Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal Na retention leading to oedema. This Na retention is usually attributed to epithelial sodium channel (ENaC) activation following plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We, then, identified a truncated variant of acid sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when co-expressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that expression of the truncated ASIC2b in kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but not in any of the patients with other renal diseases. We have, therefore, identified a novel variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.
Marc Fila, Ali Sassi, Gaelle Brideau, Lydie Cheval, Luciana Morla, Pascal Houillier, Christine Walter, Michel Gennaoui, Laure Collignon, Mathilde Keck, Gabrielle Planelles, Naziha Bakouh, Michel Peuchmaur, Georges Deschenes, Ignacio Anegon, Séverine Remy, Bruno Vogt, Gilles Crambert, Alain Doucet
Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatic vessels in bone, gradually lose bone, and can have life-threatening complications such as chylothorax. The etiology of GSD is poorly understood and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a GSD patient and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.
Nassim Homayun Sepehr, Anna L. McCarter, Raphaël Helaers, Christine Galant, Laurence M. Boon, Pascal Brouillard, Miikka Vikkula, Michael T. Dellinger
The majority of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPSIIIC), the major burdens are progressive and severe neuropsychiatric problems and dementia primarily thought to stem from neurodegeneration. Using the MPSIIIC mouse model we studied whether clinical manifestations preceding massive neurodegeneration arise from synaptic dysfunction. Reduced levels or abnormal distribution of multiple synaptic proteins were revealed in cultured hippocampal and CA1 pyramidal MPSIIIC neurons. These defects were rescued by virus-mediated gene correction. Dendritic spines were reduced in pyramidal neurons of mouse models of MPSIIIC and other (Tay-Sachs, sialidosis) LSD as early as postnatal day 10. MPSIIIC neurons also presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganised microtubule networks and partially impaired axonal transport of synaptic proteins. Furthermore, postsynaptic densities were reduced in post-mortem cortices of human MPS patients suggesting that the pathology is a common hallmark for neurological LSD. Together, our results demonstrate that lysosomal storage defects cause early alterations in synaptic structure and abnormalities in neurotransmission originating from impaired synaptic vesicular transport, and suggest that synaptic defects could be targeted to treat behavioral and cognitive defects in neurological LSD patients.
Camila Pará, Poulomee Bose, Luigi Bruno, Erika Freemantle, Mahsa Taherzadeh, Xuefang Pan, Chanshuai Han, Peter S. McPherson, Jean-Claude Lacaille, Éric Bonneil, Pierre Thibault, Claire O'Leary, Brian Bigger, Carlos Ramon Morales, Graziella Di Cristo, Alexey V. Pshezhetsky
SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to autism spectrum disorders (ASDs). Most ASD-associated mutations in SCN2A are loss-of-function, but studies examining how such mutations affect neuronal function and whether Scn2a mutant mice display ASD endophenotypes have been inconsistent. We generated a protein truncation variant Scn2a mouse model (Scn2aΔ1898/+) by CRISPR that eliminates the NaV1.2 channel’s distal intracellular C-terminal domain and analyzed the molecular and cellular consequences of this variant in a heterologous expression system, in neuronal culture, in brain slices, and in vivo. We also analyzed multiple behaviors in wild type and Scn2aΔ1898/+ mice and correlated behaviors with clinical data obtained in human subjects with SCN2A variants. Expression of the NaV1.2 mutant in a heterologous expression system revealed decreased NaV1.2 channel function and cultured pyramidal neurons isolated from Scn2aΔ1898/+ forebrain showed correspondingly reduced voltage-gated Na+ channel currents without compensation from other CNS voltage-gated Na+ channels. Na+ currents in inhibitory neurons were unaffected. Consistent with loss of voltage-gated Na+ channel currents, Scn2aΔ1898/+ pyramidal neurons displayed reduced excitability in forebrain neuronal culture and reduced excitatory synaptic input onto the pyramidal neurons in brain slices. Scn2aΔ1898/+ mice displayed several behavioral abnormalities, including abnormal social interactions that reflect behavior observed in humans with ASD and with harboring loss-of-function SCN2A variants. This model and its cellular electrophysiological characterizations provide a framework for tracing how a SCN2A loss-of-function variant leads to cellular defects that result in ASD-associated behaviors.
Hong-Gang Wang, Charlotte C. Bavley, Anfei Li, Rebecca M. Jones, Jonathan E. Hackett, Yared Bayleyen, Francis S. Lee, Anjali M. Rajadhyaksha, Geoffrey S. Pitt
Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVβ1 and NaVβ3 subunits on the functional effects of two well-known Class-Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, Nav1.5, expressed in mammalian heart. We show that both drugs stabilize the activated conformation of the voltage-sensor of in Domain-III (DIII-VSD) in NaV1.5. In the presence of NaVβ1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main Class-Ib drug binding site, F1760, affected but did not abolish, the modulation of drug block by Navβ1/β3. Recordings from adult mouse ventricular myocytes demonstrated that Scn1b (Navβ1) loss of differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to intraperitoneal injection of ranolazine or lidocaine in WT and Scn1b null animals, suggesting that NaVβ1 modulates drug responses at the whole heart level. In human heart, we found that SCN1B transcript expression is three times higher in atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically impact patient response to Class-Ib antiarrhythmic therapies.
Wandi Zhu, Wei Wang, Paweorn Angsutararux, Rebecca L. Mellor, Lori L. Isom, Jeanne M. Nerbonne, Jonathan R. Silva
The stimulator of interferon genes (STING) protein senses cyclic di-nucleotides released in response to double stranded DNA, and functions as an adaptor molecule for type I interferon (IFN-I) signaling by activating IFN-I stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared to control EC, whereas T cells adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell expressed molecules ICAM-1 and VCAM-1 compared to WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC and genetic loss or pharmacologic antagonism of IFN-type I interferon receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC STING during T cell TEM that is dependent on the ISG CXCL10 and on IFN-I-IFNAR signaling.
Marina Anastasiou, Gail A. Newton, Kuljeet Kaur, Francisco J. Carrillo-Salinas, Sasha A. Smolgovsky, Abraham L. Bayer, Vladimir Ilyukha, Shruti Sharma, Alexander Poltorak, Francis W. Luscinskas, Pilar Alcaide
Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in human resulting in a novel anomaly complex syndrome manifested with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos is required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 (Tasp1-/-) disrupted embryonic hematopoietic stem cell self-renewal and quiescence states, and axial skeleton fates. Strikingly, mice carrying knockin non-cleavable mutations of TFIIAα-β (Gtf2a1nc/nc), a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with non-cleavable MLL1 and MLL2 (Mll1nc/nc;2nc/nc), two trithorax group (Trx-G) histone H3 trimethyl transferases. Our study offers molecular insights concerning TASP1-loss human syndrome and discovers unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.
Hidetaka Niizuma, Adam C. Searleman, Shugaku Takeda, Scott A. Armstrong, Christopher Y. Park, Emily H. Cheng, James J. Hsieh
The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STI). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue resident memory T cells (TRM) in human FRT is lacking. We have taken single-cell RNA sequencing approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There are significantly more CD8 than CD4 T cells. Unsupervised clustering and trajectory analysis identify distinct populations of CD8 T cells with IFNGhiGZMBlowCD69hiCD103low or IFNGlowGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescent staining shows that CD103+ CD8 TRM cells preferentially localize in epithelium while CD69+ CD8 TRM distribute evenly in epithelium and stroma. Ex vivo assays indicate up to 14% of cervical CD8 TRM clonotypes are HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identify subgroups of CD8 TRM in the human ectocervix that exhibit distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRM is an important approach for improving host defenses to STI.
Tao Peng, Khamsone Phasouk, Emily Bossard, Alexis Klock, Lei Jin, Kerry J. Laing, Christine Johnston, Noel A. Williams, Julie L. Czartoski, Dana Varon, Annalyssa N. Long, Jason H. Bielas, Thomas M. Snyder, Harlan Robins, David M. Koelle, M Juliana McElrath, Anna Wald, Lawrence Corey, Jia Zhu
Obesity is a risk factor for gallbladder cancer (GBC) development and correlates with shorter overall survival. Leptin, derived from adipocytes, has been suggested to contribute to the growth of cancer cells. However, the detailed mechanism of leptin in GBC drug resistance remains uninvestigated. In this study, it is clinically relevant that GBC patients with a higher BMI (BMI ≥ 24 kg/m2) (n=30) were associated with increased GBC risks, including survival. Moreover, obese NOD/SCID mice exhibited a higher circulating concentration of leptin, which is associated with GBC growth and attenuated gemcitabine efficacy. We further revealed that leptin can inhibit gemcitabine-induced GBC cell death through myeloid cell leukemia 1 (MCL1) activation. The transcription factor CCAAT/enhancer-binding protein delta (CEBPD) is responsive to activated signal transducers and activators of transcription 3 (pSTAT3) and contributes to MCL1 transcriptional activation upon leptin treatment. In addition, MCL1 mediates leptin-induced mitochondrial fusion and is associated with GBC cell survival. This study suggests the involvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrial fusion and survival. It provides a new therapeutic target to improve the efficacy of gemcitabine in GBC patients.
Wei-Jan Wang, Hong-Yue Lai, Fei Zhang, Wan-Jou Shen, Pei-Yu Chu, Hsin-Yin Liang, Ying-Bin Liu, Ju-Ming Wang
Estrogen-related receptor gamma (Esrrg) is a murine lupus susceptibility gene associated with T cell activation. Here, we report that Esrrg controls regulatory T cells (Treg) through mitochondria homeostasis. Esrrg deficiency impaired the maintenance and function of Treg cells, leading to global T cell activation and autoimmunity in aged mice. Further, Esrrg-deficient Treg cells presented an impaired differentiation into follicular Treg (Tfr) cells that enhanced follicular helper T cells (Tfh) responses. Mechanistically, Esrrg-deficient Treg cells presented with dysregulated mitochondria with decreased oxygen consumption as well as ATP and NAD+ production. In addition, Esrrg-deficient Treg cells exhibited decreased phosphatidylinositol and TGF-β signaling pathways and increased mTORC1 activation. We found that the expression of human ESRRG, which is high in Treg cells, was lower in CD4+ T cells from lupus patients than in healthy controls. Finally, knocking down ESRRG in Jurkat T cells decreased their metabolism. Together, our results reveal a critical role of Esrrg in the maintenance and metabolism of Treg cells, which may provide a genetic link between lupus pathogenesis and mitochondrial dysfunction in T cells.
Wei Li, Minghao Gong, Yuk-Pheel Park, Ahmed S. Elshikha, Seung-Chul Choi, Josephine Brown, Nathalie Kanda, Wen-I Yeh, Leeana Peters, Anton A. Titov, Xiangyu Teng, Todd M. Brusko, Laurence Morel
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