Identification of a neutrophil-specific PIK3R1 mutation facilitates targeted treatment in a patient with Sweet syndrome

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Abstract

BACKGROUND. Acute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. Disease pathophysiology remains elusive, and current dogma suggests Sweet syndrome is a “reactive” process to an unknown antigen. Corticosteroids and steroid-sparing agents remain front-line therapies, but refractory cases pose a clinical challenge. METHODS. A 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole genome sequencing, transcriptomic profiling, and cellular assays of patient’s skin and neutrophils were performed. RESULTS. We identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration towards IL-1β and neutrophil respiratory burst. Targeted treatment with an IL-1R1 antagonist in the patient resulted in a dramatic therapeutic response and enabled tapering of corticosteroids. CONCLUSIONS. Dysregulated PI3K-AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests Sweet syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogenous disease that resulted in a rational and successful clinical intervention. Future cases will benefit from efforts to identify potential mutations. The ability to directly interrogate diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for challenging patients. FUNDING Berstein Foundation, NIH, VA, Moseley Foundation, and H.T. Leung Foundation.

Authors

Shreya Bhattacharya, Sayon Basu, Emily Sheng, Christina M. Murphy, Jenny Wei, Anna E. Kersh, Caroline A. Nelson, Joshua S. Bryer, Hovik A. Ashchyan, Katherine T. Steele, Amy K. Forrestel, John T. Seykora, Robert G. Micheletti, William D. James, Misha Rosenbach, Thomas H. Leung

Integration of ER protein quality control mechanisms defines β-cell function and ER architecture

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Abstract

Three principal ER quality-control mechanisms, namely, unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the three pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β-cells. SEL1L-HRD1 ERAD deficiency in β-cells triggers activation of autophagy via IRE1α [an endogenous ERAD substrate]. In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β-cells leads to diabetes in mice shortly after weaning, with premature death by ~11 weeks of age, associated with marked ER retention of proinsulin and β-cell loss. Using focus-ion beam scanning electron microscopy (FIB-SEM) powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β-cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the three ER quality-control mechanisms in the dynamic regulation of organellar architecture and β-cell function.

Authors

Neha Shrestha, Mauricio Torres, Jason Zhang, You Lu, Leena Haataja, Rachel B. Reinert, Jeffrey Knupp, Yu-Jie Chen, Gunes Parlakgul, Ana Paula Arruda, Billy Tsai, Peter Arvan, Ling Qi

Molecular identification of bulbospinal ON neurons by GPER which drives pain and morphine tolerance

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Abstract

The rostral ventromedial medulla (RVM) exerts bi-directional descending modulation of pain, attributable to the activity of electrophysiologically-identified pro-nociceptive ON and anti-nociceptive OFF neurons. Here we report that GABAergic ON neurons specifically express G protein-coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, whilst their ablation abrogated pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain and ameliorated morphine analgesia through desensitizing μ-type opioid receptor (MOR)-mediated activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly support GPER as a marker for GABAergic ON cells and also illuminate the mechanisms underlying hormonal regulation of pain and analgesia, highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

Authors

Yingfu Jiao, Po Gao, Li Dong, Xiaowei Ding, Youqiang Meng, Jiahong Qian, Ting Gao, Ruoxi Wang, Tao Jiang, Yunchun Zhang, Dexu Kong, Yi Wu, Sihan Chen, Saihong Xu, Dan Tang, Ping Luo, Meimei Wu, Li Meng, Daxiang Wen, Changhao Wu, Guohua Zhang, Xueyin Shi, Weifeng Yu, Weifang Rong

One-year follow-up of the CAPSID randomized trial for high-dose convalescent plasma in severe COVID-19 patients

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Abstract

BACKGROUND. Results of many randomized trials on COVID-19 convalescent plasma (CCP) have been reported but information on long-term outcome after CCP treatment is limited. The objectives of this extended observation of the randomized CAPSID trial are to assess long-term outcome and disease burden in patients initially treated with or without CCP. METHODS. Of 105 randomized patients, 50 participated in the extended observation. Quality of life (QoL) was assessed by questionnaires and a structured interview. CCP-donors (n=113) with asymptomatic to moderate COVID-19 were included as a reference group.RESULTS. The median follow-up of patients was 396 days, the estimated 1-year survival was 78.7% in the CCP and 60.2% in the control group (p=0.08). The subgroup treated with a higher cumulative amount of neutralizing antibodies showed a better 1-year survival compared to the control group (91.5% versus 60.2%; p=0.01). Medical events and QoL assessments showed a consistent trend for better results in the CCP group without reaching statistical significance. There was no difference in the increase of neutralizing antibodies after vaccination between CCP and the control group. CONCLUSION. The trial demonstrated a trend towards better outcome in the CCP group without reaching statistical significance. A pre-defined subgroup analysis showed a significant better outcome (long-term survival; time to discharge from ICU and time to hospital discharge) among those who received a higher amount of neutralizing antibodies compared to the control group. A substantial long-term disease burden remains after severe COVID-19. TRIAL REGISTRATION. EudraCT number 2020-001310-38 FUNDING. Bundesministerium für Gesundheit (German Federal Ministry of Health): ZMVI1-2520COR802/ZMI1-2521COR802

Authors

Sixten Körper, Beate Grüner, Daniel Zickler, Thomas Wiesmann, Patrick Wuchter, Rainer Blasczyk, Kai Zacharowski, Peter Spieth, Torsten Tonn, Peter Rosenberger, Gregor Paul, Jan Pilch, Joachim Schwäble, Tamam Bakchoul, Thomas Thiele, Julian Knoerlein, Matthias M. Dollinger, Joerg Krebs, Martin Bentz, Victor M. Corman, Dzenan Kilalic, Gerlinde Schmidtke-Schrezenmeier, Philipp M. Lepper, Lucas Ernst, Hinnerk Wulf, Alexandra Ulrich, Manfred Weiss, Jan Kruse, Thomas Burkhardt, Rebecca Müller, Harald Klüter, Michael Schmidt, Bernd Jahrsdörfer, Ramin Lotfi, Markus Rojewski, Thomas Appl, Benjamin Mayer, Philipp Schnecko, Erhard Seifried, Hubert Schrezenmeier

SCF-SKP2 E3 ubiquitin ligase links mTORC1-ER stress-ISR with YAP activation in murine renal cystogenesis

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Abstract

The Hippo pathway nuclear effector Yes-associated protein 1 (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1) driven by ERK1/2 and PI3K/AKT cascades induced endoplasmic reticulum (ER) proteotoxic stress. To reduce it by reprogramming translation, the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF-S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α-phosphorylation-deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress-ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan, impeded these processes. Restoring ER homeostasis, and/or interfering with the SKP2-YAP interaction represent novel potential therapeutic avenues for stemming the progression of renal cystogenesis.

Authors

Dibyendu K. Panda, Xiuying Bai, Yan Zhang, Nicholas A. Stylianesis, Antonis E. Koromilas, Mark L. Lipman, Andrew C. Karaplis

Mitochondrial dysfunction reactivates α-fetoprotein expression that drives copper-dependent immunosuppression in mitochondrial disease models

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Abstract

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identify a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that results in atrophy of the spleen and thymus and causes a peripheral white blood cell deficiency. We demonstrate that the leukopenia is caused by α-fetoprotein, which requires copper and the cell surface receptor CCR5 to promote white blood cell death. We further show that α-fetoprotein expression is upregulated in several cell types upon inhibition of oxidative phosphorylation, including a muscle cell model of Barth syndrome. Collectively, our data argue that α-fetoprotein secreted by bioenergetically stressed tissue suppresses the immune system, an effect which may explain the recurrent infections that are observed in a subset of mitochondrial diseases or in other disorders with mitochondrial involvement.

Authors

Kimberly A. Jett, Zakery N. Baker, Amzad Hossain, Aren Boulet, Paul A. Cobine, Sagnika Ghosh, Philip Ng, Orhan Yilmaz, Kris Barreto, John DeCoteau, Karen Mochoruk, George N. Ioannou, Christopher Savard, Sai Yuan, Osama H.M.H. Abdalla, Christopher Lowden, Byung-Eun Kim, Hai-Ying Mary Cheng, Brendan J. Battersby, Vishal M. Gohil, Scot C. Leary

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity

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Abstract

The SARS-CoV-2 spike (S) glycoprotein is synthesized as large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/2 cleavage site and K986→P and V987→P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of S broadly reactive antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Authors

Christian Meyer zu Natrup, Alina Tscherne, Christine Dahlke, Malgorzata Ciurkiewicz, Dai-Lun Shin, Anahita Fathi, Cornelius Rohde, Georgia Kalodimou, Sandro Halwe, Leonard Limpinsel, Jan H. Schwarz, Martha Klug, Meral Esen, Nicole Schneiderhan-Marra, Alex Dulovic, Alexandra Kupke, Katrin Brosinski, Sabrina Clever, Lisa-Marie Schünemann, Georg Beythien, Federico Armando, Leonie Mayer, Leonie M. Weskamm, Sylvia Jany, Astrid Freudenstein, Tamara Tuchel, Wolfgang Baumgärtner, Peter Kremsner, Rolf Fendel, Marylyn M. Addo, Stephan Becker, Gerd Sutter, Asisa Volz

A SARM1/mitochondrial feedback loop drives neuropathogenesis in a Charcot-Marie-Tooth disease type 2A rat model

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Abstract

Charcot-Marie-Tooth disease (CMT) type 2A is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying axonal pathology is unknown. SARM1, the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, NMJ abnormalities, muscle atrophy, and mitochondrial abnormalities, all hallmarks of the human disease. We generated Sarm1 knockout and Mfn2H361Y, Sarm1 double mutant rats and find that deletion of Sarm1 rescues axonal, synaptic, muscle, and functional phenotypes, demonstrating that SARM1 is responsible for much of the neuropathology in this model. Despite the presence of mutant MFN2 protein in these double mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activate SARM1, and activated SARM1 feeds back on mitochondria to exacerbate mitochondrial pathology. As such, this work identifies SARM1 inhibition as an exciting therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

Authors

Yurie Yamada, Amy Strickland, Yo Sasaki, A. Joseph Bloom, Aaron DiAntonio, Jeffrey Milbrandt

Macrophage depletion blocks congenital SARM1-dependent neuropathy

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Abstract

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions, V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Authors

Caitlin B. Dingwall, Amy Strickland, Sabrina W. Yum, Aldrin Kay-Yuen Yim, Jian Zhu, Peter L. Wang, Yurie Yamada, Robert E. Schmidt, Yo Sasaki, A. Joseph Bloom, Aaron DiAntonio, Jeffrey Milbrandt

Autosomal recessive progeroid syndrome due to homozygosity for a TOMM7 variant

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Abstract

Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here we report a 21-year-old man of Chinese origin who had a novel autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia and partial lipodystrophy. Analyses of exome sequencing data of the entire family revealed only one rare homozygous missense variant, (c.86C>T; p.Pro29Leu), in TOMM7 in the proband, while the parents and two unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex constitutes the outer membrane pore for import of several preproteins into mitochondria. Proteomics analyses of mitochondria from cultured fibroblasts of the proband, as compared to control fibroblasts, revealed increases in several proteins involved in oxidative phosphorylation, but reduced abundance of proteins involved in the phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared to control fibroblasts. We conclude that altered mitochondrial protein import due to loss of function bi-allelic variant in TOMM7 can cause severe growth retardation and progeroid features.

Authors

Abhimanyu Garg, Wee-Teik Keng, Zhenkang Chen, Adwait Amod Sathe, Chao Xing, Pavithira Devi Kailasam, Yanqiu Shao, Nicholas P. Lesner, Claire B. Llamas, Anil K. Agarwal, Prashant Mishra