Synovial fibroblasts (SFs) are key pathogenic drivers in rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models, and TNF blockade proved efficacious for a high percentage of patients with RA albeit coinducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF–transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely amisulpride, which reduced SFs’ inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that amisulpride function was neither through its known targets dopamine receptors D2 and D3 and serotonin receptor 7 nor through TNF–TNF receptor I binding inhibition. Through a click chemistry approach, potentially novel targets of amisulpride were identified, which were further validated to repress hTNFtg SFs’ inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, amisulpride could prove beneficial to patients experiencing RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its antidepressive activity, serving further as a “lead” compound for the development of novel therapeutics against fibroblast activation.
Dimitra Papadopoulou, Fani Roumelioti, Christos Tzaferis, Panagiotis Chouvardas, Anna-Kathrine Pedersen, Filippos Charalampous, Eleni Christodoulou-Vafeiadou, Lydia Ntari, Niki Karagianni, Maria C. Denis, Jesper V. Olsen, Alexios N. Matralis, George Kollias
RNA-binding protein 47 (RBM47) is required for embryonic endoderm development, but a role in adult intestine is unknown. We studied intestine-specific Rbm47-knockout mice (Rbm47-IKO) following intestinal injury and made crosses into ApcMin/+ mice to examine alterations in intestinal proliferation, response to injury, and tumorigenesis. We also interrogated human colorectal polyps and colon carcinoma tissue. Rbm47-IKO mice exhibited increased proliferation and abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapted to radiation injury and were protected against chemical-induced colitis, with Rbm47-IKO intestine showing upregulation of antioxidant and Wnt signaling pathways as well as stem cell and developmental genes. Furthermore, Rbm47-IKO mice were protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice developed spontaneous polyposis, and Rbm47-IKO ApcMin/+ mice manifested an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue, along with alternative splicing of tight junction protein 1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer associated independently with decreased overall survival. These findings implicate RBM47 as a cell-intrinsic modifier of intestinal growth, inflammatory, and tumorigenic pathways.
Saeed Soleymanjahi, Valerie Blanc, Elizabeth A. Molitor, David M. Alvarado, Yan Xie, Vered Gazit, Jeffrey W. Brown, Kathleen Byrnes, Ta-Chiang Liu, Jason C. Mills, Matthew A. Ciorba, Deborah C. Rubin, Nicholas O. Davidson
Although the expression of Mex3 RNA-binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HNECs) predominately in the eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) subtype, its functions as an RNA binding protein in airway epithelial cells remain unknown. Here, we revealed the role of MEX3B based on different subtypes of CRS and demonstrated that MEX3B decreased the TGF-β receptor III (TGFBR3) mRNA level by binding to its 3′ UTR and reducing its stability in HNECs. TGF-βR3 was found to be a TGF-β2–specific coreceptor in HNECs. Knocking down or overexpressing MEX3B promoted or inhibited TGF-β2–induced phosphorylation of SMAD2 in HNECs, respectively. TGF-βR3 and phosphorylated SMAD2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps with a more prominent downregulation in the eosinophilic CRSwNP. TGF-β2 promoted collagen production in HNECs. Collagen abundance decreased and edema scores increased in CRSwNP compared with control, again more prominently in the eosinophilic type. Collagen expression in eosinophilic CRSwNP was negatively correlated with MEX3B but positively correlated with TGF-βR3. These results suggest that MEX3B inhibits tissue fibrosis in eosinophilic CRSwNP by downregulating epithelial cell TGFBR3 expression; consequently, MEX3B might be a valuable therapeutic target against eosinophilic CRSwNP.
Jin-Xin Liu, Ao-Nan Chen, Qihong Yu, Ke-Tai Shi, Yi-Bo Liu, Cui-Lian Guo, Zhe-Zheng Wang, Yin Yao, Li Pan, Xiang Lu, Kai Xu, Heng Wang, Ming Zeng, Chaohong Liu, Robert P. Schleimer, Ning Wu, Bo Liao, Zheng Liu
Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.
James N. Sleigh, David Villarroel-Campos, Sunaina Surana, Tahmina Wickenden, Yao Tong, Rebecca L. Simkin, Jose Norberto S. Vargas, Elena R. Rhymes, Andrew P. Tosolini, Steven J. West, Qian Zhang, Xiang-Lei Yang, Giampietro Schiavo
GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) can cross the blood-brain barrier to induce widespread, long-term gene expression in the CNS and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector using a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived induced pluripotent stem cell neurons and brain tissue from St3gal5-KO mice but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either the intracerebroventricular or i.v. route at P1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.
Huiya Yang, Robert H. Brown Jr., Dan Wang, Kevin A. Strauss, Guangping Gao
Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.
Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron
Vascular smooth muscle–derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM–derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM–to–myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1–induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM–to–myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 — an inwardly rectifying potassium channel that regulates membrane hyperpolarization — contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.
Claire A. Ruddiman, Richard Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, P. Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which — when combined with other genetic lesions — result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a–/– HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a–/– LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation–driven myeloid malignancies.
Lakshmi Reddy Palam, Baskar Ramdas, Katelyn Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce Selman, Utpal P. Dave, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur
Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2′-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.
Alexandre Derre, Noelian Soler, Valentine Billoux, Sebastien Benizri, Brune Vialet, Cyril Rivat, Philippe Barthélémy, Patrick Carroll, Alexandre Pattyn, Stephanie Venteo
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