The increase in internalized D 1 receptors induced by GRK4␣ phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D 1 receptor by GRK4␣ does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4␣. These observations shift the attenuation of D 1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization. Dopamine signaling in mammals is mediated by five G protein-coupled receptor (GPCR) proteins divided into two groups based upon sequence homology, G protein coupling, signaling pathways, pharmacological profiles, and desensitization kinetics (Sibley and Monsma, 1992;Missale et al., 1998 Upon agonist activation, GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response under continued agonist stimulation (Ferguson et al., 1996;Gainetdinov et al., 2004). Desensitization involves phosphorylation of the receptor by GRKs and/or second messenger-activated kinases (cAMP-dependent protein kinase or protein kinase C). Homologous desensitization of GPCRs involves only activated receptors and is primarily mediated by GRKs. GRKs are serine/threonine-directed protein kinases composed of seven isoforms divided into three families (Penela et al., 2003). GRK1 and GRK7 compose the rhodopsin kinase/visual family, are expressed exclusively in retina, and participate in desensitization of opsins in rods and cones (Somers and Klein, 1984;Hisatomi et al., 1998;Weiss et al., 1998). GRK2 (␤ARK1) and GRK3 (␤ARK2) were originally identified as regulating the ␤-adrenergic receptor and com- ABBREVIATIONS: GPCR, G protein-coupled receptor; ␤ARK, ␤-adrenergic ...

Homologous desensitization of D 1 dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D 1 receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.