GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation

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Abstract

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause AAMR syndrome, which is characterized by achalasia, alacrima, mental retardation, and muscle weakness. Here we show that Gmppa KO mice recapitulate cognitive and motor deficits. As structural correlates we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identify GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhances mannose incorporation into glycoproteins including α-Dg in mice and men. This increases α-Dg turnover and thereby lowers α-Dg abundance. In mice dietary mannose restriction beginning after weaning corrects α-DG hyperglycosylation and abundance, normalizes skeletal muscle morphology, and prevents neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, are not improved. We thus identify GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, unravel underlying disease mechanisms and point to potential dietary treatment options.

Authors

Patricia Franzka, Henriette Henze, M. Juliane Jung, Svenja C. Schüler, Sonnhild Mittag, Karina Biskup, Lutz Liebmann, Takfarinas Kentache, José Morales, Braulio Martínez, Istvan Katona, Tanja Herrmann, Antje-Kathrin Huebner, J. Christopher Hennings, Susann Groth, Lennart J. Gresing, Rüdiger Horstkorte, Thorsten Marquardt, Joachim Weis, Christoph Kaether, Osvaldo M. Mutchinick, Alessandro Ori, Otmar Huber, Véronique Blanchard, Julia von Maltzahn, Christian A. Hübner

Pathogenic variants in TNNC2 cause congenital myopathy due to an impaired force response to calcium

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Abstract

Troponin C (TnC) is a critical regulator of skeletal muscle contraction: it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report two families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggest that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients’ biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, healthy TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients’ myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.

Authors

Martijn van de Locht, Sandra Donkervoort, Josine M. de Winter, Stefan Conijn, Leon Begthel, Benno Kusters, Payam Mohassel, Ying Hu, Livija Medne, Colin Quinn, Steven A. Moore, A. Reghan Foley, Gwimoon Seo, Darren T. Hwee, Fady I. Malik, Thomas Irving, Weikang Ma, Henk Granzier, Erik-Jan Kamsteeg, Kalyan Immadisetty, Peter Kekenes-Huskey, Jose Renato Pinto, Nicol Voermans, Carsten G. Bönnemann, Coen A.C. Ottenheijm

Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvβ6 upregulation following liver injury

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Abstract

Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. By using microdissection and next generation RNA sequencing, we identified five genes that were most upregulated in proliferating BECs after acute injury including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2. Immunohistochemistry analyses confirmed robust upregulation of integrin αvβ6 (ITGβ6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of Itgb6 gene attenuated BEC proliferation post-acute bile duct injury. Macrophage depletion or Ccr2-deficiency impaired ITGβ6 expression and BEC proliferation. In vitro experiments revealed that bile-acid activated monocytes promoted BEC proliferation through ITGβ6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGβ6, which work together to promote BEC proliferation, and that therefore represent potential therapeutic targets for cholangiopathies.

Authors

Adrien Guillot, Lucia Guerri, Dechun Feng, Seung-Jin Kim, Yeni Ait Ahmed, Janos Paloczi, Yong He, Kornel Schuebel, Shen Dai, Fengming Liu, Pal Pacher, Tatiana Kisseleva, Xuebin Qin, David Goldman, Frank Tacke, Bin Gao

HLA class I-associated expansion of TRBV11-2 T cells in Multisystem Inflammatory Syndrome in Children

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Abstract

Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vβ-chains results in Vβ-skewing, whereby T cells with specific Vβ-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Βeta Variable gene (TRBV)11-2, with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion showed HLA class I allele restriction to HLA-I A02, C35 and C04, indicating a novel mechanism for CDR3-independent T cell expansion. In silico modelling indicated that polyacidic residues in the Vβ chain encoded by TRBV11-2 strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Authors

Rebecca A. Porritt, Lisa Paschold, Magali Noval Rivas, Mary Hongying Cheng, Lael M. Yonker, Harsha Chandnani, Merrick Lopez, Donjete Simnica, Christoph Schultheiß, Chintda Santiskulvong, Jennifer van Eyk, John K. McCormick, Alessio Fasano, Ivet Bahar, Mascha Binder, Moshe Arditi

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates anti-tumor immune responses

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Abstract

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance, however, the mechanisms are not completely understood and thus therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and MDSCs, and increased CD8+ T-cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable anti-tumor immunity, was detected in mice administered a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a new strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

Authors

Fan Huang, Christophe Goncalves, Margarita Bartish, Joelle Rémy-Sarrazin, Mark E. Issa, Brendan Cordeiro, Qianyu Guo, Audrey Emond, Mikhael Attias, William Yang, Dany Plourde, Jie Su, Marina Godoy Gimeno, Yao Zhan, Alba Galán, Tomasz Rzymski, Milena Mazan, Magdalena Masiejczyk, Jacek Faber, Elie Khoury, Alexandre Benoit, Natascha Gagnon, David Dankort, Fabrice Journe, Ghanem Ghanem, Connie M. Krawczyk, H. Uri Saragovi, Ciriaco A. Piccirillo, Nahum Sonenberg, Ivan Topisirovic, Christopher E. Rudd, Wilson H. Miller Jr., Sonia V. del Rincón

Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation

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Abstract

Although cancer cells are frequently faced with nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anti-cancer strategy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) was downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low glucose condition. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo UTP synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

Authors

Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang

Metabolic rerouting via SCD1 induction impacts X-linked adrenoleukodystrophy

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Abstract

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift towards mono-unsaturated fatty acids relieved toxicity. In human ALD fibroblasts chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts whereas mono-unsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated towards mono-unsaturated VLCFA, and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to mono-unsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.

Authors

Quentin Raas, Malu-Clair van de Beek, Sonja Forss-Petter, Inge M.E. Dijkstra, Abigail DeSchiffart, Briana C. Freshner, Tamara J. Stevenson, Yorrick R.J. Jaspers, Liselotte M. Nagtzaam, Ronald J.A. Wanders, Michel van Weeghel, Joo-Yeon Engelen-Lee, Marc Engelen, Florian Eichler, Johannes Berger, Joshua L. Bonkowsky, Stephan Kemp

Age-specific effects of vaccine egg-adaptation and immune priming on A(H3N2) antibody responses following influenza vaccination

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Abstract

A(H3N2) Influenza vaccine effectiveness (VE) were low during 2016-2019 seasons and varied by age. We analyzed neutralizing antibody responses to egg- and cell-propagated vaccine and circulating viruses following vaccination in 375 individuals (aged 7 months to 82 years) across all vaccine eligible age groups in 3 influenza seasons. Antibody responses to cell- compared to egg-propagated vaccine viruses were significantly reduced due to egg-adapted changes T160K, D225G, and L194P in the vaccine hemagglutinins. Vaccine egg-adaptation had differential impact on antibody responses across different age groups. Immunologically naive children immunized with egg-adapted vaccines mostly mounted antibodies targeting egg-adapted epitopes, whereas those previously primed with infection produced broader responses even when vaccinated with egg-based vaccines. In elderly, repeated boost of vaccine egg-adapted epitopes significantly reduced antibody responses to the wild type cell-grown viruses. Analysis with reverse genetics viruses suggested that the response to each egg-adapted substitution varied by age. Antibody responses did not differ in male versus female vaccinees. Here, the combination of age-specific responses to vaccine egg-adapted substitutions, diverse host immune priming histories and virus antigenic drift impacted antibody responses following vaccination and may have led to the low and variable VE against A(H3N2) viruses across different age groups.

Authors

Feng Liu, F. Liaini Gross, Stacie N. Jefferson, Crystal Holiday, Yaohui Bai, Li Wang, Bin Zhou, Min Z. Levine

SUMOylation promotes extracellular vesicle-mediated transmission of lncRNA ELNAT1 and lymph node metastasis in bladder cancer

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Abstract

SUMOylation emerged as the inducer for the sorting of bioactive molecules into extracellular vesicles (EVs) triggering lymphangiogenesis, further driving tumor lymph node (LN) metastasis, but the precise mechanisms remain largely unclear. Herein, we identified that bladder cancer (BCa) cell-secreted EVs mediated the intercellular communication with human lymphatic endothelial cells (HLECs) through the transmission of a long noncoding RNA ELNAT1, and promoted lymphangiogenesis and LN metastasis in a SUMOylation-dependent manner in both cultured BCa cell lines and mouse models. Mechanistically, ELNAT1 induced UBC9 overexpression to catalyze the SUMOylation of hnRNPA1 at lysine-113 residue, which mediated the recognition of ELNAT1 by endosomal sorting complex required for transport (ESCRT) and facilitated their packaging into EVs. EV-mediated ELNAT1 was specifically transmitted into HLECs and epigenetically activated SOX18 transcription to induce lymphangiogenesis. Importantly, blocking the SUMOylation of tumor by downregulating UBC9 expression markedly reduced lymphatic metastasis in EV-mediated ELNAT1-treated BCa in vivo. Clinically, EV-mediated ELNAT1 was correlated with LN metastasis and poor prognosis of patients with BCa. These findings highlight a molecular mechanism that EV-mediated ELNAT1/UBC9/SOX18 regulatory axis promotes the lymphangiogenesis and LN metastasis of BCa in a SUMOylation-dependent manner, and implicate ELNAT1 as an attractive therapeutic target for LN metastatic BCa.

Authors

Changhao Chen, Hanhao Zheng, Yuming Luo, Yao Kong, Mingjie An, Yuting Li, Wang He, Bowen Gao, Yue Zhao, Hao Huang, Jian Huang, Tianxin Lin

Macrophage SR-BI modulates autophagy via VPS34 complex and PPARα transcription of Tfeb in atherosclerosis

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Abstract

Autophagy modulates lipid turnover, cell survival, inflammation and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34/Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescues the defective autophagy in Sr-b1-/- macrophages. Taken together, macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34/Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and novel targets for the treatment of atherosclerosis.

Authors

Huan Tao, Patricia G. Yancey, John L. Blakemore, Youmin Zhang, Lei Ding, W. Gray Jerome, Jonathan D. Brown, Kasey C. Vickers, MacRae F. Linton