Spinal muscular atrophy (SMA) is a neuromuscular disorder due to degeneration of spinal cord motor neurons caused by the deficiency of the ubiquitously expressed SMN protein. Here, we present a retinal vascular defect in patients, recapitulated in SMA transgenic mice, driven by failure of angiogenesis and maturation of blood vessels. Importantly, the retinal vascular phenotype was rescued by early, systemic SMN restoration therapy in SMA mice. We also demonstrate in patients an unfavourable imbalance between endothelial injury and repair, as indicated by increased circulating endothelial cell counts and decreased endothelial progenitor cell counts in blood circulation. The cellular markers of endothelial injury were associated with disease severity and improved following SMN restoration treatment in cultured endothelial cells from patients. Finally, we demonstrated autonomous defects in angiogenesis and blood vessel formation, secondary to SMN deficiency in cultured human and mouse endothelial cells, as the underlying cellular mechanism of microvascular pathology. Our cellular and vascular biomarkers findings indicate microvasculopathy as a fundamental feature of SMA. Our findings provide mechanistic insights into previously described SMA microvascular complications, and highlight the functional role of SMN in the periphery, including the vascular system, where deficiency of SMN can be addressed by systemic SMN-restoring treatment.
Haiyan Zhou, Ying Hong, Mariacristina Scoto, Alison Thomson, Emma Pead, Tom MacGillivray, Elena Hernandez-Gerez, Francesco Catapano, Jinhong Meng, Qiang Zhang, Gillian Hunter, Hannah K. Shorrock, Thomas K. Ng, Abedallah Hamida, Mathilde Sanson, Giovanni Baranello, Kevin Howell, Thomas H. Gillingwater, Paul Brogan, Dorothy A. Thompson, Simon H. Parson, Francesco Muntoni
Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we described a single nucleotide mutation in exon 2 of the methyltransferase like 23 (METTL23) gene identified in a three-generation Japanese NTG family. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23 knock-in (Mettl23+/G & Mettl23G/G) and knockout (Mettl23+/- & Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced Mettl23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that Mettl23 catalyzed the dimethylation of H3R17 in the retina, and was required for the transcription of pS2, an estrogen receptor α target gene that was critical to RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α/IL-1β feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.
Yang Pan, Akiko Suga, Itaru Kimura, Chojiro Kimura, Yuriko Minegishi, Mao Nakayama, Kazutoshi Yoshitake, Daisuke Iejima, Naoko Minematsu, Megumi Yamamoto, Fumihiko Mabuchi, Mitsuko Takamoto, Yukihiro Shiga, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Toru Nakazawa, Takeshi Iwata
The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells, to suppress MAPK pathway signaling that in turn blocked indirect (gamma interferon release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ regulated gene transcription and lead to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL that resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell specific immune checkpoint, which is responsible for NK cell associated poor outcomes in many cancers.
Huw J. Morgan, Elise Rees, Simone Lanfredini, Kate A. Powell, Jasmine Gore, Alex Gibbs, Charlotte Lovatt, Gemma E. Davies, Carlotta Olivero, Boris Y. Shorning, Giusy Tornillo, Alex Tonks, Richard Darley, Eddie C.Y. Wang, Girish K. Patel
Graft-versus-host disease (GVHD), manifesting in either acute (aGVHD) or chronic (cGVHD), presents significant life-threatening complications following allogeneic hematopoietic cell transplantation. Here, we investigated Friend Virus Leukemia Integration 1 (Fli-1) in GVHD pathogenesis and validated Fli-1 as a therapeutic target. Using genetic approaches, we found that Fli-1 dynamically regulates different T-cell subsets in allogeneic responses and pathogenicity in the development of aGVHD and cGVHD. Compared to homozygous Fli1-deficient or WT T cells, heterozygous Fli1-deficient T cells induced the mildest GVHD supported by lowest Th1 and Th17 differentiation. Single-cell RNA sequencing analysis revealed that Fli-1 differentially regulated CD4 versus CD8 T-cell response. Fli-1 promoted the transcription of Th1/Th17-pathways and TCR-inducible transcription factors in CD4 T cells, while suppressing activation and function-related gene pathways in CD8 T cells. Importantly, low-dose of camptothecin, topotecan or etoposide exhibited action as potent Fli-1 inhibitors and significantly attenuated GVHD severity while preserving the graft-versus-leukemia (GVL) effect. The observation was extended to a xenograft model where GVHD was induced by human T cells. In conclusion, we provide the evidence that Fli-1 plays a crucial role in alloreactive CD4+ T-cell activation and differentiation, and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising GVL effect.
Steven Schutt, Yongxia Wu, Arjun Kharel, David Bastian, Hee-Jin Choi, M. Hanief Sofi, Corey Mealer, Brianyell McDaniel Mims, Hung Nguyen, Chen Liu, Kris Helke, Weiguo Cui, Xian Zhang, Yaacov Ben-David, Xue-Zhong Yu
SAMD9 and SAMD9L germline mutations have recently emerged as a new class of predispositions to pediatric myeloid neoplasms. Patients commonly have impaired hematopoiesis, hypocellular marrows, and a greater risk of developing clonal chromosome 7 deletions leading to MDS and AML. We recently demonstrated that expressing SAMD9 or SAMD9L mutations in hematopoietic cells suppresses their proliferation and induces cell death. Here we generated a mouse model that conditionally expresses mutant Samd9l to assess the in vivo impact on hematopoiesis. Using a range of in vivo and ex vivo assays, we showed that cells with heterozygous Samd9l mutations have impaired stemness relative to wild-type counterparts, which was exacerbated by inflammatory stimuli, and ultimately led to bone marrow hypocellularity. Genomic and phenotypic analyses recapitulated many of the hematopoietic cellular phenotypes observed in patients with SAMD9 or SAMD9L mutations, including lymphopenia, and pinpointed TGF-β as a potential targetable pathway. Further, we observed non-random genetic deletion of the mutant Samd9l locus on mouse chromosome 6, mimicking chromosome 7 deletions observed in patients. Collectively, our study has enhanced our understanding of mutant Samd9l hematopoietic phenotypes, emphasized the synergistic role of inflammation in exaggerating the associated hematopoietic defects, and provided insights into potential therapeutic options for patients.
Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco
Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis; yet, little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMC), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both AngII- and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMC resulted in the loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its co-activator P300 and the SWI/SNF complex, suppressed VSMC inflammation by promoting a repressive chromatin state of the NFκB-target genes, as well as prevented VSMC apoptosis through transcriptional activation of KLF5-dependent BCL2 expression. Together, our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.
Guizhen Zhao, Yang Zhao, Haocheng Lu, Ziyi Chang, Hongyu Liu, Huilun Wang, Wenying Liang, Yuhao Liu, Tianqing Zhu, Oren Rom, Yanhong Guo, Lin Chang, Bo Yang, Minerva T. Garcia-Barrio, Jiandie D. Lin, Y. Eugene Chen, Jifeng Zhang
The molecular mechanisms underlying obesity-induced increase in β cell mass, and the resulting β cell dysfunction need to be elucidated further. Our study revealed that GPR92, expressed in islet macrophages, is modulated by dietary interventions in metabolic tissues. Therefore, we aimed to define the role of GPR92 in islet inflammation by using high fat diet (HFD)-induced obese mouse model. GPR92 knockout mice exhibited glucose intolerance and reduced insulin level, despite the enlarged pancreatic islets, and increased islet macrophage content and inflammation level (compared to those in wild type mice). These results indicate that the lack of GPR92 in islet macrophages can cause β cell dysfunction, leading to disrupted glucose homeostasis. Alternatively, GPR92 agonist, farnesyl pyrophosphate (FPP) stimulation results in the inhibition of HFD-induced islet inflammation and increased insulin secretion in WT mice, but not in GPR92 KO mice. Thus, our study suggests that GPR92 can be a potential target to alleviate β cell dysfunction via the inhibition of islet inflammation associated with the progression of diabetes.
Camila O. de Souza, Vivian A. Paschoal, Xue-Nan Sun, Lavanya Vishvanath, Qianbin Zhang, Mengle Shao, Toshiharu Onodera, Shiuhwei Chen, Nolwenn Joffin, Lorena M.A. Bueno, Rana K. Gupta, Da Young Oh
Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed a significant CD69 overexpression on Treg cells after MI. Our results in mice showed that CD69 expression on Treg cells increased survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Treg cells, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from two independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of re-hospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Treg cells as a potential prognostic factor and a therapeutic option to prevent HF after MI.
Rafael Blanco-Domínguez, Hortensia de la Fuente, Cristina Rodríguez, Laura Martín-Aguado, Raquel Sánchez-Díaz, Rosa Jiménez-Alejandre, Iker Rodriguez-Arabaolaza, Andrea Curtabbi, Marcos M. Garcia-Guimaraes, Alberto Vera, Fernando Rivero, Javier Cuesta, Luis Jesus Jimenez-Borreguero, Alberto Cecconi, Albert Duran-Cambra, Manel Taurón, Judith Alonso, Héctor Bueno, María Villalba-Orero, José Antonio Enriquez, Simon C. Robson, Fernando Alfonso, Francisco Sánchez-Madrid, José Martínez-González, Pilar Martín
Although first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy is effective for treating EGFR-mutant non-small cell lung cancer (NSCLC), it is now understood that drug-tolerant persister (DTP) cells escaping from initial treatment eventually drives drug resistance. Here, through integration of metabolomics and transcriptomics, we found that the neurotransmitter acetylcholine (ACh) was specifically accumulated in DTP cells, and illustrated that treatment with EGFR-TKI heightens the expression of the rate-limiting enzyme choline acetyltransferase (ChAT) in ACh biosynthesis via YAP mediation. Genetic and pharmacological manipulation of ACh biosynthesis or ACh signaling could predictably regulate the extent of DTP formation in vitro and in vivo. Strikingly, pharmacologically targeting ACh/M3R signaling with an FDA-approved drug, Darifenacin, retarded tumor relapse in vivo. Mechanistically, upregulated ACh metabolism mediated drug tolerance in part through activating WNT signaling via ACh muscarinic receptor-3 (M3R). Importantly, aberrant ACh metabolism in NSCLC patients represented a potential role in predicting EGFR-TKI response rate and progression-free survival. Our study therefore defines a new therapeutic strategy—targeting ACh-M3R-WNT axis—for manipulating EGFR TKI drug tolerance in the treatment of NSCLC.
Meng Nie, Na Chen, Huanhuan Pang, Tao Jiang, Wei Jiang, Panwen Tian, LiAng Yao, Yangzi Chen, Ralph J. DeBerardinis, Weimin Li, Qitao Yu, Caicun Zhou, Zeping Hu
CLN1 disease is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by delivering monthly infusions of recombinant human PPT1 (rhPPT1) in PPT1-deficient mice (Cln1−/−), and CLN1R151X sheep to assess scale up for translation. In Cln1−/− mice, intracerebroventricular rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1 treated-mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, intracerebroventricular infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural magnetic resonance imaging and neuropathology. These findings demonstrate the feasibility and therapeutic efficacy of intracerebroventricular rhPPT1 enzyme replacement therapy. This represents a key step towards clinical testing of ERT in children with CLN1 disease and highlights the importance of a cross-species approach to developing a successful treatment strategy.
Hemanth R. Nelvagal, Samantha L. Eaton, Sophie H. Wang, Elizabeth M. Eultgen, Keigo Takahashi, Steven Q. Le, Rachel Nesbitt, Joshua T. Dearborn, Nicholas Siano, Ana C. Puhl, Patricia I. Dickson, Gerard Thompson, Fraser Murdoch, Paul M. Brennan, Mark Gray, Stephen N. Greenhalgh, Peter Tennant, Rachael Gregson, Eddie Clutton, James Nixon, Chris Proudfoot, Stefano Guido, Simon G. Lillico, C. Bruce A. Whitelaw, Jui-Yun Lu, Sandra L. Hofmann, Sean Ekins, Mark S. Sands, Thomas M. Wishart, Jonathan D. Cooper
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