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Abstract
Huntington’s disease (HD) is a progressive autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that HD patients also exhibit cardiac and respiratory dysfunction including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via post-translationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated and leaky in brain, heart and diaphragm in both HD patients and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum (ER) Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor and respiratory functions were ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac and diaphragmatic pathophysiology in HD and identify RyRs as a potential novel therapeutic target.
Authors
Haikel Dridi, Xiaoping Liu, Qi Yuan, Steve Reiken, Yehya Mohamad, Leah R. Sittenfeld, Panagiota Apostolou, Julie Buron, Pierre Sicard, Stefan Matecki, Jérôme Thireau, Clement Menuet, Alain Lacampagne, Andrew R. Marks
Abstract
In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these two compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas 24-month-old control mice had lower cortical bone compared to 8-month-old controls, mice lacking RANKL in osteocytes gained cortical bone from 8 to 24 months of age. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice, but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Over-expression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Lastly, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.
Authors
Ha-Neui Kim, Jinhu Xiong, Ryan S. MacLeod, Srividhya Iyer, Yuko Fujiwara, Keisha M. Cawley, Li Han, Yonghan He, Jeff D. Thostenson, Elisabeth Ferreira, Robert L. Jilka, Daohong Zhou, Maria Almeida, Charles A. O'Brien
Essential role and therapeutic targeting of the glomerular endothelial glycocalyx in lupus nephritis
Abstract
Lupus Nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in new mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx play central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted it. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs, and may be therapeutically targeted for SLE complications including LN.
Authors
Hiroyuki Kadoya, Ning Yu, Ina Maria Schiessl, Anne Riquier-Brison, Georgina Gyarmati, Dorinne Desposito, Kengo Kidokoro, Matthew J. Butler, Chaim O. Jacob, János Peti-Peterdi
Abstract
The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine-substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias, and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for IQ/AA mutant, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We find FGF13 diminishes late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.
Authors
Jeffrey M. Abrams, Daniel Roybal, Nourdine Chakouri, Alex N. Katchman, Richard L. Weinberg, Lin Yang, Bi-Xing Chen, Sergey I. Zakharov, Jessica A. Hennessey, Uma Mahesh R. Avula, Johanna Diaz, Chaojian Wang, Elaine Y. Wan, Geoffrey S. Pitt, Manu Ben-Johny, Steven O. Marx
Abstract
Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.
Authors
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
Abstract
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Authors
Muchaala J. Yeboah, Charys Papagregoriou, Des C. Jones, H. T. Claude Chan, Guangan Hu, Justine S. McPartlan, Torbjörn Schiött, Ulrika T. Mattson, C. Ian Mockridge, Ulla-Carin Tornberg, Björn Hambe, Anne Ljungars, Mikael Mattsson, Ivo Tews, Martin J. Glennie, Stephen M. Thirdborough, John Trowsdale, Björn Frendéus, Jianzhu Chen, Mark S. Cragg, Ali Roghanian
Abstract
Hidradenitis Suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Utilizing proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered around IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with BTK and SYK pathway activation as a central signal transduction networks in HS. These data provide preclinical evidence to accelerate the path towards clinical trials targeting BTK and SYK signaling in moderate to severe HS.
Authors
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, Kelsey R. van Straalen, Allard R.J.V. Vossen, H.H. Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed Monem Rizvi, Xianying Xing, Enze Xing, Olesya Plazyo, Chang Zeng, Matthew T. Patrick, Margaret M. Lowe, Richard E. Burney, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, Yanyun Jiang, Joseph Kirma, Stephan Weidinger, Kelly C. Cushing, Michael D. Rosenblum, Celine C. Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
Abstract
Based on its clinical benefits, Trikafta, the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) was FDA-approved for treatment of cystic fibrosis (CF) patients carrying deletion of phenylalanine 508 (F508del) of the CF Transmembrane Conductance Regulator (CFTR) on at least one allele. Neither the mechanism of action of VX-445, nor the susceptibility of rare CF folding mutants to Trikafta are known. Here we show that in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1)-membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661+VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to ~62% of wild-type CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K and N1303K), confined to MSD1, MSD2, NBD1 and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.
Authors
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
Abstract
African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically down-regulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant cells with T-helper functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process we performed genome-wide transcriptional analysis on T cells induced to down-regulate CD4 in vitro from AGMs and closely-related Patas monkeys, and T cells that maintain CD4 expression from rhesus macaques. In T cells that down-regulated CD4, pathway analysis revealed an atypical regulation ofthe DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2deoxycytidine. This signature was driven largely by the dioxygenase TET3 that became down-regulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4-CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and human immunodeficiency virus (HIV)-1 entry receptor expression in hosts that become progressively SIV-infected, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.
Authors
Joseph C. Mudd, Stephen Lai, Sanjana Shah, Andrew R. Rahmberg, Jacob K. Flynn, Carly E. Starke, Molly R. Perkins, Amy Ransier, Samuel Darko, Daniel Douek, Vanessa Hirsch, Mark J. Cameron, Jason M. Brenchley
Abstract
Lynch syndrome is the most common colorectal cancer (CRC) hereditary form and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM, nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a LLS male patient with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR-Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than the MMR genes. We suggest MCM8 as a new gene involved in CRC germline predisposition with a recessive pattern of inheritance.
Authors
Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Díaz-Gay, Jenifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Soledad Iseas, Guillermo Mendez, Daniel Cisterna, Stephanie A. Schubert, Maartje Nielsen, Tom van Wezel, Yael Goldberg, Eli Pikarsky, Juan Robbio, Enrique Roca, Antoni Castells, Francesc Balaguer, Marina Antelo, Sergi Castellví-Bel
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