Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize UDP-GlcNAc. In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-L-norleucine or DON). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cell. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM, and an increased infiltration CD8+ T-cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy resulting in tumor regression and prolonged survival.
Nikita S. Sharma, Vineet K. Gupta, Vanessa T. Garrido, Roey Hadad, Brittany C. Durden, Kousik Kesh, Bhuwan Giri, Anthony Ferrantella, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee
Background: Proteinuria is considered as an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear if all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS) featured by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding. Methods: We used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling and epidemiological methods. Results: We identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene are associated with chronic isolated proteinuria with childhood onset. Since the proteinuria displayed a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and proteinuria-lowering treatments. Yet, renal function was normal in all cases. By contrast, we did not find any biallelic pathogenic CUBN variants in patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 out of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that four C-terminal CUBN variants are associated with albuminuria and moderately increased GFR in meta-analyses of large population-based cohorts. Conclusions: Collectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsies.
Mathilda Bedin, Olivia Boyer, Aude Servais, Yong Li, Laure Villoing-Gaudé, Marie-Josephe Tête, Alexandra Cambier, Julien Hogan, Veronique Baudouin, Saoussen Krid, Albert Bensman, Florie Lammens, Ferielle Louillet, Bruno Ranchin, Cecile Vigneau, Iseline Bouteau, Corinne Isnard-Bagnis, Christoph J. Mache, Tobias Schäfer, Lars Pape, Markus Gödel, Tobias B. Huber, Marcus Benz, Günter Klaus, Matthias Hansen, Kay Latta, Olivier Gribouval, Vincent Morinière, Carole Tournant, Maik Grohmann, Elisa Kuhn, Timo Wagner, Christine Bole-Feysot, Fabienne Jabot-Hanin, Patrick Nitschké, Tarunveer S. Ahluwalia, Anna Köttgen, Christian Brix Folsted Andersen, Carsten Bergmann, Corinne Antignac, Matias Simons
Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell–specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling and anti–PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.
Heather A. Himburg, Martina Roos, Tiancheng Fang, Yurun Zhang, Christina M. Termini, Lauren Schlussel, Mindy M. Kim, Amara Pang, Jenny Kan, Liman Zhao, Hyung Suh, Joshua P. Sasine, Gopal Sapparapu, Peter M. Bowers, Gary Schiller, John P. Chute
Novel approaches for adjunctive therapy are urgently needed for infections complicated by antibiotic-resistant pathogens and for patients with compromised immunity. Necrotizing fasciitis (NF) is a destructive skin and soft tissue infection. Despite treatment with systemic antibiotics and radical debridement of necrotic tissue, lethality remains high. The key iron regulatory hormone hepcidin was originally identified as a cationic antimicrobial peptide (AMP), but its putative expression and role in the skin, a major site of AMP production, has never been investigated. We report here that hepcidin production is induced in the skin of patients with Group A Streptococcal (GAS) NF. In a GAS-induced NF model, mice lacking hepcidin in keratinocytes failed to restrict systemic spread of infection from an initial tissue focus. Unexpectedly, this effect was due its ability to promote production of the CXCL1 chemokine by keratinocytes resulting in neutrophil recruitment. Unlike CXCL1, hepcidin is resistant to degradation by major GAS proteases and could therefore serve as a reservoir to maintain steady state levels of CXCL1 in infected tissue. Finally, injection of synthetic hepcidin at the site of infection can limit or completely prevent systemic spread of GAS infection suggesting that hepcidin agonists could have a therapeutic role in NF.
Mariangela Malerba, Sabine Louis, Sylvain Cuvellier, Srikanth Mairpady Shambat, Camille Hua, Camille Gomart, Agnès Fouet, Nicolas Ortonne, Jean-Winoc Decousser, Annelies S. Zinkernagel, Jacques R.R. Mathieu, Carole Peyssonnaux
Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context-dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell-independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.
Karim Mahiddine, Adam Blaisdell, Stephany Ma, Amandine Créquer-Grandhomme, Clifford A. Lowell, Adrian Erlebacher
Patients with bladder cancer (BCa) with clinical lymph node (LN) metastasis have extremely poor prognosis. VEGF-C has been demonstrated to play vital roles in LN metastasis in BCa. However, approximately 20% of BCa with LN metastasis exhibits low VEGF-C expression, suggesting a VEGF-C-independent mechanism for LN metastasis of BCa. Herein, we demonstrated that BCa cell-secreted exosomes-mediated lymphangiogenesis promoted LN metastasis in BCa, which was in a VEGF-C-independent manner. We identified an exosomal long noncoding RNA (lncRNA), termed lymph node metastasis-associated transcript 2 (LNMAT2), stimulated HLEC tube formation and migration in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo. Mechanistically, LNMAT2 was loaded to BCa cell-secreted exosomes by directly interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). Subsequently, exosomal LNMAT2 was internalized by HLECs and epigenetically upregulated prospero homeobox 1 (PROX1) expression by recruitment of hnRNPA2B1 and increasing the H3K4 trimethylation level in the PROX1 promoter, ultimately resulting in lymphangiogenesis and lymphatic metastasis. Therefore, our findings highlight a VEGF-C-independent mechanism of exosomal lncRNA-mediated LN metastasis and identify LNMAT2 as a therapeutic target for LN metastasis in BCa.
Changhao Chen, Yuming Luo, Wang He, Yue Zhao, Yao Kong, Hongwei Liu, Guangzheng Zhong, Yuting Li, Jun Li, Jian Huang, Rufu Chen, Tianxin Lin
Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly-identified highly-selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted-radiotherapy on human orthotopic lung tumors without influencing acute DNA-damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and toxicity of a parenterally-administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.
Catherine E. Willoughby, Yanyan Jiang, Huw D. Thomas, Elaine Willmore, Suzanne Kyle, Anita Wittner, Nicole Phillips, Yan Zhao, Susan J. Tudhope, Lisa Prendergast, Gesa Junge, Luiza Madia Lourenco, M. Raymond V. Finlay, Paul Turner, Joanne M. Munck, Roger J. Griffin, Tommy Rennison, James Pickles, Celine Cano, David R. Newell, Helen L. Reeves, Anderson J. Ryan, Stephen R. Wedge
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40% to 50% of adults with CF. The age-at-onset of CF-related diabetes (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age-at-onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that two common DNA haplotypes formed by the risk variants account for the association with diabetes (high risk, P-value: 4.34E-3; low risk, P-value: 1.14E-3). Single-cell RNA (scRNA) sequencing indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells, and frequently co-expressed with CFTR along with transcription factors that have binding sites 5′ of SLC26A9. These findings replicated upon re-analysis of scRNA data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9 bearing variants from the low risk haplotype generated 12% to 20% higher levels of expression in PANC-1 and CFPAC-1 cells compared to the high risk haplotype (P-values: 2.00E-3 to 5.15E-9). Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age-at-onset of diabetes, thereby suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Although CD4 T cells are implicated in MS pathogenesis and have been the main focus of MS research using the animal model experimental autoimmune encephalomyelitis (EAE), substantial evidence from patients with MS points to a role for CD8 T cells in disease pathogenesis. We previously showed that an MHC class I-restricted epitope of myelin basic protein (MBP) is presented in the CNS during CD4 T cell-initiated EAE. Here, we investigated whether naïve MBP-specific CD8 T cells recruited to the CNS during CD4 T cell-initiated EAE engaged in determinant-spreading and influenced disease. We found that the MBP-specific CD8 T cells exacerbated brain but not spinal cord inflammation. We show that a higher frequency of monocytes and monocyte-derived cells presented the MHC class I-restricted MBP ligand in the brain compared to the spinal cord. Infiltration of MBP-specific CD8 T cells enhanced ROS production in the brain only in these cell-types and only when the MBP-specific CD8 T cells expressed Fas ligand (FasL). These results suggest that myelin-specific CD8 T cells may contribute to disease pathogenesis via a FasL-dependent mechanism that preferentially promotes lesion formation in the brain.
Catriona A. Wagner, Pamela J. Roqué, Trevor R. Mileur, Denny Liggitt, Joan M. Goverman
Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing, and 3D tissue profiling, we discovered a new brown adipocyte subpopulation with low thermogenic activity co-existing with the classical high thermogenic brown adipocytes within the BAT. These low thermogenic brown adipocytes had significantly lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that the low thermogenic brown adipocytes have significant lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the two brown adipocyte subpopulations underwent dynamic inter-conversions. Cold exposure converted low thermogenic brown adipocytes into high thermogenic cells, and a thermoneutral environment had the opposite effect. This recruitment of high thermogenic brown adipocytes by cold stimulation is not affected by high fat diet feeding, but significantly declined with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.
Anying Song, Wenting Dai, Min Jee Jang, Leonard Medrano, Zhuo Li, Hu Zhao, Mengle Shao, Jiayi Tan, Aimin Li, Tinglu Ning, Marcia M. Miller, Brian Armstrong, Janice M. Huss, Yi Zhu, Yong Liu, Viviana Gradinaru, Xiwei Wu, Lei Jiang, Philipp E. Scherer, Qiong A. Wang
Catecholamines released by sympathetic nerves can activate adrenergic receptors present on nearly every cell type, including myeloid derived suppressor cells (MDSCs). Using in vitro systems and murine tumor models, in wild-type mice and genetically modified (β2-AR–/–) mice, as well adoptive transfer approaches, we found that the degree of β2-AR signaling significantly influences MDSC frequency and survival in tumors and other tissues, modulates their expression of immunosuppressive molecules such as arginase-I and PDL-1 and alters their ability to suppress the proliferation of T cells. The regulatory functions of β-AR signaling in MDSCs were found to be dependent upon STAT3 phosphorylation. Moreover, we observed that the β2-AR-mediated increase in survival of MDSCs is dependent upon Fas-FasL interactions, and this is consistent with gene expression analyses which reveal a greater expression of apoptosis-related genes in β2-AR–/– MDSCs. Our data reveals the potential of β2-AR signaling to increase the generation of MDSCs from both murine and human peripheral blood cells and that the immunosuppressive function of MDSCs could be mitigated by treatment with β-AR antagonists, or enhanced by β-AR agonists, strongly supporting the possibility that reducing stress-induced activation of β2-ARs could help to overcome immune suppression and enhance the efficacy of immunotherapy and other cancer therapies.
Hemn Mohammadpour, Cameron R. MacDonald, Guanxi Qiao, Minhui Chen, Bowen Dong, Bonnie L. Hylander, Philip L. McCarthy, Scott I. Abrams, Elizabeth A. Repasky
Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly-inherited diseases—where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function (GoF) allele—is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapeutic for Charcot-Marie-Tooth type 2D (CMT2D), caused by dominant mutations in glycyl tRNA-synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks-of-age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least one year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof-of-concept for gene therapy approaches for dominant neuromuscular diseases.
Kathryn H. Morelli, Laurie B. Griffin, Nettie K. Pyne, Lindsay M. Wallace, Allison M. Fowler, Stephanie N. Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O. Kitzman, Samuel G. Kocen, Timothy J. Hines, Emily L. Spaulding, James R. Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J. Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q. Harper, Robert W. Burgess
Arcuate nucleus agouti-related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs and other behaviors implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. Activity of these neurons increased upon food availability and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST nociceptin/GABAergic neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and non-homeostatic neuronal inputs.
Mark A. Smith, Agharul I. Choudhury, Justyna A. Glegola, Paulius Viskaitis, Elaine E. Irvine, Pedro Caldas Custodio de Campos Silva, Sanjay Khadayate, Hanns Ulrich Zeilhofer, Dominic J. Withers
Currently, an effective targeted therapy for pancreatitis is still lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1R122Hgene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1R122H protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1R122H mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1R122H mice with PRSS1WT mice as well as enzymatically inactivated Dead-PRSS1R122H mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anti-coagulation therapy, synergistically prevented the progression to CP in PRSS1R122H mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.
Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji
Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type (AT)1 cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein (EMP)2, a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells, but has no known function in lung biology. Here, we show that Emp2–/– mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2–/– mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labelling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2–/– lungs. Emp2–/– AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.
Wan-Chi Lin, Kymberly M. Gowdy, Jennifer H. Madenspacher, Rachel L. Zemans, Kazuko Yamamoto, Miranda R. Lyons-Cohen, Hideki Nakano, Kyathanahalli Janardhan, Carmen J. Williams, Donald N. Cook, Joseph P. Mizgerd, Michael B. Fessler
Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDS), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in SSBP1 in five unrelated families, leading to the R38Q and R107Q amino-acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a new revised SSBP1 crystal structure. Structural analysis suggests that both mutations affect dimer interactions and presumably distort the DNA binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication and induces mtDNA depletion. Our study, showing that mutations in SSBP1 cause a novel form of dominant optic atrophy frequently accompanied with foveopathy, brings new insights into mtDNA maintenance disorders.
Camille Piro-Mégy, Emmanuelle Sarzi, Aleix Tarrés-Solé, Marie Péquignot, Fenna Hensen, Mélanie Quilès, Gaël Manes, Arka Chakraborty, Audrey Sénéchal, Béatrice Bocquet, Chantal Cazevieille, Agathe Roubertie, Agnès Müller, Majida Charif, David Goudenège, Guy Lenaers, Helmut Wilhelm, Ulrich Kellner, Nicole Weisschuh, Bernd Wissinger, Xavier Zanlonghi, Christian Hamel, Johannes N. Spelbrink, Maria Solà, Cécile Delettre
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single strand binding protein (SSBP1) in four families with dominant and one with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect its amount and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids and 7S-DNA amounts as well as mtDNA replication, impacting replisome machinery. The variable mtDNA depletion in cells reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits and complexes amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex-vivo in biopsies of affected tissues, like kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by wild-type mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as cause of human pathology.
Valentina Del Dotto, Farid Ullah, Ivano Di Meo, Pamela Magini, Mirjana Gusic, Alessandra Maresca, Leonardo Caporali, Flavia Palombo, Francesca Tagliavini, Evan H. Baugh, Bertil Macao, Zsolt Szilagyi, Camille Péron, Margaret A. Gustafson, Kamal Khan, Chiara La Morgia, Piero Barboni, Michele Carbonelli, Maria Lucia Valentino, Rocco Liguori, Vandana Shashi, Jennifer A. Sullivan, Shashi Nagaraj, Mays El-Dairi, Alessandro Iannaccone, Ioana Cutcutache, Enrico Bertini, Rosalba Carrozzo, Francesco Emma, Francesca Diomedi-Camassei, Claudia Zanna, Martin Armstrong, Matthew J Page, Sylvia Boesch, Saskia B. Wortmann, Robert Kopajtich, Nicholas Stong, Wolfgang Sperl, Erica Davis, William C. Copeland, Marco Seri, Maria Falkenberg, Holger Prokisch, Nicholas Katsanis, Valeria Tiranti, Tommaso Pippucci, Valerio Carelli
Manganese (Mn), an essential metal and nutrient, is toxic in excess. Toxicity classically results from inhalational exposures in individuals working in industrial settings. Identified in 2012, the first known disease of inherited Mn excess is caused by mutations in the metal exporter SLC30A10 and is characterized by Mn excess, dystonia, cirrhosis, and polycythemia. To investigate the role of SLC30A10 in Mn homeostasis, we first generated mice with whole body Slc30a10 deficiency, which developed severe Mn excess and impaired systemic and biliary Mn excretion. Slc30a10 localized to canalicular membrane of hepatocytes, but mice with liver Slc30a10 deficiency developed minimal Mn excess despite impaired biliary Mn excretion. Slc30a10 also localized to the apical membrane of enterocytes, but mice with Slc30a10 deficiency in small intestines developed minimal Mn excess despite impaired Mn export into the lumen of the small intestines. Finally, mice with Slc30a10 deficiency in liver and small intestines developed Mn excess less severe than that observed in mice with whole body Slc30a10 deficiency, suggesting that additional sites of Slc30a10 expression contribute to Mn homeostasis. Overall, these results indicated that Slc30a10 is essential for Mn excretion and could be an effective target for pharmacological intervention for Mn toxicity.
Courtney J. Mercadante, Milankumar Prajapati, Heather L. Conboy, Miriam E. Dash, Carolina Herrera, Michael A. Pettiglio, Layra Cintron-Rivera, Madeleine A. Salesky, Deepa B. Rao, Thomas B. Bartnikas
The Microphthalmia family of transcription factors (MiT/TFE) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via up-regulated expression and activity of MiT/TFEs, while genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE down-regulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling.
Kaushal Asrani, Sanjana Murali, Brandon Lam, Chan-Hyun Na, Pornima Phatak, Akshay Sood, Harsimar Kaur, Zoya Khan, Michaël Noë, Ravi K. Anchoori, C. Conover Talbot Jr., Barbara Smith, Michael Skaro, Tamara L. Lotan
Myocardin (MYOCD) is the founding member of a class of transcriptional co-activators that bind serum response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies and, to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by co-segregation of MYOCD variants with the phenotype in four unrelated families, by in vitro transactivation studies where pathogenic variants resulted in abrogated SM gene expression, and finding megabladder in two distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.
Arjan C. Houweling, Glenda M. Beaman, Alex V. Postma, T. Blair Gainous, Klaske D. Lichtenbelt, Francesco Brancati, Filipa M. Lopes, Ingeborg van der Made, Abeltje M. Polstra, Michael L. Robinson, Kevin D. Wright, Jamie M. Ellingford, Ashley R. Jackson, Eline Overwater, Rita Genesio, Silvio Romano, Letizia Camerota, Emanuela D'Angelo, Elizabeth J. Meijers-Heijboer, Vincent M. Christoffels, Kirk M. McHugh, Brian L. Black, William G. Newman, Adrian S. Woolf, Esther E. Creemers