PKC and Ras are Involved in M1 Muscarinic Receptor-Mediated Modulation of AMPA Receptor GluA1 Subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) have long been an attractive target for the treatment of Alzheimer's disease (AD), the most common cause of dementia in the elderly. M1 mAChR agonists show desirably preclinical activities; however, most have not gone further into late clinical trials due to ineffectiveness or side effects. Thus, to understand the signaling pathways involved in M1 mAChR-mediated memory improvement may be important for design of biased agonists with on-target therapeutic effects. M1 mAChRs are classically coupled to Gαq or ectopically to Gαs to activate multiple kinases such as protein kinase C (PKC), Ras and protein kinase A (PKA). Our previous studies have found that M1 mAChRs could improve learning and memory through modulating AMPA receptor GluA1 subunit via PKA-PI3K-Akt signaling. Here, we further investigated whether PKC and Ras were involved in M1 mAChR-mediated modulation of GluA1. We demonstrated the role of PKC and Ras in the signaling pathway, as both PKC inhibitors Ro-31-8425 or Gö6983 and Ras inhibitor salirasib abolished the membrane insertion of GluA1 and enhancement of its phosphorylation at Ser845 induced by M1 mAChRs in the primary cultured neurons and hippocampus in vivo. We further showed that PKC and Ras modulated PKA-PI3K-Akt signaling since the increases of PKA, Akt and mTOR activities by M1 mAChR activation were blocked by PKC and Ras inhibitors. These data demonstrated the detailed mechanism underlying M1 mAChR-mediated modulation of GluA1 through Gαq/11 coupling, broadening the knowledge of the downstream signaling after M1 mAChR-Gαq/11 coupling.

M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt.

M1 muscarinic acetylcholine receptors are highly expressed in key areas that control cognition, such as the cortex and hippocampus, representing one potential therapeutic target for cognitive dysfunctions of Alzheimer's disease and schizophrenia. We have reported that M1 receptors facilitate cognition by promoting membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPA receptor subunit 1 (GluA1) through phosphorylation at Ser845. However, the signaling pathway is still unclear. Here we showed that adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720 inhibited enhancement of phosphorylation of Ser845 and membrane insertion of GluA1 induced by M1 receptor activation. Furthermore, PI3K inhibitor LY294002 and protein kinase B (Akt) inhibitor IV blocked the effects of M1 receptors as well. Remarkably, the increase of the activity of PI3K-Akt signaling induced by M1 receptor activation could be abolished by cAMP-PKA inhibitors. Moreover, inhibiting the mammalian target of rapamycin (mTOR) complex 1, an important downstream effector of PI3K-Akt, by short-term application of rapamycin attenuated the effects of M1 receptors on GluA1. Furthermore, such effect was unrelated to possible protein synthesis promoted by mTOR. Taken together, these data demonstrate that M1 receptor activation induces membrane insertion of GluA1 via a signaling linking cAMP-PKA and PI3K-Akt-mTOR pathways but is irrelevant to protein synthesis.-Zhao, L.-X., Ge, Y.-H., Li, J.-B., Xiong, C.-H., Law, P.-Y., Xu, J.-R., Qiu, Y., Chen, H.-Z. M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt.

M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

In vitro and in vivo activities of pterostilbene against Candida albicans biofilms.

Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 µg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 µg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 µg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway.

Fluconazole assists berberine to kill fluconazole-resistant Candida albicans.

It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.

ECM17-dependent methionine/cysteine biosynthesis contributes to biofilm formation in Candida albicans.

Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17Δ/Δ mutant failed to catalyze the biochemical reaction from sulfite to H(2)S and hardly grew in media lacking Met and Cys. NaSH, the donor of H(2)S, dose-dependently improved the growth of ecm17Δ/Δ in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17Δ/Δ mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP-protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation.

Using Galleria mellonella-Candida albicans infection model to evaluate antifungal agents.

Candida albicans is the most common fungal pathogen. Galleria mellonella is widely used as an infection model host. Nevertheless, the G. mellonella-C. albicans infection model had not been optimized for drug evaluation before this study. In this work, we revealed that 5 × 10(5) colony forming unit (CFU)/larva was a suitable inoculum to optimize the G. mellonella-C. albicans infection model in order to evaluate antifungal agents. Using our optimized model, the antifungal effect of fluconazole, amphotericin B and flucytosine, and the synergy between amphotericin B and flucytosine were successfully verified. Thus, this study provides a rapid, inexpensive and reliable way to evaluate antifungals in vivo.

p85S6K sustains synaptic GluA1 to ameliorate cognitive deficits in Alzheimer's disease.

BACKGROUND: Ribosomal protein S6 kinase 1 (S6K1) is a serine-threonine kinase that has two main isoforms: p70S6K (70-kDa isoform) and p85S6K (85-kDa isoform). p70S6K, with its upstream mammalian target of rapamycin (mTOR), has been shown to be involved in learning and memory and participate in the pathophysiology of Alzheimer's disease (AD). However, the function of p85S6K has long been neglected due to its high similarity to p70S6k. The role of p85S6K in learning and memory is still largely unknown. METHODS: We fractionated the postsynaptic densities to illustrate the differential distribution of p85S6K and p70S6K. Coimmunoprecipitation was performed to unveil interactions between p85S6K and the GluA1 subunit of AMPA receptor. The roles of p85S6K in synaptic targeting of GluA1 and learning and memory were evaluated by specific knockdown or overexpression of p85S6K followed by a broad range of methodologies including immunofluorescence, Western blot, in situ proximity ligation assay, morphological staining and behavioral examination. Further, the expression level of p85S6K was measured in brains from AD patients and AD model mice. RESULTS: p85S6K, but not p70S6K, was enriched in the postsynaptic densities. Moreover, knockdown of p85S6K resulted in defective spatial and recognition memory. In addition, p85S6K could interact with the GluA1 subunit of AMPA receptor through synapse-associated protein 97 and A-kinase anchoring protein 79/150. Mechanistic studies demonstrated that p85S6K could directly phosphorylate GluA1 at Ser845 and increase the amount of GluA1 in synapses, thus sustaining synaptic function and spine densities. Moreover, p85S6K was found to be specifically decreased in the synaptosomal compartment in the brains of AD patients and AD mice. Overexpression of p85S6K ameliorated the synaptic deficits and cognitive impairment in transgenic AD model mice. CONCLUSIONS: These results strongly imply a significant role for p85S6K in maintaining synaptic and cognitive function by interacting with GluA1. The findings provide an insight into the rational targeting of p85S6K as a therapeutic potential for AD.

M1 Muscarinic Receptor Activation Rescues ß-Amyloid-Induced Cognitive Impairment through AMPA Receptor GluA1 Subunit.

M1 muscarinic receptors have long been identified as a potential therapeutic target for the treatment of cognitive impairment in Alzheimer's disease (AD). Our previous study has shown that M1 receptors promote membrane insertion and synaptic delivery of AMPA receptor GluA1 subunit. In this study, we sought to determine whether activation of M1 receptor would rescue the cognitive impairment in AD model mice through modulation of GluA1 subunit. For the mice injected with aggregated ß-amyloid (Aß) fragments to impair learning and memory, activation of M1 receptors could rescue it by reducing the latency to find the platform and spending more time in the target quadrant in the probe test in the Morris water maze. However, such an effect was ablated in mice with Ser845 residue of GluA1 mutated to alanine. Furthermore, the activation of M1 receptors enhanced the expression of GluA1 and its phosphorylation at Ser845 and drove GluA1 to incorporate with PSD95, a postsynaptic marker, in the hippocampi from Aß-injected wild type mice but not from the mutant mice. Moreover, for 9-month-old APP/PS1 transgenic AD model mice, which may resemble the late AD, M1 receptor activation could not improve the cognitive impairment significantly. In addition, the enhancement of GluA1 expression and its phosphorylation at Ser845 were not observed in their hippocampi. Taken together, the study indicated that M1 receptor activation rescued the cognitive deficit through modulating the trafficking of GluA1-containing AMPA receptors and the therapeutics targeting M1 receptors should aim at mild AD or even pre-AD.

Integrated Genomic Analysis Revealed Associated Genes for Alzheimer's Disease in APOE4 Non-Carriers.

BACKGROUND: APOE4 is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD). LOAD patients carrying or not carrying APOE4 manifest distinct clinico-pathological characteristics. APOE4 has been shown to play a critical role in the pathogenesis of AD by affecting various aspects of pathological processes. However, the pathogenesis involved in LOAD not-carrying APOE4 remains elusive. OBJECTIVE: We aimed to identify the associated genes involved in LOAD not-carrying APOE4. METHODS: An integrated genomic analysis of datasets of genome-wide association study, genome-wide expression profiling and genome-wide linkage scan and protein-protein interaction network construction were applied to identify associated gene clusters in APOE4 non-carriers. The role of one of hub gene of an APOE4 non-carrier-associated gene cluster in tau phosphorylation was studied by knockdown and western blot. RESULTS: We identified 12 gene clusters associated with AD APOE4 non-carriers. The hub genes associated with AD in these clusters were MAPK8, POU2F1, XRCC1, PRKCG, EXOC6, VAMP4, SIRT1, MME, NOS1, ABCA1 and LDLR. The associated genes for APOE4 non-carriers were enriched in hereditary disorder, neurological disease and psychological disorders. Moreover, knockdown of PRKCG to reduce the expression of protein kinase Cγ isoform enhanced tau phosphorylation at Thr181 and Thr231 and the expression of glycogen synthase kinase 3ß and cyclin-dependent kinase 5 in the presence of APOE3 but not APOE4. CONCLUSION: The study provides new insight into the mechanism of distinct pathogenesis of LOAD not carrying APOE4 and prompts the functional exploration of identified genes based on APOE genotypes.

M1 muscarinic receptors facilitate hippocampus-dependent cognitive flexibility via modulating GluA2 subunit of AMPA receptors.

Cognitive flexibility is an important aspect of executive function. The cholinergic system, an important component of cognition, has been shown to modulate cognitive flexibility mainly through the striatum and prefrontal cortex. The role of M1 muscarinic receptors (M1 mAChRs), an important therapeutic target in the cholinergic system, in hippocampus-dependent cognitive flexibility is unclarified. In the present study, we demonstrated that selective activation of M1 mAChRs promoted extinction of initial learned response and facilitated acquisition of reversal learning in the Morris water maze, a behavior test that is mainly dependent on the hippocampus. However, these effects were abolished in GluA2 mutant mice with deficiency in phosphorylation of Ser880 by protein kinase C (PKC). Further long-term depression (LTD) in the hippocampal CA1 area induced by M1 mAChR activation was shown to be dependent on AMPA receptor subunit GluA2 but not GluA1. M1 mAChRs increased GluA2 endocytosis through phosphorylation of Ser880 by PKC. Inhibition of PKC blocked M1 mAChR-mediated LTD, memory switching and reversal learning facilitation. Moreover, the slow memory extinction observed in GluA2 mutant mice and PKC inhibitor-treated mice appeared to affect the consolidation and retrieval of reversal learning. Thus, these results demonstrate that M1 mAChRs mainly facilitate acquisition in spatial reversal learning and further elucidate that such an effect is dependent on the phosphorylation of GluA2 by PKC. The study helps clarify the role of M1 mAChRs in cognitive flexibility and may prompt the earlier prevention of cognitive inflexibility.

α-Mangostin decreases ß-amyloid peptides production via modulation of amyloidogenic pathway.

AIMS: ß-amyloid (Aß) aggregation and deposition play a central role in the pathogenic process of Alzheimer's disease (AD). α-Mangostin (α-M), a polyphenolic xanthone, have been shown to dissociate Aß oligomers. In this study, we further investigated the effect of α-M on Aß production and its molecular mechanism. METHODS: The Aß and soluble amyloid precursor protein α (sAPPα) in culture medium of cortical neurons were measured by ELISA. The activities of α-, ß-, and γ-secretases were assayed, and the interaction between α-M and ß- or γ-secretases was simulated by molecular docking. RESULTS: α-M significantly decreased Aß40 and Aß42 production. α-M did not affect the expression of enzymes involved in nonamyloidogenic and amyloidogenic pathways, but significantly decreased the activities of ß-secretase and likely γ-secretase with IC50 13.22 nmol·L-1 and 16.98 nmol·L-1 , respectively. Molecular docking demonstrated that α-M interacted with ß-site amyloid precursor protein cleaving enzyme 1 and presenilin 1 to interfere with their active sites. CONCLUSIONS: Our data demonstrate that α-M decreases Aß production through inhibiting activities of ß-secretase and likely γ-secretase in the amyloidogenic pathway. The current data together with previous study indicated that α-M could be a novel neuroprotective agent through intervention of multiple pathological processes of AD.

Role of FK506 binding protein 12 in morphine-induced µ-opioid receptor internalization and desensitization.

Agonist-activated µ-opioid receptor (OPRM1) undergoes robust receptor phosphorylation by G protein-coupled receptor kinases and subsequent ß-arrestin recruitment, triggering receptor internalization and desensitization. Morphine, a widely prescribed opioid, induces receptor phosphorylation inefficiently. Previously we reported that FK506 binding protein 12 (FKBP12) specifically interacts with OPRM1 and such interaction attenuates receptor phosphorylation and facilitates morphine-induced recruitment and activation of protein kinase C. In the current study, we demonstrated that the association of FKBP12 with OPRM1 also affects morphine-induced receptor internalization and G protein-dependent adenylyl cyclase desensitization. Morphine induced faster receptor internalization and adenylyl cyclase desensitization in cells expressing OPRM1 with Pro(353) mutated to Ala (OPRM1P353A), which does not interact with FKBP12, or in the presence of FK506 which dissociates the receptor-FKBP12 interaction. Furthermore, knockdown of cellular FKBP12 level by siRNA accelerated morphine-induced receptor internalization and adenylyl cyclase desensitization. Our study further demonstrated that peptidyl prolyl cis-trans isomerase activity of FKBP12 probably plays a role in inhibition of receptor phosphorylation. In the view that internalized receptor recycles and thus counteracts the development of analgesic tolerance, receptor's association with FKBP12 could also contribute to the development of morphine tolerance through modulation of receptor trafficking.

Effect of tetrandrine against Candida albicans biofilms.

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.

Cap1p attenuates the apoptosis of Candida albicans.

Candida albicans is the most common opportunistic fungal pathogen and its apoptosis is inducible by environmental stress. Based on our previous finding that transcription factor Cap1p was involved in baicalein-induced apoptosis, the present study aimed to further clarify the role of Cap1p in apoptosis by observing the impact of CAP1 deletion on cell fate. It was found that apoptotic stimulation with amphotericin B, acetic acid and hydrogen peroxide increased the number of apoptotic and necrotic cells, caspase activity and the accumulation of reactive oxygen species, whereas it decreased the mitochondrial membrane potential and intracellular ATP level in the cap1Δ/Δ mutant. The cell fate was, at least partly, caused by glutathione depletion and attenuation of the expression of the glutathione reductase gene in the cap1Δ/Δ mutant. Collectively, our data suggest that Cap1p participated in the apoptosis of C. albicans by regulating the expression of the glutathione reductase gene and glutathione content.