MuSK cysteine-rich domain antibodies are pathogenic in a mouse model of autoimmune myasthenia gravis

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Abstract

The neuromuscular junction (NMJ), synapse between the motor neuron terminal and a skeletal muscle fiber is crucial, throughout life, in maintaining the reliable neurotransmission required for functional motricity. Disruption of this system leads to neuromuscular disorders, such as auto-immune myasthenia gravis (MG), the most common form of NMJ diseases. MG is caused by autoantibodies directed mostly against the acetylcholine receptor (AChR) or the muscle-specific kinase MuSK. Several studies report immunoreactivity to the Frizzled-like cysteine-rich Wnt-binding domain of MuSK (CRD) in patients, although the pathogenicity of the antibodies involved remains unknown. We showed here that the immunoreactivity to MuSK CRD induced by the passive transfer of anti-MuSKCRD antibodies in mice led to typical MG symptoms, characterized by a loss of body weight and a locomotor deficit. The functional and morphological integrity of the NMJ was compromised with a progressive decay of neurotransmission and disruption of the structure of pre- and post-synaptic compartments. We found that anti-MuSKCRD antibodies completely abolished Agrin-mediated AChR clustering by decreasing the Lrp4-MuSK interaction. These results provide the first demonstration of the role of the MuSK CRD in MG pathogenesis and improve our understanding of the underlying pathophysiological mechanisms.

Authors

Marius Halliez, Steve Cottin, Axel You, Céline Buon, Antony Grondin, Léa S. Lippens, Megane Lemaitre, Jérome Ezan, Charlotte Isch, Yann Rufin, Mireille Montcouquiol, Nathalie Sans, Bertrand Fontaine, Julien Messéant, Rozen Le Panse, Laure Strochlic

Wilms’ tumor 1 impairs apoptotic clearance of fibroblasts in distal fibrotic lung lesions

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease characterized by impaired fibroblast clearance and excessive extracellular matrix (ECM) protein production. Wilms' Tumor 1 (WT1), a transcription factor, is selectively upregulated in IPF fibroblasts. However, the mechanisms by which WT1 contributes to fibroblast accumulation and ECM production remain unknown. Here, we investigated the heterogeneity of WT1-expressing mesenchymal cells using single-nucleus RNA sequencing of distal lung tissues from IPF patients and control donors. WT1 was selectively upregulated in a subset of IPF fibroblasts that co-expressed several pro-survival and ECM genes. The results of both loss-of-function and gain-of-function studies are consistent with a role for WT1 as a positive regulator of pro-survival genes to impair apoptotic clearance and promote ECM production. Fibroblast-specific overexpression of WT1 augmented fibroproliferation, myofibroblast accumulation, and ECM production during bleomycin-induced pulmonary fibrosis in young and aged mice. Together, these findings suggest that targeting WT1 is a promising strategy for attenuating fibroblast expansion and ECM production during fibrogenesis.

Authors

Harshavardhana H. Ediga, Chanukya P. Vemulapalli, Vishwaraj Sontake, Pradeep K. Patel, Hikaru Miyazaki, Dimitry Popov, Martin B. Jensen, Anil G. Jegga, Steven K. Huang, Christoph Englert, Andreas Schedl, Nishant Gupta, Francis X. McCormack, Satish K. Madala

Kidney-specific WNK1 amplifies kidney tubule responsiveness to potassium via WNK body condensates

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Abstract

To maintain potassium homeostasis, the kidney’s distal convoluted tubule (DCT) evolved to convert small changes in blood [K+] into robust effects on salt reabsorption. This process requires NaCl cotransporter (NCC) activation by the With-No-Lysine (WNK) kinases. During hypokalemia, the Kidney-Specific WNK1 isoform (KS-WNK1) scaffolds the DCT-expressed WNK signaling pathway within biomolecular condensates of unknown function termed WNK bodies. Here, we show that KS-WNK1 amplifies kidney tubule reactivity to blood [K+], in part via WNK bodies. Genetically modified mice with targeted condensate disruption trap the WNK pathway, causing renal salt wasting that is more pronounced in females. In humans, WNK bodies accumulate as plasma potassium falls below 4.0 mmol/L, suggesting that the human DCT experiences the stress of potassium deficiency even when [K+] is in the low-normal range. These data identify WNK bodies as kinase signal amplifiers that mediate tubular [K+] responsiveness, nephron sexual dimorphism, and blood pressure salt-sensitivity. Our results illustrate how biomolecular condensate specialization can optimize a mammalian physiologic stress response that impacts human health.

Authors

Cary R. Boyd-Shiwarski, Rebecca T. Beacham, Jared A. Lashway, Katherine E. Querry, Shawn E. Griffiths, Daniel J. Shiwarski, Sophia A. Knoell, Nga H. Nguyen, Lubika J. Nkashama, Melissa N. Valladares, Anagha Bandaru, Allison L. Marciszyn, Jonathan Franks, Mara Sullivan, Simon C. Watkins, Aylin R. Rodan, Chou-Long Huang, Sean D. Stocker, Ossama B. Kashlan, Arohan R. Subramanya

TNFα impairs platelet function by inhibiting autophagy and disrupting metabolism via Syntaxin-17 downregulation

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Abstract

Platelets play a dual role in hemostasis and inflammation-associated thrombosis and hemorrhage. While the mechanisms linking inflammation to platelet dysfunction remain poorly understood, our previous work demonstrated that TNFα alters mitochondrial mass, platelet activation, and autophagy-related pathways in megakaryocytes. Here, we hypothesized that TNFα impairs platelet function by disrupting autophagy, a process critical for mitochondrial health and cellular metabolism. Using human and murine models of TNFα-driven diseases, including myeloproliferative neoplasms and rheumatoid arthritis, we found that TNFα downregulates STX17, a key mediator of autophagosome–lysosome fusion. This disruption inhibited autophagy, leading to the accumulation of dysfunctional mitochondria and reduced mitochondrial respiration. These metabolic alterations compromised platelet-driven clot contraction, a process linked to thrombotic and hemorrhagic complications. Our findings reveal a mechanism by which TNFα disrupts hemostasis through autophagy inhibition, highlighting TNFα as a critical regulator of platelet metabolism and function. This study provides new insights into inflammation-associated pathologies and suggests autophagy-targeting strategies as potential therapeutic avenues to restore hemostatic balance.

Authors

Guadalupe Rojas-Sanchez, Jorge Calzada-Martinez, Brandon McMahon, Aaron C. Petrey, Gabriela Dveksler, Gerardo P. Espino-Solis, Orlando Esparza, Giovanny Hernandez, Dennis Le, Eric P. Wartchow, Ken Jones, Lucas H. Ting, Catherine Jankowski, Marguerite R. Kelher, Marilyn Manco-Johnson, Marie L. Feser, Kevin D. Deane, Travis Nemkov, Angelo D'Alessandro, Andrew Thorburn, Paola Maycotte, José A. López, Pavel Davizon-Castillo

Expression of full-length dystrophin reverses muscular dystrophy defects in young and old mdx^4cv mice

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Abstract

Gene replacement therapies mediated by adeno-associated viral (AAV) vectors represent a promising approach for treating genetic diseases. However, their modest packaging capacity (~4.7 kb) remains an important constraint and significantly limits their application for genetic disorders involving large genes. A prominent example is Duchenne muscular dystrophy (DMD), whose protein product dystrophin is generated from an 11.2 kb segment of the DMD mRNA. Here, we explored methods that enable efficient expression of full-length dystrophin via triple AAV co-delivery. This method exploits the protein trans-splicing mechanism mediated by split inteins. We identified a combination of efficient and specific split intein pairs that enables the reconstitution of full-length dystrophin from three dystrophin fragments. We show that systemic delivery of low doses of the myotropic AAVMYO1 in mdx4cv mice leads to efficient expression of full-length dystrophin in the hindlimb, diaphragm, and heart muscles. Notably, muscle morphology and physiology were significantly improved in triple AAV-treated mdx4cv mice versus saline-treated controls. This method shows the feasibility of expressing large proteins from several fragments that are delivered using low doses of myotropic AAV vectors. It can be adapted to other large genes involved in disorders for which gene replacement remains challenged by the modest AAV cargo capacity.

Authors

Hichem Tasfaout, Timothy S. McMillen, Theodore R. Reyes, Christine L. Halbert, Rong Tian, Michael Regnier, Jeffrey S. Chamberlain

B cell deficiency induces cytotoxic memory CD8⁺ T cells during influenza-associated bacterial pneumonia

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Abstract

Influenza-associated bacterial super-infections in the lung lead to increased morbidity and mortality. Nearly all people have pre-existing memory to influenza virus, which can protect against subsequent infection in the lung. This study explored the role B cells play in protection against bacterial (Staphylococcus aureus or Klebsiella pneumoniae) super-infection with previous heterotypic influenza memory. B cell deficiency resulted in an increased inflammatory lung environment and lung tissue injury during super-infection. Loss of B cells increased populations of memory CD8+ T cells in the lung and these CD8+ T cells were transcriptionally and functionally distinct from WT mice. Use of antibody-deficient mouse models showed that this phenotype was specifically due to loss of antibody production from B cells. Passive immunization with influenza-antibody serum in B cell deficient mice rescued the CD8+ T cell phenotype. CD8+ T cell depletion and lethal super-infection challenge experiments showed that the cytotoxic memory CD8+ T cells from B cell deficient mice protect against super-infection bacterial burden and mortality. These findings provide insight into the importance of B cells for regulating immune responses against infection.

Authors

Leigh M. Miller, Alexis M. Duray, Ellyse M. Cipolla, Flavia Rago, Brooke P. Dresden, Kristen L. Parenteau, Abhigya Gupta, John F. Alcorn

Patterns of intra- and inter-tumor phenotypic heterogeneity in lethal prostate cancer

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Abstract

Metastatic prostate cancer (mPC) is a clinically and molecularly heterogeneous disease. While there is increasing recognition of diverse tumor phenotypes across patients, less is known about the molecular and phenotypic heterogeneity present within an individual. In this study, we aimed to define the patterns, extent, and consequences of inter- and intra-tumoral heterogeneity in lethal prostate cancer. By combining and integrating in situ tissue-based and sequencing approaches, we analyzed over 630 tumor samples from 52 mPC patients. Our efforts revealed phenotypic heterogeneity at the patient, metastasis, and cellular levels. We observed that intra-patient, inter-tumoral molecular subtype heterogeneity was common in mPC and showed associations with genomic and clinical features. Additionally, cellular proliferation rates varied within a given patient across molecular subtypes and anatomic sites. Single-cell sequencing studies revealed features of morphologically and molecularly divergent tumor cell populations within a single metastatic site. These data provide a deeper insight into the complex patterns of tumoral heterogeneity in mPC with implications for clinical management and the future development of diagnostic and therapeutic approaches.

Authors

Martine P. Roudier, Roman Gulati, Erolcan Sayar, Radhika A. Patel, Micah Tratt, Helen M. Richards, Paloma Cejas, Miguel Munoz Gomez, Xintao Qiu, Yingtian Xie, Brian Hanratty, Samir Zaidi, Jimmy L. Zhao, Mohamed Adil, Chitvan Mittal, Yibai Zhao, Ruth Dumpit, Ilsa Coleman, Jin-Yih Low, Thomas Persse, Patricia C. Galipeau, John K. Lee, Maria Tretiakova, Meagan Chambers, Funda Vakar-Lopez, Lawrence D. True, Marie Perrone, Hung-Ming Lam, Lori A. Kollath, Chien-Kuang C. Ding, Stephanie Harmon, Heather H. Cheng, Evan Y. Yu, Robert B. Montgomery, Jessica E. Hawley, Daniel W. Lin, Eva Corey, Michael T. Schweizer, Manu Setty, Gavin Ha, Charles L. Sawyers, Colm Morrissey, Henry W. Long, Peter S. Nelson, Michael C. Haffner

The macrophage-intrinsic MDA5-IRF5 axis drives HIV-1 intron-containing RNA-induced inflammatory responses

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Abstract

Despite effective antiretroviral therapy (ART), transcriptionally competent HIV-1 reservoirs persist and contribute to persistent immune activation in people living with HIV (PWH). HIV-1-infected macrophages are important mediators of chronic innate immune activation, though mechanisms remain unclear. We previously reported that nuclear export and cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) activates mitochondrial antiviral signaling protein (MAVS)-mediated type I interferon (IFN) responses in macrophages. In this study, we demonstrate an essential role of melanoma differentiation-associated protein 5 (MDA5) in sensing HIV-1 icRNA and promoting MAVS-dependent IRF5 activation in macrophages. Suppression of MDA5, but not RIG-I expression nor disruption of endosomal TLR pathway, abrogated HIV-1 icRNA-induced type I IFN responses and IP-10 expression in macrophages. Furthermore, induction of IP-10 in macrophages upon HIV-1 icRNA sensing by MDA5 was dependent on IRF5. Additionally, monocytes and MDMs from older (>50 years) individuals exhibit constitutively higher levels of IRF5 expression compared to younger (<35 years) individuals, and HIV-1 icRNA induced IP-10 expression was significantly enhanced in older macrophages, which was attenuated upon ablation of IRF5 expression suggesting that IRF5 functions as a major mediator of pro-inflammatory response downstream of MDA5-dependent HIV-1 icRNA sensing, dysregulation of which might contribute to chronic inflammation in older PWH.

Authors

Sita Ramaswamy, Hisashi Akiyama, Jacob Berrigan, Andrés A. Quiñones-Molina, Alex J. Olson, Yunhan Chen, YanMei Liang, Andrew J. Henderson, Archana Asundi, Manish Sagar, Suryaram Gummuluru

Disruption of KLHL37-N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

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Abstract

MYCN amplification accounts for the most common genetic aberration in neuroblastoma and strongly predicts the aggressive progression and poor clinical prognosis. However, clinically effective therapies that directly target N-Myc activity are limited. N-Myc is a transcription factor, and its stability are tightly controlled by ubiquitination-dependent proteasomal degradation. Here, we discovered that Kelch-like protein 37 (KLHL37) played a crucial role in enhancing the protein stability of N-Myc in neuroblastoma. KLHL37 directly interacted with N-Myc to disrupt the N-Myc/FBXW7 interaction, thereby stabilizing N-Myc and enabling tumor progression. Suppressing KLHL37 effectively induced the degradation of N-Myc and exhibited a profound inhibitory effect on the growth of MYCN-amplified neuroblastoma. Notably, we identified RTA-408 as an inhibitor of KLHL37 to disrupt KLHL37-N-Myc complex, promoting the degradation of N-Myc and suppressing neuroblastoma in vivo and in vitro. Moreover, we elucidated the therapeutic potential of RTA-408 for neuroblastoma by utilizing the PDC and PDX tumor models. RTA408's anti-tumor effects may not be exclusively via KLHL37, and specific KLHL37 inhibitors are expected to be developed in the future. These findings not only uncover the biological function of KLHL37 in regulating N-Myc stability, but also indicate that KLHL37 inhibition is a promising therapeutic regimen for neuroblastoma, especially in MYCN-amplified patients.

Authors

Senfeng Xiang, Pengfei Chen, Xiaoxian Shi, Hanqi Cai, Zihan Shen, Luyang Liu, Aixiao Xu, Jianhua Zhang, Xingya Zhang, Shaowei Bing, Jinhu Wang, Xuejing Shao, Ji Cao, Bo Yang, Qiaojun He, Meidan Ying

Diet-induced obesity promotes endothelial cell desensitization to VEGF-A and permanent islet vessel dysfunction in mice

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Abstract

Pancreatic islet microvasculature is essential for optimal islet function and glucose homeostasis. However, islet vessel pathogenesis in obesity and its role in the manifestation of metabolic disorders remain understudied. Here, we depict the time-resolved decline of intra-islet endothelial cell responsiveness to vascular endothelial cell growth factor A (VEGF-A) and islet vessel function in a mouse model of diet-induced obesity. Longitudinal imaging of sentinel islets transplanted into mouse eyes revealed substantial vascular remodeling and diminished VEGF-A response in islet endothelial cells after 12 weeks of western diet (WD) feeding. This led to islet vessel barrier dysfunction and hemodynamic dysregulation, delaying transportation of secreted insulin into the blood. Notably, islet vessels exhibited a metabolic memory of previous WD feeding. Neither VEGF-A sensitivity nor the other vascular alterations was fully restored by control diet (CD) refeeding, resulting in modest yet significant impairment in glucose clearance despite normalized insulin sensitivity. Mechanistic analysis implicated hyperactivation of atypical protein kinase C (aPKC) under both WD and recovery conditions, which inhibited VEGF receptor 2 (VEGFR2) internalization and blunted VEGF-A triggered signal transduction in endothelial cells. In summary, prolonged WD feeding causes irreversible islet endothelial cell desensitization to VEGF-A and islet vessel dysfunction, directly undermining glucose homeostasis.

Authors

Yan Xiong, Andrea Dicker, Montse Visa, Erwin Ilegems, Per-Olof Berggren