Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurological disorder caused by a deleterious CAG repeat expansion in the coding region of the ataxin-7 gene. Infantile onset SCA7 leads to severe clinical manifestation of respiratory distress, but the exact cause of respiratory impairment remains unclear. Using the infantile SCA7 mouse model, the SCA7266Q/5Q mouse, we examined the impact of pathological poly-Q-ataxin-7 mutant ataxin-7 on hypoglossal (XII) and phrenic motor units. We identified the transcript profile of the medulla and cervical spinal cord and, investigated the XII and phrenic nerve and the neuromuscular junctions in the diaphragm and tongue. SCA-7 astrocytes showed significant intranuclear inclusions of ataxin-7 in the XII and putative phrenic motor nuclei. Transcriptomic analysis revealed dysregulation of genes involved in amino acid and neurotransmitter transportation and myelination. Additionally, SCA7 mice demonstrated blunted efferent output of the XII nerve and demyelination in both XII and phrenic nerves. Finally, there was an increased number of NMJ clusters with higher expression of synaptic markers in SCA7 mice compared to WT controls. These pre-clinical findings elucidate the underlying pathophysiology responsible for impaired glial cell function and death leading to dysphagia, aspiration and respiratory failure in infantile SCA7.
Debolina Dipankar Biswas, Yihan Shi, Léa El Haddad, Ronit Sethi, Meredith L. Huston, Sean Kehoe, Evelyn R. Scarrow, Laura M. Strickland, Logan A. Pucci, Justin S. Dhindsa, Ani Hunanyan, Albert R. La Spada, Mai K. ElMallah
The clinical therapy for treating acute myocardial infarction is primary percutaneous coronary intervention (PPCI). PPCI is effective at reperfusing the heart, however the rapid re-introduction of blood can cause ischemia-reperfusion (I/R). Reperfusion injury is responsible for up to half of the final myocardial damage, but there are no pharmacological interventions to reduce I/R. We previously demonstrated that inhibiting monocarboxylate transporter 4 (MCT4) and re-directing pyruvate towards oxidation can blunt hypertrophy. We hypothesized this pathway might be important during I/R. Here, we establish that the pyruvate-lactate axis plays a role in determining myocardial salvage following injury. Post-I/R, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium. In cardiomyocytes lacking the MPC, there was increased cell death and less salvage after I/R, which was associated with an upregulation of MCT4. To determine the importance of pyruvate oxidation, we inhibited MCT4 with a small-molecule drug (VB124) at reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψ), and Ca2+, increased pyruvate entry to TCA cycle, increased oxygen consumption, improved myocardial salvage and functional outcomes following I/R. Our data suggests normalizing pyruvate-lactate metabolism by inhibiting MCT4 is a promising therapy to mitigate I/R injury.
Joseph R. Visker, Ahmad A. Cluntun, Jesse N. Velasco-Silva, David R. Eberhardt, Luis Cedeno-Rosario, Thirupura S. Shankar, Rana Hamouche, Jing Ling, Hyoin Kwak, J. Yanni Hillas, Ian Aist, Eleni Tseliou, Sutip Navankasattusas, Dipayan Chaudhuri, Gregory S. Ducker, Stavros G. Drakos, Jared Rutter
Over 200,000 individuals are diagnosed with lung cancer in the U.S. every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from post-menopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound impact of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.
Sitapriya Moorthi, Amy Paguirigan, Pushpa Itagi, Minjeong Ko, Mary Pettinger, Anna C. Hoge, Anwesha Nag, Neil A. Patel, Feinan Wu, Cassie Sather, Kevin M. Levine, Matthew P. Fitzgibbon, Aaron R. Thorner, Garnet L. Anderson, Gavin Ha, Alice H. Berger
Loss of NADPH oxidase (NOX2) exacerbates systemic lupus erythematosus (SLE) in mice and humans, but the mechanisms underlying this effect remain unclear. To identify the cell lineages in which NOX2 deficiency drives SLE, we employed conditional knockout (KO) and chimera approaches to delete Cybb in several hematopoietic cell lineages of MRL.Faslpr lupus-prone mice. Deletion of Cybb in macrophages/monocytes exacerbated lupus nephritis, though not to the degree observed in the Cybb global KOs. Unexpectedly, the absence of Cybb in B cells resulted in profound glomerulonephritis and interstitial nephritis, rivaling that seen with global deletion. Further, we identified that NOX2 is a key regulator of TLR7, a driver of SLE pathology, both globally and specifically in B cells. This is mediated in part through suppression of TLR7-mediated NF-kB signaling in B cells. Thus, NOX2's immunomodulatory effect in SLE is orchestrated not only by its function in the myeloid compartment, but through a pivotal role in B cells by selectively inhibiting TLR7 signaling.
Rachael A. Gordon, Haylee A. Cosgrove, Anthony Marinov, Sebastien Gingras, Jeremy S. Tilstra, Allison M. Campbell, Sheldon I. Bastacky, Michael Kashgarian, Andras Perl, Kevin M. Nickerson, Mark J. Shlomchik
Alveolar macrophages (AMs) act as gatekeepers of the lung’s immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) Early Growth Response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA-sequencing, ATAC-sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes, and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmarker in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.
Zsuzsanna Kolostyak, Dora Bojcsuk, Viktoria Baksa, Zsuzsa Mathene Szigeti, Krisztian Bene, Zsolt Czimmerer, Pal Boto, Lina Fadel, Szilard Poliska, Laszlo Halasz, Petros Tzerpos, Wilhelm K. Berger, Andres Villabona-Rueda, Zsofia Varga, Tunde Kovacs, Andreas Patsalos, Attila Pap, György Vámosi, Peter Bai, Balazs Dezso, Matthew Spite, Franco R. D'Alessio, Istvan Szatmari, Laszlo Nagy
The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system (ENS), restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb knock-out (KO) mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colon of recipient Ednrb KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in “neo-ganglia”. Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared to sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.
Ahmed A. Rahman, Takahiro Ohkura, Sukhada Bhave, Weikang Pan, Kensuke Ohishi, Leah Ott, Christopher Han, Abigail Leavitt, Rhian Stavely, Alan J. Burns, Allan M. Goldstein, Ryo Hotta
Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA sequencing, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, healthy individuals, and induced pluripotent stem (iPS) cells generated from a PCD patient. Transcriptomic analysis revealed unique signatures in PCD airway cells compared to their mothers and healthy individuals. Gene expression in heterozygous mothers’ cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases, GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism, Chlamydomonas reinhardtii, demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.
Jeffrey R. Koenitzer, Deepesh Kumar Gupta, Wang Kyaw Twan, Huihui Xu, Nicholas Hadas, Finn J. Hawkins, Mary Lou Beermann, Gervette M. Penny, Nathan T. Wamsley, Andrew Berical, Michael B. Major, Susan K. Dutcher, Steven L. Brody, Amjad Horani
Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy linked to high-risk Human papillomavirus (HPV) infection, which develops from precursor lesions like Low-Grade Squamous Intraepithelial Lesions (LGSIL) and High-Grade Squamous Intraepithelial Lesions (HGSIL). ASCC incidence varies across populations, posing increased risk for People Living with HIV (PLWH). Our investigation focused on transcriptomic and metatranscriptomic changes from Squamous Intraepithelial Lesions (SILs) to ASCC. Metatranscriptomic analysis highlighted specific bacterial species (e.g., Fusobacterium nucleatum, Bacteroides fragilis) more prevalent in ASCC than precancerous lesions. These species correlated with gene encoding enzymes (Acca, glyQ, eno, pgk, por) and oncoproteins (FadA, dnaK), presenting potential diagnostic or treatment markers. Unsupervised transcriptome analysis identified distinct sample clusters reflecting histological diagnosis, immune infiltrate, HIV/HPV status, and pathway activities, recapitulating anal cancer progression's natural history. Our study unveiled molecular mechanisms in anal cancer progression, aiding in stratifying HGSIL cases based on low- or high-risk progression to malignancy.
Ezequiel Lacunza, Valeria Fink, María E. Salas, Ana M. Gun, Jorge A. Basiletti, María Alejandra Picconi, Mariano Golubicki, Juan Robbio, Mirta Kujaruk, Soledad Iseas, Sion L. Williams, María Figueroa, Omar Coso, Pedro Cahn, Juan C. Ramos, Martin C. Abba
HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors (TCRs) in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyze ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8 T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4β7, and CCR6. In addition, we find an expansion of YeiH-specific CD8 T cells in the blood of axSpA and B27AAU over healthy controls, whereas influenza-specific CD8 T cells were equivalent across groups. Lastly, we demonstrate the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8 T cells may occur in enteric organs.
Michael A. Paley, Xinbo Yang, Lynn M. Hassman, Frank Penkava, Lee I. Garner, Grace L. Paley, Nicole Linskey, Ryan Agnew, Paulo Henrique Arantes de Faria, Annie Feng, Sophia Y. Li, Davide Simone, Elisha D.O. Roberson, Philip A. Ruzycki, Ekaterina Esaulova, Jennifer Laurent, Lacey Feigl-Lenzen, Luke E. Springer, Chang Liu, Geraldine M. Gillespie, Paul Bowness, K. Christopher Garcia, Wayne M. Yokoyama
Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation STING V154M (VM) similarly develop ILD, but exhibit severe T and B cell lymphopenia, low serum Ig titers, and lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with wildtype (WT) stem cells (WT→VM) still develop ILD. In this study, we find that WT→VM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in SAVI patients. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+Tg→VM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WTVM chimeras were adoptively transferred to naïve Rag1-deficient 2º hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.
Kevin MingJie Gao, Kristy Chiang, Sharon Subramanian, Xihui Yin, Paul J. Utz, Kerstin Nündel, Kate A. Fitzgerald, Ann Marshak-Rothstein
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