The clinical spectrum of thyrotropin receptor (TSHR)-mediated diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR, and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with one CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in two families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants, as well as distinct common non-coding TSHR SNPs significantly associated with thyroid phenotypes, but no other significant association between TSHR variants and over 2,000 non-thyroid disease endpoints. Finally, our TSHR M453T knock-in model revealed that the phenotype was dependent on the mutation´s signaling properties and was ameliorated by increased iodine intake. In summary, our data shows that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region, and by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.
Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli M. Linnossuo, Johanna O. Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi J. Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, Finngen Finngen, Mary Pat Reeve, Jukka Kero
Neonatal gene therapy has been shown to prevent inner ear dysfunction in mouse models of Usher syndrome type I (USH1), the most common genetic cause of combined deafness-blindness and vestibular dysfunction. However, hearing onset occurs after birth in mice and in utero in humans, making it questionable how to transpose murine gene therapy outcomes to clinical settings. Here, we sought to extend the therapeutic time window in a mouse model for USH1G to periods corresponding to human neonatal stages, more suitable for intervention in patients. Mice defective for Ush1g (Ush1g-/-) were subjected to gene therapy after the hearing onset. The rescue of inner ear hair-cell structure was evaluated by confocal imaging and electron microscopy. Hearing and vestibular function were assessed by recordings of the auditory brainstem response and vestibulo-ocular reflex, and by locomotor tests. Up to P21, gene therapy significantly restored both the hearing and balance deficits of Ush1g-/- mice. However, beyond this age and up to P30, vestibular function was restored, but not hearing. Our data shows that effective gene therapy is possible in Ush1g-/- mice well beyond neonatal stages, implying that the therapeutic window for USH1G may be wide enough to be transposable to newborn human patients.
Ghizlene Lahlou, Charlotte Calvet, François Simon, Vincent Michel, Lauranne Alciato, Baptiste Plion, Jacques Boutet de Monvel, Marie-José Lecomte, Mathieu Beraneck, Christine Petit, Saaid Safieddine
Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Neurofibromatosis Type 1 (NF1) individuals may develop thousands of cNFs, greatly affecting their quality of life. cNF growth is driven by the proliferation of NF1(-/-) SCs and their interaction with NF1(+/-) microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP-elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by Ogerin in combination with the MEKi Selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an iPSC-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with Selumetinib. Interestingly, whereas primary SC cultures reset proliferation after removal of single Selumetib treatment, Ogerin-Selumetinib combo elicited a permanent halt on SC expansion, also after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators arises as a potential treatment for cNFs.
Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra
Diabetic patients have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild type mice also developed diabetic bone disease, but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independent of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.
Jennifer M. Brazill, Ivana R. Shen, Clarissa S. Craft, Kristann L. Magee, Jay S. Park, Madelyn Lorenz, Amy Strickland, Natalie K. Wee, Xiao Zhang, Alec T. Beeve, Gretchen A. Meyer, Jeffrey Milbrandt, Aaron DiAntonio, Erica L. Scheller
The Murphy Roths Large (MRL) mouse strain has “super-healing” properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-β signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-β1 and TGF-β3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg–/– MRL and Sgcg–/– DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared to dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.
Joseph G. O'Brien, Alexander B. Willis, Ashlee M. Long, Jason M. Kwon, GaHyun Lee, Frank W. Li, Patrick G.T. Page, Andy H. Vo, Michele Hadhazy, Melissa J. Spencer, Rachelle H. Crosbie, Alexis R. Demonbreun, Elizabeth M. McNally
Immunoglobulin (IG) replacement products are used routinely in patients with immune deficiency and other immune dysregulation disorders, who have poor immune responses to vaccination and require passive immunity conferred by commercial antibody products. The binding, neutralizing, and protective activity of intravenously administered immunoglobulin (IG) against SARS-CoV-2 emerging variants remains unknown. Here, we tested 198 different IG products manufactured from December 2019 to August 2022. We show that pre-pandemic IG had no appreciable cross-reactivity or neutralizing activity against SARS-CoV-2. Anti-spike antibody titers and neutralizing activity against SARS-CoV-2 WA1/2020 D614G increased gradually after the pandemic started and reached levels comparable with vaccinated healthy donors 18 months after the diagnosis of the first COVID-19 case in the United States in January 2020. The average time between production to infusion of IG products was 8 months, which resulted in poor neutralization of the variant strain circulating at the time of infusion. Despite limited neutralizing activity, IG prophylaxis with clinically relevant dosing protected susceptible K18-hACE2 transgenic mice against clinical disease, lung infection, and lung inflammation caused by the XBB.1.5 Omicron variant. Moreover, following IG prophylaxis, levels of XBB.1.5 infection in the lung were higher in FcγR KO mice than in wild-type mice. Thus, IG replacement products with poor neutralizing activity against evolving SARS-CoV-2 variants likely confer protection to patients with immune deficiency disorders through Fc-effector function mechanisms.
Ofer Zimmerman, Alexa Michelle Altman Doss, Baoling Ying, Chieh-Yu Liang, Samantha R. Mackin, Hannah G. Davis-Adams, Lucas J. Adams, Laura A. VanBlargan, Rita E. Chen, Suzanne M. Scheaffer, Pritesh Desai, Saravanan Raju, Tarisa L. Mantia, Caitlin C. O'Shaughnessy, Jennifer M. Monroy, H. James Wedner, Christpher J. Rigell, Andrew L. Kau, Tiffany B. Dy, Zhen Ren, Jackson S. Turner, Jane A. O’Halloran, Rachel M. Presti, Peggy L. Kendall, Daved H. Fremont, Ali H. Ellebedy, Michael S. Diamond
Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte derived macrophages. The receptor for advanced glycation end products (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared to healthy controls, RAGE expression was increased in liver biopsies from human NASH patients. In a high -fat, -fructose, and -cholesterol (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE knockout mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomic analysis suggested that pathways of macrophage and T-cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated bone marrow derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.
Gopanandan Parthasarathy, Amy S. Mauer, Naresh Golla, P. Vineeth Daniel, Lily H. Kim, Guneet S. Sidhu, George W. Marek 3rd, Emilien Loeuillard, Anuradha Krishnan, Hyun Se Kim Lee, Kevin D. Pavelko, Michael Charlton, Petra Hirsova, Sumera I. Ilyas, Harmeet Malhi
Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease characterized by the expansion of the aortic wall. One of the most significant features is the infiltration of macrophages in the adventitia, which drives vasculature remodeling. The macrophage-derived interferon regulatory factor 5 (IRF5) in macrophage infiltration and AAA formation remains unknown. RNA sequencing of AAA adventitia identifies Irf5 as the top significantly increased transcriptional factor, which is predominantly expressed in macrophages. Global and myeloid specific deficiency of Irf5 reduces AAA progression, with marked reduction of macrophage infiltration. Further cellular investigations indicate that IRF5 promotes macrophage migration by direct regulation of downstream phosphoinositide 3-kinase gamma (PI3Kγ, Pik3cg). Pik3cg ablation hinders AAA progression, and myeloid-specific salvage of Pik3cg restores AAA progression and macrophage infiltration derived from Irf5 deficiency. Finally, we discovered that the IRF5 and PI3Kγ expression in the adventitia are significantly increased in AAA patients. These findings uncover that the IRF5-dependent regulation of PI3Kγ is essential for AAA formation.
Yidong Wang, Zhenjie Liu, Shen Song, Jianfang Wang, Chunna Jin, Liangliang Jia, Yuankun Ma, Tan Yuan, Zhejun Cai, Meixiang Xiang
Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of five tumor sites and regions from each of 23 patients (n=108 samples) based on deep whole-exome sequencing. Polyclonal cancer origin was detected in one patient. Ovarian tumors had more complex subclonal architectures than other intra-peritoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal (27%) or polyclonal dissemination of monophyletic (linear; 50%) or polyphyletic (branched; 23%) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in seven cancer-critical genes, such as TP53, BRCA1, BRCA2, DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.
Anita Sveen, Bjarne Johannessen, Solveig M.K. Klokkerud, Sigrid M. Kraggerud, Leonardo A. Meza-Zepeda, Merete Bjørnslett, Katharina Bischof, Ola Myklebost, Kjetil Taskén, Rolf I. Skotheim, Anne Dørum, Ben Davidson, Ragnhild A. Lothe
The impairment of left ventricular (LV) diastolic function with inadequate increase in myocardial relaxation velocity directly results in lower LV compliance, increased LV filling pressures and heart failure symptoms. The development of agents facilitating the relaxation of human cardiomyocytes requires a better understanding of the underlying regulatory mechanisms. We performed a high-content microscopy-based screening in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a library of 2565 human miRNA mimics and measured relaxation kinetics via high-computing analyses of motion movies. We identified hsa-miR-548v, a primate specific miRNA, as the miRNA producing the largest increase in relaxation velocities. This positive lusitropic effect was reproduced in engineered cardiac tissues generated with healthy and BRAF T599R mutant hiPSC-CMs, and was independent of changes in calcium transients. Consistent with improvements in viscoelastic responses to mechanical stretch, RNA-sequencing showed that hsa-miR-548v down-regulated multiple targets, especially components of the mechano-sensing machinery. The exogenous administration of hsa-miR-548v in hiPSC-CMs notably resulted in a significant reduction of ANKRD1/CARP1 expression and localization at the sarcomeric I-band. This study suggests that the sarcomere I-band is a critical control center of the ability of cardiomyocytes to relax and a target for improving relaxation and diastolic dysfunction.
Eva Vermersch, Salomé Neuvendel, Charlene Jouve, Andrea Ruiz-Velasco, Céline Pereira, Magali Seguret, Marie-Elodie Cattin-Messaoudi, Sofia Lotfi, Thierry Dorval, Pascal Berson, Jean-Sébastien Hulot
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