TREM2 drives microglia response to amyloid-ß via SYK-dependent and -independent pathways.

Genetic studies have highlighted microglia as pivotal in orchestrating Alzheimer's disease (AD). Microglia that adhere to Aß plaques acquire a transcriptional signature, "disease-associated microglia" (DAM), which largely emanates from the TREM2-DAP12 receptor complex that transmits intracellular signals through the protein tyrosine kinase SYK. The human TREM2R47H variant associated with high AD risk fails to activate microglia via SYK. We found that SYK-deficient microglia cannot encase Aß plaques, accelerating brain pathology and behavioral deficits. SYK deficiency impaired the PI3K-AKT-GSK-3ß-mTOR pathway, incapacitating anabolic support required for attaining the DAM profile. However, SYK-deficient microglia proliferated and advanced to an Apoe-expressing prodromal stage of DAM; this pathway relied on the adapter DAP10, which also binds TREM2. Thus, microglial responses to Aß involve non-redundant SYK- and DAP10-pathways. Systemic administration of an antibody against CLEC7A, a receptor that directly activates SYK, rescued microglia activation in mice expressing the TREM2R47H allele, unveiling new options for AD immunotherapy.

Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

Supramolecular assembly of GSK3α as a cellular response to amino acid starvation.

The tolerance of amino acid starvation is fundamental to robust cellular fitness. Asparagine depletion is lethal to some cancer cells, a vulnerability that can be exploited clinically. We report that resistance to asparagine starvation is uniquely dependent on an N-terminal low-complexity domain of GSK3α, which its paralog GSK3ß lacks. In response to depletion of specific amino acids, including asparagine, leucine, and valine, this domain mediates supramolecular assembly of GSK3α with ubiquitin-proteasome system components in spatially sequestered cytoplasmic bodies. This effect is independent of mTORC1 or GCN2. In normal cells, GSK3α promotes survival during essential amino acid starvation. In human leukemia, GSK3α body formation predicts asparaginase resistance, and sensitivity to asparaginase combined with a GSK3α inhibitor. We propose that GSK3α body formation provides a cellular mechanism to maximize the catalytic efficiency of proteasomal protein degradation in response to amino acid starvation, an adaptive response co-opted by cancer cells for asparaginase resistance.

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in ß-catenin capture, phosphorylation, and ubiquitylation.

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.

Multi-signal regulation of the GSK-3ß homolog Rim11 controls meiosis entry in budding yeast.

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3ß kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

DNA Damage Promotes TMPRSS2-ERG Oncoprotein Destruction and Prostate Cancer Suppression via Signaling Converged by GSK3ß and WEE1.

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.

ppGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During Starvation.

(p)ppGpp is a nucleotide messenger universally produced in bacteria following nutrient starvation. In E. coli, ppGpp inhibits purine nucleotide synthesis by targeting several different enzymes, but the physiological significance of their inhibition is unknown. Here, we report the structural basis of inhibition for one target, Gsk, the inosine-guanosine kinase. Gsk creates an unprecedented, allosteric binding pocket for ppGpp by restructuring terminal sequences, which restrains conformational dynamics necessary for catalysis. Guided by this structure, we generated a chromosomal mutation that abolishes Gsk regulation by ppGpp. This mutant strain accumulates abnormally high levels of purine nucleotides following amino-acid starvation, compromising cellular fitness. We demonstrate that this unrestricted increase in purine nucleotides is detrimental because it severely depletes pRpp and essential, pRpp-derived metabolites, including UTP, histidine, and tryptophan. Thus, our results reveal the significance of ppGpp's regulation of purine nucleotide synthesis and a critical mechanism by which E. coli coordinates biosynthetic processes during starvation.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism.

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.

Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3ß signalling.

ER-mitochondria contact sites (ERMCSs) regulate processes, including calcium homoeostasis, energy metabolism and autophagy. Previously, it was shown that during growth factor signalling, mTORC2/Akt gets recruited to and stabilizes ERMCSs. Independent studies showed that GSK3ß, a well-known Akt substrate, reduces ER-mitochondria connectivity by disrupting the VAPB-PTPIP51 tethering complex. However, the mechanisms that regulate ERMCSs are incompletely understood. Here we find that annulate lamellae (AL), relatively unexplored subdomains of ER enriched with a subset of nucleoporins, are present at ERMCSs. Depletion of Nup358, an AL-resident nucleoporin, results in enhanced mTORC2/Akt activation, GSK3ß inhibition and increased ERMCSs. Depletion of Rictor, a mTORC2-specific subunit, or exogenous expression of GSK3ß, was sufficient to reverse the ERMCS-phenotype in Nup358-deficient cells. We show that growth factor-mediated activation of mTORC2 requires the VAPB-PTPIP51 complex, whereas, Nup358's association with this tether restricts mTORC2/Akt signalling and ER-mitochondria connectivity. Expression of a Nup358 fragment that is sufficient for interaction with the VAPB-PTPIP51 complex suppresses mTORC2/Akt activation and disrupts ERMCSs. Collectively, our study uncovers a novel role for Nup358 in controlling ERMCSs by modulating the mTORC2/Akt/GSK3ß axis.

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

The inflammatory response mediated by nuclear factor κB (NF-κB) signaling is essential for host defense against pathogens. Although the regulatory mechanism of NF-κB signaling has been well studied, the molecular basis for epigenetic regulation of the inflammatory response is poorly understood. Here we identify a new signaling axis of PKCα-LSD1-NF-κB, which is critical for activation and amplification of the inflammatory response. In response to excessive inflammatory stimuli, PKCα translocates to the nucleus and phosphorylates LSD1. LSD1 phosphorylation is required for p65 binding and facilitates p65 demethylation, leading to enhanced stability. In vivo genetic analysis using Lsd1SA/SA mice with ablation of LSD1 phosphorylation and chemical approaches in wild-type mice with inhibition of PKCα or LSD1 activity show attenuated sepsis-induced inflammatory lung injury and mortality. Together, we demonstrate that the PKCα-LSD1-NF-κB signaling cascade is crucial for epigenetic control of the inflammatory response, and targeting this signaling could be a powerful therapeutic strategy for systemic inflammatory diseases, including sepsis.

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1.

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells in vivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRß-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRß-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRß-sufficient Tfh cells. Loss of LXRß confers inactivation of GSK3ß induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/ß-catenin pathway, leading to elevated TCF-1 expression in CD4+ T cells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ T cells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3ß-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

Immunometabolic crosstalk in Aedes fluviatilis and Wolbachia pipientis symbiosis.

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase ß (GSK3ß) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population.

Comparative analysis of BZR1/BES1 family transcription factors in Arabidopsis.

Brassinazole Resistant 1 (BZR1) and bri1 EMS Suppressor 1 (BES1) are key transcription factors that mediate brassinosteroid (BR)-responsive gene expression in Arabidopsis. The BZR1/BES1 family is composed of BZR1, BES1, and four BES1/BZR1 homologs (BEH1-BEH4). However, little is known about whether BEHs are regulated by BR signaling in the same way as BZR1 and BES1. We comparatively analyzed the functional characteristics of six BZR1/BES1 family members and their regulatory mechanisms in BR signaling using genetic and biochemical analyses. We also compared their subcellular localizations regulated by the phosphorylation status, interaction with GSK3-like kinases, and heterodimeric combination. We found that all BZR1/BES1 family members restored the phenotypic defects of bri1-5 by their overexpression. Unexpectedly, BEH2-overexpressing plants showed the most distinct phenotype with enhanced BR responses. RNA-Seq analysis indicated that overexpression of both BZR1 and BEH2 regulates BR-responsive gene expression, but BEH2 has a much greater proportion of BR-independent gene expression than BZR1. Unlike BZR1 and BES1, the BR-regulated subcellular translocation of the four BEHs was not tightly correlated with their phosphorylation status. Notably, BEH1 and BEH2 are predominantly localized in the nucleus, which induces the nuclear accumulation of other BZR1/BES1 family proteins through heterodimerization. Altogether, our comparative analyses suggest that BEH1 and BEH2 play an important role in the functional interaction between BZR1/BES1 family transcription factors.

Drosophila GSK3ß promotes microtubule disassembly and dendrite pruning in sensory neurons.

The evolutionarily conserved Glycogen Synthase Kinase 3ß (GSK3ß), a negative regulator of microtubules, is crucial for neuronal polarization, growth and migration during animal development. However, it remains unknown whether GSK3ß regulates neuronal pruning, which is a regressive process. Here, we report that the Drosophila GSK3ß homologue Shaggy (Sgg) is cell-autonomously required for dendrite pruning of ddaC sensory neurons during metamorphosis. Sgg is necessary and sufficient to promote microtubule depolymerization, turnover and disassembly in the dendrites. Although Sgg is not required for the minus-end-out microtubule orientation in dendrites, hyperactivated Sgg can disturb the dendritic microtubule orientation. Moreover, our pharmacological and genetic data suggest that Sgg is required to promote dendrite pruning at least partly via microtubule disassembly. We show that Sgg and Par-1 kinases act synergistically to promote microtubule disassembly and dendrite pruning. Thus, Sgg and Par-1 might converge on and phosphorylate a common downstream microtubule-associated protein(s) to disassemble microtubules and thereby facilitate dendrite pruning.

Sperm-specific glycogen synthase kinase 3 is required for sperm motility and the post-fertilization signal for female meiosis II in Caenorhabditis elegans.

In most sexually reproducing animals, sperm entry provides the signal to initiate the final stages of female meiosis. In Caenorhabditis elegans, this signal is required for completion of female anaphase I and entry into meiosis II (MII). memi-1/2/3 (meiosis-to-mitosis) encode maternal components that facilitate this process; memi-1/2/3(RNAi) results in a skipped-MII phenotype. Previously, we used a gain-of-function mutation, memi-1(sb41), to identify genetic suppressors that represent candidates for the sperm-delivered signal. Herein, we characterize two suppressors of memi-1(sb41): gskl-1 and gskl-2. Both genes encode functionally redundant sperm glycogen synthase kinase, type 3 (GSK3) protein kinases. Loss of both genes causes defects in male spermatogenesis, sperm pseudopod treadmilling and paternal-effect embryonic lethality. The two kinases locate within the pseudopod of activated sperm, suggesting that they directly or indirectly regulate the sperm cytoskeletal polymer major sperm protein (MSP). The GSK3 genes genetically interact with another memi-1(sb41) suppressor, gsp-4, which encodes a sperm-specific PP1 phosphatase, previously proposed to regulate MSP dynamics. Moreover, gskl-2 gsp-4; gskl-1 triple mutants often skip female MII, similar to memi-1/2/3(RNAi). The GSK3 kinases and PP1 phosphatases perform similar sperm-related functions and work together for post-fertilization functions in the oocyte that involve MEMI.

GSK3ß: A ray of hope for the treatment of diabetic kidney disease.

Diabetic kidney disease (DKD), a major microvascular complication of diabetes, is characterized by its complex pathogenesis, high risk of chronic renal failure, and lack of effective diagnosis and treatment methods. GSK3ß (glycogen synthase kinase 3ß), a highly conserved threonine/serine kinase, was found to activate glycogen synthase. As a key molecule of the glucose metabolism pathway, GSK3ß participates in a variety of cellular activities and plays a pivotal role in multiple diseases. However, these effects are not only mediated by affecting glucose metabolism. This review elaborates on the role of GSK3ß in DKD and its damage mechanism in different intrinsic renal cells. GSK3ß is also a biomarker indicating the progression of DKD. Finally, the protective effects of GSK3ß inhibitors on DKD are also discussed.

GSK805 inhibits alpha-smooth muscle expression and modulates liver inflammation without impairing the well-being of mice.

Cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), lead to inflammation and severe hepatic damage with limited therapeutic options. This study assessed the efficacy of the inverse RORγt agonist, GSK805, both in vitro using the hepatic stellate cell-line LX-2 and in vivo using male bile duct-ligated BALB/c mice. In vitro, 0.3 µM GSK805 reduced alpha-smooth muscle actin expression in LX-2 cells. In vivo, GSK805 significantly decreased IL-23R, TNF-α, and IFN-γ expression in cholestatic liver. Despite high concentrations of GSK805 in the liver, no significant reduction in fibrosis was noticed. GSK805 significantly increased aspartate aminotransferase and alanine aminotransferase activity in the blood, while levels of glutamate dehydrogenase, alkaline phosphatase, and bilirubin were not substantially increased. Importantly, GSK805 did neither increase an animal distress score nor substantially reduce body weight, burrowing activity, or nesting behavior. These results suggest that a high liver concentration of GSK805 is achieved by daily oral administration and that this drug modulates inflammation in cholestatic mice without impairing animal well-being.

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3ß decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.