Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of Growth Arrest and DNA Damage Inducible Alpha (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MEKK4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.
George R. Marcotte, Matthew J. Miller, Hawley E. Kunz, Zachary C. Ryan, Matthew D. Strub, Patrick M. Vanderboom, Carrie J. Heppelmann, Sarah Chau, Zachary D. Von Ruff, Sean P. Kilroe, Andrew T. McKeen, Jason M. Dierdorff, Jennifer I. Stern, Karl A. Nath, Chad E. Grueter, Vitor A. Lira, Andrew R. Judge, Blake B. Rasmussen, K. Sreekumaran Nair, Ian R. Lanza, Scott M. Ebert, Christopher M. Adams
Ventricular arrhythmias (VAs) in heart failure are enhanced by sympathoexcitation. However, radiotracer studies of catecholamine uptake in failing human hearts demonstrate a proclivity for VAs in patients with reduced cardiac sympathetic innervation. We hypothesized that this counterintuitive finding is explained by heterogeneous loss of sympathetic nerves in the failing heart. In a murine model of dilated cardiomyopathy (DCM), delayed PET imaging of sympathetic nerve density using the catecholamine analog [11C]meta-Hydroxyephedrine ([11C]-mHED) demonstrated global hypoinnervation in ventricular myocardium. Although reduced, sympathetic innervation in two distinct DCM models invariably exhibited transmural (epicardial to endocardial) gradients, with the endocardium being devoid of sympathetic nerve fibers vs. controls. Further, the severity of transmural innervation gradients was correlated with VAs (r = 0.6, P < 0.05). Transmural innervation gradients were also identified in human left ventricular free wall samples from DCM vs controls. We investigated mechanisms underlying this relationship by in silico studies in 1-D, 2-D, and 3-D models of failing and normal human hearts, finding that arrhythmogenesis increased as heterogeneity in sympathetic innervation worsened. Specifically, both DCM-induced myocyte electrical remodeling and spatially inhomogeneous innervation gradients synergistically worsened arrhythmogenesis. Thus, heterogeneous innervation gradients in DCM promoted arrhythmogenesis. Restoration of homogeneous sympathetic innervation in the failing heart may reduce VAs.
Al-Hassan J. Dajani, Michael B. Liu, Michael A. Olaopa, Lucian Cao, Carla Valenzuela Ripoll, Timothy J. Davis, Megan D. Poston, Elizabeth H. Smith, Jaime Contreras, Marissa Pennino, Christopher M. Waldmann, Donald B. Hoover, Jason T. Lee, Patrick Y. Jay, Ali Javaheri, Roger Slavik, Zhilin Qu, Olujimi A. Ajijola
Although SARS-CoV-2 evolution seeds a continuous stream of antibody-evasive viral variants, COVID-19 mRNA vaccines provide robust protection against severe disease and hospitalization. Here, we asked whether mRNA vaccine-induced memory T cells limits lung SARS-CoV-2 replication and severe disease. We show that mice and humans receiving booster BioNTech mRNA vaccine developed potent CD8 T-cell responses and show similar kinetics of expansion and contraction of granzyme B/perforin-expressing effector CD8 T cells. Both monovalent and bivalent mRNA vaccines elicited strong expansion of a heterogeneous pool of terminal effectors and memory precursor effector CD8 T cells in spleen, inguinal and mediastinal lymph nodes, pulmonary vasculature, and most surprisingly in the airways, suggestive of systemic and regional surveillance. Further, we document that: (1) CD8 T-cell memory persists in multiple tissues for > 200 days; (2) following challenge with pathogenic SARS-CoV-2, circulating memory CD8 T cells rapidly extravasate to the lungs and promote expeditious viral clearance, by mechanisms that require CD4 T cell help; (3) adoptively transferred splenic memory CD8 T cells traffic to the airways, and promote lung SARS-CoV-2 clearance. These findings provide new insights into the critical role of memory T cells in preventing severe lung disease following breakthrough infections with antibody-evasive SARS-CoV-2 variants.
Brock Kingstad-Bakke, Thomas Cleven, Hailey Bussan, Boyd L. Yount Jr., Ryuta Uraki, Kiyoko Iwatsuki-Horimoto, Michiko Koga, Shinya Yamamoto, Hiroshi Yotsuyanagi, Hongtae Park, Jay S. Mishra, Sathish Kumar, Ralph Baric, Peter J. Halfmann, Yoshihiro Kawaoka, M. Suresh
Osteogenesis imperfecta (OI), brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/- mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used scRNAseq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same UPR pathway as previously demonstrated in osteoblasts. Aga2+/- proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has a previously unappreciated impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.
Jennifer Zieba, Lisette Nevarez, Davis Wachtell, Jorge H. Martin, Alexander Kot, Sereen Wong, Daniel H. Cohn, Deborah Krakow
MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in two siblings with microcephaly, epileptic encephalopathy and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13-, Mad2- and Rev7-binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated a newly identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early deceased patient with low-level aneuploidy, severe epileptic encephalopathy and frequent blood glucose elevations likely corresponds to complete loss-of-function, as in Mad2l1bp–/– mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.
Ghada M. H. Abdel-Salam, Susanne Hellmuth, Elise Gradhand, Stephan Käseberg, Jennifer Winter, Ann-Sophie Pabst, Maha M. Eid, Holger Thiele, Peter Nürnberg, Birgit S. Budde, Mohammad Reza Toliat, Ines B. Brecht, Christopher Schroeder, Axel Gschwind, Stephan Ossowski, Friederike Häuser, Heidi Rossmann, Mohamed S. Abdel-Hamid, Ibrahim Hegazy, Ahmed G. Mohamed, Dominik T. Schneider, Aida M. Bertoli-Avella, Peter Bauer, Jillian N. Pearring, Rolph Pfundt, Alexander Hoischen, Christian Gilissen, Dennis Strand, Ulrich Zechner, Soha A. Tashkandi, Eissa A. Faqeih, Olaf Stemmann, Susanne Strand, Hanno J. Bolz
Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.
Glenn Carrington, Hoi Ting Abbi Hau, Sarah Kosta, Hannah F. Dugdale, Francesco Muntoni, Adele D'Amico, Peter Y. K. Van den Bergh, Norma B. Romero, Edoardo Malfatti, Juan J. Vilchez, Anders Oldfors, Sander Pajusalu, Katrin Õunap, Marta Giralt-Pujol, Edmar Zanoteli, Kenneth S. Campbell, Hiroyuki Iwamoto, Michelle Peckham, Julien Ochala
MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in CD8+ T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T cells expressing a phospho-silencing mutant TSC2-S1365A (SA) retain normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, SA CD8+ T cells display greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T cells used as adoptive cell therapy display greater anti-tumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhances both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.
Chirag H. Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L. Dunkerly-Eyring, Jiayu Wen, Mark J. Ranek, Laura M. Bartle, Daniel B. Henderson, Jason G. Sagert, David A. Kass, Jonathan D. Powell
Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence, thus understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that Semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the central nervous system, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor Neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF receptor 1 (TGFR1), which in turn leads to activation of canonical TGF signaling in both GSCs and NPCs. TGF signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A-NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A-NRP1-TGFR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.
Hye-Min Jeon, Yong Jae Shin, Jaehyun Lee, Nakho Chang, Dong-Hun Woo, Won Jun Lee, Dayna Nguyen, Wonyoung Kang, Hee Jin Cho, Heekyoung Yang, Jin-Ku Lee, Jason K. Sa, Yeri Lee, Donggeon Kim, Benjamin W. Purow, Yeup Yoon, Do-Hyun Nam, Jeongwu Lee
Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independent of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.
Jesse Santos, Ping Wang, Avishai Shemesh, Fengchun Liu, Tasha Tsao, Oscar A. Aguilar, Simon J. Cleary, Jonathan P. Singer, Ying Gao, Steven R. Hays, Jeffrey Golden, Lorriana E. Leard, Mary Ellen Kleinhenz, Nicholas A. Kolaitis, Rupal J. Shah, Aida Venado, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Lewis L. Lanier, Mark R. Looney, John R. Greenland, Daniel R. Calabrese
Postictal apnea is thought to be a major cause of sudden unexpected death in epilepsy (SUDEP). However, the mechanisms underlying postictal apnea are unknown. To understand causes of postictal apnea, we used a multimodal approach to study brain mechanisms of breathing control in 20 patients (ranging from pediatric to adult) undergoing intracranial electroencephalography (iEEG) for intractable epilepsy. Our results indicate that amygdala seizures can cause postictal apnea. Moreover, we identified a distinct region within the amygdala where electrical stimulation was sufficient to reproduce prolonged breathing loss persisting well beyond the end of stimulation. The persistent apnea was resistant to rising CO2 levels, and air hunger failed to occur, suggesting impaired CO2 chemosensitivity. Using es-fMRI, a novel approach combining electrical stimulation with functional MRI, we found amygdala stimulation altered BOLD activity in the pons/medulla and ventral insula. Together, these findings suggest that seizure activity in a focal subregion of the amygdala is sufficient to suppress breathing and air hunger for prolonged periods of time in the postictal period, likely via brainstem and insula sites involved in chemosensation and interoception. They further provide new insights into SUDEP, may help identify those at greatest risk, and may lead to treatments to prevent SUDEP.
Gail I.S. Harmata, Ariane E. Rhone, Christopher K. Kovach, Sukhbinder Kumar, Md Rakibul Mowla, Rup K. Sainju, Yasunori Nagahama, Hiroyuki Oya, Brian K. Gehlbach, Michael A. Ciliberto, Rashmi N. Mueller, Hiroto Kawasaki, Kyle T.S. Pattinson, Kristina Simonyan, Paul W. Davenport, Matthew A. Howard III, Mitchell Steinschneider, Aubrey C. Chan, George B. Richerson, John A. Wemmie, Brian J. Dlouhy
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