HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that Caspase 6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) leads to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment can restrain p65 NF-κB transcriptional activity by cytoplasmic sequestration NF-κB signaling component, p65 NF-κB, and attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidney of mice with unilateral ureteral obstruction and lipopolysaccharide-induced acute kidney injury. Collectively, our findings indicate that the C-terminal region of HIPK2 is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential anti-inflammatory agents to treat kidney disease.
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.
Lisa Duvick, W. Michael Southern, Kellie A. Benzow, Zoe N. Burch, Hillary P. Handler, Jason S. Mitchell, Hannah Kuivinen, Udaya Gadiparthi, Praseuth Yang, Alyssa Soles, Carrie A. Sheeler, Orion Rainwater, Shannah Serres, Erin B. Lind, Tessa Nichols-Meade, Brennon O'Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement of cholesterol for activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and non-responsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells detects T cell activation and has potential to assess the success of ICI therapy.
Nicholas G. Ciavattone, Jenny Nan Guan, Alex Farfel, Jenelle Stauff, Timothy J. Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), were dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression in a destabilization of the medial meniscus (DMM) mouse model, while genetic deficiency of plasmin activator inhibitor (PAI-1), or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans, induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, PDK1, AKT, and ERK signaling cascades, and activates matrix metalloproteinases (pro-MMPs) to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
Qian Wang, Guoqiang Shao, Xiaoyi Zhao, Heidi H. Wong, Kate Chin, Mackenzie Zhao, Audrey Bai, Michelle S. Bloom, Zelda Z. Love, Constance R. Chu, Zhen Cheng, William H. Robinson
BACKGROUND. Survivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans. METHODS. We measured the levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from a cohort of 341 patients with respiratory failure and 13 healthy control patients, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We flow-cytometry sorted neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing. RESULTS. Microglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines. CONCLUSIONS. Prolonged lung inflammation results in sustained elevations in circulating cytokines patients with SARS-CoV-2 pneumonia compared to those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction. FUNDING. SCRIPT U19AI135964.
Rogan A. Grant, Taylor A. Poor, Lango Sichizya, Estefani Diaz, Joseph I. Bailey, Sahil Soni, Karolina J. Senkow, Xóchitl G. Pérez-Leonor, Hiam Abdala-Valencia, Ziyan Lu, Helen K. Donnelly, Lacy M. Simons, Egon A. Ozer, Robert M. Tighe, Jon W. Lomasney, Richard G. Wunderink, Benjamin D. Singer, Alexander V. Misharin, G.R. Scott Budinger
BACKGROUND COVID-19 convalescent plasma (CCP) viral specific antibody levels that translate into recipient post-transfusion antibody levels sufficient to prevent disease progression is not defined. METHODS This secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low post-transfusion antibody levels was established by two methods: 1) analyzing virus neutralization-equivalent anti-Spike-receptor-binding-domain immunoglobulin G (anti-S-RBD IgG) responses in donors or 2) receiver operated curve (ROC) analysis. RESULTS SARS-CoV-2 anti-S-RBD IgG antibody was volume diluted 21.3 fold into post-transfusion seronegative recipients from matched donor units. Viral specific antibody delivered approximated 1.2 mg. The high antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma) with antibody cutoffs established by both methods-donor-based virus neutralization cutoff in post-transfusion recipients: (0/85; 0% versus 15/276; 5.6%) p=0.03 or ROC based cutoff: (0/94; 0% versus 15/267; 5.4%) p=0.01. CONCLUSION In unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use. Trial registration: NCT04373460 FUNDING Defense Health Agency and others.
Han-Sol Park, Anna Yin, Caelan Barranta, John S. Lee, Christopher A. Caputo, Jaiprasath Sachithanandham, Maggie Li, Steve Yoon, Ioannis Sitaras, Anne Jedlicka, Yolanda Eby, Malathi Ram, Reinaldo E. Fernandez, Owen R. Baker, Aarthi G. Shenoy, Giselle S. Mosnaim, Yuriko Fukuta, Bela Patel, Sonya L. Heath, Adam C. Levine, Barry R. Meisenberg, Emily S. Spivak, Shweta Anjan, Moises A. Huaman, Janis E. Blair, Judith S. Currier, James H. Paxton, Jonathan M. Gerber, Joann R. Petrini, Patrick B. Broderick, William Rausch, Marie Elena Cordisco, Jean Hammel, Benjamin Greenblatt, Valerie C. Cluzet, Daniel Cruser, Kevin Oei, Matthew Abinante, Laura L. Hammitt, Catherine G. Sutcliffe, Donald N. Forthal, Martin S. Zand, Edward R. Cachay, Jay S. Raval, Seble G. Kassaye, Christi E. Marshall, Anusha Yarava, Karen Lane, Nichol A. McBee, Amy L. Gawad, Nicky Karlen, Atika Singh, Daniel E. Ford, Douglas A. Jabs, Lawrence J. Appel, David M. Shade, Bryan Lau, Stephan Ehrhardt, Sheriza N. Baksh, Janna R. Shapiro, Jiangda Ou, Yu Bin Na, Maria D. Knoll, Elysse Ornelas-Gatdula, Netzahualcóyotl Arroyo-Currás, Thomas J. Gniadek, Patrizio Caturegli, Jinke Wu, Nelson Ndahiro, Michael J. Betenbaugh, Alyssa Ziman, Daniel F. Hanley, Arturo Casadevall, Shmuel Shoham, Evan M. Bloch, Kelly A. Gebo, Aaron A.R. Tobian, Oliver Laeyendecker, Andrew Pekosz, Sabra L. Klein, David J. Sullivan
The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected >5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may impact cardiac contractility.
Colleen M. Kelly, Jody L. Martin, Michael J. Previs
Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the alpha-ketoglutarate dehydrogenase (OGDH) gene, which encodes for an enzyme that is part of the tricarboxylic acid cycle (TCA cycle) as essential for GBM growth. Moreover, by combining a transcriptome and metabolite screening analyses we discovered that loss of function of OGDH by the clinically validated drug compound, CPI-613, was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3-mimetic, ABT263) in patient-derived xenograft as well neurosphere GBM cultures. CPI-613 mediated energy deprivation drove an integrated stress response with an up-regulation of the BH3-only domain protein, Noxa in an ATF4 dependent manner as demonstrated by genetic loss of function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with interference of the TCA-cycle might be a treatment strategy for GBM.
Trang T.T. Nguyen, Consuelo Torrini, Enyuan Shang, Chang Shu, Jeong-Yeon Mun, Qiuqiang Gao, Nelson Humala, Hasan O. Akman, Guoan Zhang, Mike-Andrew Westhoff, Georg Karpel-Massler, Jeffrey N. Bruce, Peter Canoll, Markus D. Siegelin
Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we utilized single-cell RNA sequencing (scRNA-seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment (TME) components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Lastly, murine experiments and scRNA-seq analysis of a treated patient’s tumor demonstrated that Famitinib plus anti-PD-1 antibody could advance TLS in thyroid cancer. Conclusively, we displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.
Pei-Zhen Han, Wei-Dong Ye, Peng-Cheng Yu, Li-Cheng Tan, Xiao Shi, Xu-Feng Chen, Cong He, Jia-Qian Hu, Wen-Jun Wei, Zhong-Wu Lu, Ning Qu, Yu Wang, Qing-Hai Ji, Dong-Mei Ji, Yu-Long Wang
IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator also increased following TCF4 siRNA knockdown and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a, that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together these findings identify a role for TCF4 in the negative regulation of IL-17C, which alone and with TNF-α and IL-17A, feedback to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.
Yanyun Jiang, Dennis Gruszka, Chang Zeng, William R. Swindell, Christa Gaskill, Christian Sorensen, Whitney Brown, Roopesh Singh Gangwar, Lam C. Tsoi, Joshua Webster, Sigrun Laufey Sigurdardottir, Mrinal K. Sarkar, Ranjitha Uppala, Austin Kidder, Xianying Xing, Olesya Plazyo, Enze Xing, Allison C. Billi, Emanual Maverakis, J. Michelle Kahlenberg, Johann Gudjonsson, Nicole L. Ward
IKK2-NF𝜅B pathway mediated-inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2-NF𝜅B pathway in medial calcification remains to be elucidated. In this study, we found that CKD induces inflammatory pathways through the local activation of the IKK2-NF𝜅B pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2-NF𝜅B pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF𝜅B by SMC-specific I𝜅B-α deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2-NF𝜅B pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF𝜅B reduced CKD-dependent vascular cell death. In addition, increased calcifying extracellular vesicles through the inhibition of the IKK2-NF𝜅B pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2-NF𝜅B pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.
Shinobu Miyazaki-Anzai, Masashi Masuda, Audrey L. Keenan, Yuji Shiozaki, Jose G. Miranda, Makoto Miyazaki
Excessive lipolysis in white adipose tissues (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In human, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocytes lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicate that mice lacking P2Y13-R show a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared to their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez
Allergic Airway Disease (AAD) is an example of type 2 inflammation which leads to chronic airway eosinophilia controlled by CD4 Th2 cells. Inflammation is reinforced by mast cells and basophils armed with allergen-specific IgE made by allergen-specific B2 B cells of the adaptive immune system. Little is known about how AAD is affected by innate B1 cells which produce natural antibodies (NAbs) that facilitate apoptotic cell clearance and detect damage and pathogen associated molecular patterns (DAMPS and PAMPS). We used transgenic mouse models lacking either B cells or NAbs in distinct mouse models of AAD, that require either DAMPS or PAMPS as the initial trigger for type 2 immunity. In a DAMP-induced allergic model, driven by alum and uric acid, mouse strains lacking B cells (CD19DTA), NAbs (IgHEL MD4), or all secreted antibodies (sIgm-/-Aid-/-), displayed significant reduction in both eosinophilia and Th2 priming compared to wild-type or Aid-/- mice lacking only germinal center dependent high-affinity class switched antibodies. Replenishing B-cell deficient mice with either unimmunized B1 B cells or NAbs during sensitization restored eosinophilia, suggesting NAbs are required for licensing antigen presenting cells to prime type 2 immunity. Conversely, PAMP-dependent type 2 priming to house dust mite or Aspergillus were not dependent on NAbs. This study reveals an underappreciated role of B1 B cell-generated natural antibodies in selectively driving DAMP-induced type-2 immunity.
Arlind B. Mara, Kavita Rawat, William T. King, Claudia V. Jakubzick
Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases were shown to facilitate virus entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleave the S protein at multiple sites and abrogate virus entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with virus clearance and inflammation in COVID-19 patients.
Nathan G.F. Leborgne, Christelle Devisme, Nedim Kozarac, Inês Berenguer Veiga, Nadine Ebert, Aurélie Godel, Llorenç Grau-Roma, Melanie Scherer, Philippe Plattet, Volker Thiel, Gert Zimmer, Adriano Taddeo, Charaf Benarafa
Prior studies showed that polyQ-expanded AR is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of NAD+-dependent sirtuin 1 (SIRT1) is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating post-symptomatic transgenic SBMA mice with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cord of SBMA mice. Metabolite and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that utilize NAD+, in addition to the NAD salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of Nmrk2, which encodes a key kinase responsible for NR phosphorylation, allowing its utilization by the NAD salvage pathway. Together these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation due to decreased levels of Nmrk2.
Danielle DeBartolo, Frederick J. Arnold, Yuhong Liu, Elana Molotsky, Hsin-Yao Tang, Diane E. Merry
Mesenchymal stem cells, suffering from diverse gene hits, undergoes malignant transformation and aberrant osteochondral differentiation. Src homology region 2- (SH2-) containing protein tyrosine phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, regulates multicellular differentiation, proliferation, and transformation. However, the role of SHP2 in MSC fate determination remains unclear. Here, we showed that MSCs bearing the activating SHP2E76K mutation underwent malignant transformation into sarcoma stem-like cells (SSCs). We revealed that the SHP2E76K mutation in mouse MSCs led to hyperactive mitochondrial metabolism by activating mitochondrial complexes I and III. Inhibition of complexes I and III prevented hyperactive mitochondrial metabolism and malignant transformation of SHP2E76K MSCs. Mechanistically, we confirmed that SHP2 underwent liquid–liquid phase separation (LLPS) in SHP2E76K MSCs. SHP2 LLPS led to its dissociation from complexes I and III, causing their hyperactivation. Blockade of SHP2 LLPS by LLPS‒defective mutations or allosteric inhibitors suppressed complex I and III hyperactivation as well as malignant transformation of SHP2E76K MSCs. These findings reveal that complex I and III hyperactivation driven by SHP2 LLPS promotes malignant transformation of SHP2E76K MSCs and suggest that inhibition of SHP2 LLPS could be a potential therapeutic target for the treatment of activating SHP2‒associated cancers.
Chen Kan, Zhenya Tan, Liwei Liu, Bo Liu, Li Zhan, Jicheng Zhu, Xiaofei Li, Keqiong Lin, Jia Liu, Yakun Liu, Fan Yang, Mandy Wong, Siying Wang, Hong Zheng
Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2 - a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1 knockout cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global knockout of Spns1 caused embryonic lethality between E12.5-E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1 knockout (Spns1-KO) mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K-AKT signaling pathway. Furthermore, we identified three human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and exhibiting cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
Programmed cell death protein 1 (PD-1), a coinhibitory T-cell checkpoint, is also expressed on macrophages (Mφ) in pathogen- or tumor-driven chronic inflammation. Increasing evidence underscores the importance of PD-1 on Mφ for dampening immune responses. However, the mechanism governing PD-1 expression in Mφ in chronic inflammation remains largely unknown. TGF-β1 (transforming growth factor-β1) is abundant within chronic inflammatory microenvironments. Here, based on public databases, significant positive correlations between PDCD1 and TGFB1 gene expression were observed in most human tumors. Of note, among immune infiltrates, Mφ as the predominant infiltrate expressed higher PDCD1 and TGFBR1/TGFBR2 genes. MC38 colon cancer and S. japonicum infection were used as experimental models for chronic inflammation. PD-1hi Mφ from chronic inflammatory tissues displayed an immunoregulatory pattern and expressed a higher level of TGF-β receptors. Either TGF-β1-neutralizing antibody administration or Mφ-specific Tgfbr1 knockdown largely reduced PD-1 expression on Mφ in animal models. We further demonstrated that TGF-β1 directly induced PD-1 expression on Mφ. Mechanistically, TGF-β1-induced PD-1 expression on Mφ was dependent on SMAD3 and STAT3, which formed a complex at the Pdcd1 promoter. Collectively, our study shows that Mφ adapt to chronic inflammation through TGF-β1-triggered cooperative SMAD3-STAT3 signaling that induces PD-1 expression and modulates Mφ function.
Zhigang Lei, Rui Tang, Yu Wu, Chenxu Mao, Weijie Xue, Junyao Shen, Jiaojiao Yu, Xiaohong Wang, Xin Qi, Chuan Wei, Lei Xu, Jifeng Zhu, Yalin Li, Xiujun Zhang, Chunyan Ye, Xiaojun Chen, Xiaojun Yang, Sha Zhou, Chuan Su
Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for anti-angiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93-/- mice but metastatic dissemination was increased, associated with a disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 (MMP9) at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyper-phosphorylation of VEGFR2 in endothelial cells. Antagonistic-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93-/- mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.
Kalyani Vemuri, Beatriz de Alves Pereira, Patricia Fuenzalida, Yelin Subashi, Stefano Barbera, Luuk van Hooren, Marie Hedlund, Fredrik Pontén, Cecilia Lindskog, Anna-Karin Olsson, Roberta Lugano, Anna Dimberg
Myocardial ischemia/reperfusion (MI/R) injury is a major cause of adverse outcomes of revascularization following myocardial infarction. Anaerobic glycolysis during myocardial ischemia is well-studied, but the role of aerobic glycolysis during the early phase of reperfusion is incompletely understood. Lactylation of Histone H3 (H3) is an epigenetic indicator of the glycolytic switch. Heat shock protein A12A (HSPA12A) is an atypic member of the HSP70 family. In the present study, we report that during reperfusion following myocardial ischemia, HSPA12A was downregulated and aerobic glycolytic flux was decreased in cardiomyocytes. Notably, HSPA12A knockout in mice exacerbated MI/R-induced aerobic glycolysis decrease, cardiomyocyte death, and cardiac dysfunction. Gain- and loss-of-function studies demonstrated that HSPA12A was required to support cardiomyocyte survival upon hypoxia/reoxygenation (H/R) challenge, and that its protective effects were mediated by maintaining aerobic glycolytic homeostasis for H3 lactylation. Further analyses revealed that HSPA12A increased Smurf1-mediated Hif1α protein stability, thus increasing glycolytic gene expression to maintain appropriate aerobic glycolytic activity to sustain H3 lactylation during reperfusion, and ultimately improving cardiomyocyte survival to attenuate MI/R injury.
Wansu Yu, Qiuyue Kong, Surong Jiang, Yunfan Li, Zhaohe Wang, Qian Mao, Xiaojin Zhang, Qianhui Liu, Pengjun Zhang, Yuehua Li, Chuanfu Li, Zhengnian Ding, Li Liu