ASXL1 (Additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS, OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, characteristic facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies including heart defects and severe skeletal defects giving rise to a typical ‘BOS posture’. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia (AML). We use primary cells from BOS individuals (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multi-omics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data shows that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, a planar cell polarity pathway protein that acts through non-canonical Wnt signaling to direct tissue patterning and cell migration. This multi-omics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.
Isabella Lin, Angela Wei, Zain Awamleh, Meghna Singh, Aileen Ning, Analeyla Herrera, Bianca E. Russell, Rosanna Weksberg, Valerie A. Arboleda
Viral illnesses like SARS-CoV-2 have pathologic effects on non-respiratory organs in the absence of direct viral infection. We injected mice with cocktails of rodent equivalents of human cytokine storms resulting from SARS-CoV-2 / COVID-19 or Rhinovirus common cold infection. At low doses, COVID-19 cocktails induced glomerular injury and albuminuria in Zhx2 hypomorph and Zhx2+/+ mice to mimic COVID-19 related proteinuria. Common Cold cocktail induced albuminuria selectively in Zhx2 hypomorph mice to model relapse of Minimal Change Disease (MCD), that improved after depletion of TNF-α or sIL-4Rα or IL-6. The Zhx2 hypomorph state increased cell membrane to nuclear migration of podocyte ZHX proteins in vivo (both cocktails) and lowered pSTAT6 activation (COVID-19 cocktail) in vitro. At higher doses, COVID-19 cocktails induced acute heart injury, myocarditis, pericarditis, acute liver injury and acute kidney injury, and high mortality in Zhx2+/+ mice, whereas Zhx2 hypomorph mice were relatively protected, due in part to early asynchronous activation of STAT5 and STAT6 pathways in these organs. Dual depletion of cytokine combinations of TNF-α with IL-2 or IL-13 or IL-4 in Zhx2+/+ mice reduced multiorgan injury and eliminated mortality. Using genome sequencing and CRISPR-Cas9, an insertion upstream of ZHX2 was identified as a cause of the human ZHX2 hypomorph state.
Maria Del Nogal Avila, Ranjan Das, Joubert B. Kharlyngdoh, Eduardo Molina-Jijon, Hector Donoro-Blazquez, Stéphanie Gambut, Michael R. Crowley, David K. Crossman, Rasheed A. Gbadagesin, Sunveer S. Chugh, Sunjeet S. Chugh, Carmen Avila-Casado, Camille Macé, Lionel C. Clement, Sumant S. Chugh
To improve our limited understanding of the pathogenesis of thoracic aortic aneurysm (TAA) leading to acute aortic dissection, single-cell RNA sequencing (scRNA-seq) was employed to profile disease-relevant transcriptomic changes of aortic cell populations in a well-characterized mouse model of the most commonly diagnosed form of Marfan syndrome (MFS). As result, two discrete sub-populations of aortic cells (SMC3 and EC4) were identified only in the aorta of Fbn1mgR/mgR mice. SMC3 highly express genes related to extracellular matrix formation and nitric oxide signaling, whereas EC4 transcriptional profile is enriched in SMC, fibroblast, and immune cell-related genes. Trajectory analysis predicted close phenotypic modulation between SMC3 and EC4, which were therefore analyzed together as a discrete MFS-modulated (MFSmod) sub-population. In situ hybridizations of diagnostic transcripts located MFSmod cells to the intima of Fbn1mgR/mgR aortas. Reference-based dataset integration revealed transcriptomic similarity between MFSmod and an SMC-derived cell cluster modulated in human TAA. Consistent with angiotensin II type I receptor (At1r) contribution to TAA development, MFSmod cells were absent in the aorta of Fbn1mgR/mgR mice treated with the At1r antagonist losartan. Altogether, our findings indicate that a discrete dynamic alteration of aortic cell identity is associated with dissecting TAA in MFS mice and increased risk of aortic dissection in MFS patients.
Yifei Sun, Keiichi Asano, Lauriane Sedes, Anna Cantalupo, Jens Hansen, Ravi Iyengar, Martin J. Walsh, Francesco Ramirez
Rationale. RNA binding protein 47 (RBM47) is required for embryonic endoderm development but a role in adult intestine is unknown. Objective. 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. Findings. Rbm47-IKO mice exhibit increased proliferation, abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapt to radiation injury and are 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 are protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice develop spontaneous polyposis and Rbm47-IKO, Apcmin/+ mice manifest an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue along with alternative splicing of TJP1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer, associated independently with decreased overall survival. Conclusions. 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
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) are capable of crossing the blood-brain barrier to induce widespread, long-term gene expression in the central nervous system (CNS), and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector employing a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived iPSC neurons and brain tissue from St3gal5 knock-out mice, but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either intracerebroventricular or intravenous route at postnatal day 1, 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 Brown, Dan Wang, Kevin A. Strauss, Guangping Gao
Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the two-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and reactive oxygen species accumulation in the central nervous system (CNS) and muscle, indicating mitochondrial dysfunction. Applied Therapeutics, Inc has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, iPSC-derived motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.
Yi Zhu, Amanda G. Lobato, Adriana P. Rebelo, Tijana Canic, Natalie Ortiz Vega, Xianzun Tao, Sheyum Syed, Christopher Yanick, Mario Saporta, Michael Shy, Riccardo Perfetti, Shoshana Shendelman, Stephan L. Zuchner, R. Grace Zhai
The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCS) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulations. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that is upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulates some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring's metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.
Soyoung Park, Amine M. Belfoul, Marialetizia Rastelli, Alice Jang, Magali Monnoye, Hosung Bae, Anna Kamitakahara, Patrick Giavalisco, Shan Sun, Pierre-Yves Barelle, Jasmine Plows, Cholsoon Jang, Anthony Fodor, Michael I. Goran, Sebastien G. Bouret
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 phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data has implied PS may compete with PIP2 binding on Kir2.1. We found 83.33% of Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate PS blocks PIP2 activation of Kir2.1, and 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 suggests PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and demonstrates the intracellular lipid localization within endothelium is an important determinant of vascular function.
Claire A. Ruddiman, Richard G. Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
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 RA patients albeit co-inducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, in order 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 is neither through its known targets Dopamine receptors 2 and 3 and Serotonin Receptor 7, nor through TNF-TNFRI binding inhibition. Through a click chemistry approach, novel potential 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 suffering from RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its anti-depressive 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, Alexis Ν. Matralis, George Kollias
Although the expression of Mex3 RNA binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HENCs) 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 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 p-Smad2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps (CRSsNP), 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 to 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, Chen Ao-Nan, 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
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