SPOP mutations increase PARP inhibitor sensitivity via the CK2/PIAS1/SPOP axis in prostate cancer
Zhang et al. report that disruption of the CK2/PIAS1/SPOP signaling axis, by SPOP mutations or pharmacological blockade, increases the efficacy of the PARP inhibitor olaparib in prostate cancer models. The cover art represents DNA damage induced by PARP inhibitors, with SUMOylated SPOP promoting DNA damage repair. Cover art was generated with the Dreamina AI tool.
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Research Letter
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Abstract
Authors
Máté Sándor, Balázs Csaba Németh, Alexandra Demcsák, Miklós Sahin-Tóth
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Research Articles
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Abstract
Glycine and β-alanine activate glycine receptors (GlyRs), with glycine known to enhance insulin secretion from pancreatic islet β cells, primarily through GlyR activation. However, the effects of GlyR activation on β cell proliferation have not been examined. Here, we aim to investigate the potential proliferative effects of glycine and β-alanine on islets. In vitro experiments on mouse and human islets revealed that glycine and β-alanine, via GlyR activation, stimulated the proliferation of β cells and α cells, without affecting insulin or glucagon secretion. Further analysis indicated the involvement of the PI3K/mTORC1/p70S6K signaling pathway in this process. Inhibition of GlyRs and PI3K/mTORC1/p70S6K signaling attenuated proliferative effects of glycine and β-alanine. In vivo and ex vivo studies supported these findings, showing increased β and α cell mass after 12 weeks of oral administration of glycine and β-alanine, with no changes in insulin secretion or glucose homeostasis under normal conditions. However, during an acute insulin resistance induced by insulin receptor antagonist S961, glycine and β-alanine enhanced insulin secretion and reduced blood glucose levels by increasing β cell secretory capacity. These findings demonstrate glycine and β-alanine in vivo and in vitro promote islet cell proliferation via GlyR activation and the PI3K/mTORC1/p70S6K pathway, potentially providing a target to enhance islet capacity.
Authors
Ziyi Zhang, Wenyue W. Ye, Anthony L. Piro, Dian-Shi Wang, Ashley Untereiner, Sulayman A. Lyons, Alpana Bhattacharjee, Ishnoor Singh, Jacqueline L. Beaudry, Beverley A. Orser, Feihan F. Dai, Michael B. Wheeler
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Skin inflammation in juvenile dermatomyositis (JDM) can signal disease onset or flare, and the persistence of cutaneous disease can prevent complete disease remission. The noninvasive study of cutaneous expression signatures through tape stripping (TS) holds the potential to reveal mechanisms underlying disease heterogeneity and organ-specific inflammation. The objectives of this study were to (a) define TS expression signatures in lesional and nonlesional JDM skin, (b) analyze TS signatures to identify JDM disease endotypes, and (c) compare TS and blood signatures. Although JDM lesional skin demonstrated interferon signaling as the top upregulated pathway, nonlesional skin uniquely highlighted pathways involved in metabolism, angiogenesis, and calcium signaling. Both lesional and nonlesional skin shared inflammasome pathway dysregulation. Using unsupervised clustering of skin expression data, we identified a treatment-refractory JDM subgroup distinguished by upregulation of genes associated with mitochondrial dysfunction. The treatment-refractory JDM subgroup also demonstrated higher interferon, angiogenesis, and innate immune expression scores in skin and blood, though scores were more pronounced in skin as compared with blood. TS expression signatures in JDM provided insight into disease mechanisms and molecular subgroups. Skin, as compared with blood, transcriptional profiles served as more sensitive markers to classify disease subgroups and identify candidate treatment targets.
Authors
Jessica L. Turnier, Sarah M.H. Vandenbergen, Madison E. McClune, Christine Goudsmit, Sophia Matossian, Meredith Riebschleger, Nadine Saad, Jacqueline A. Madison, Smriti Mohan, Johann E. Gudjonsson, Lam C. Tsoi, Celine C. Berthier, J. Michelle Kahlenberg
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BACKGROUND Alterations in circulating metabolites have been described in obese metabolic dysfunction–associated steatotic liver disease (MASLD), but data on lean MASLD are lacking. We investigated serum metabolites, including microbial bile acids and short-chain fatty acids (SCFAs), and their association with lean and obese MASLD.METHODS Serum samples from 204 people of European descent were allocated to four groups: lean healthy, lean MASLD, obese healthy, and obese MASLD. Liquid chromatography–mass spectrometry–based metabolomics and linear model analysis were performed. MASLD prediction was assessed based on least absolute shrinkage and selection operator regression. Functional effects of altered molecules were verified in organotypic 3D primary human liver cultures.RESULTS Lean MASLD was characterized by elevated isobutyrate, methionine sulfoxide, propionate, and phosphatidylcholines. Patients with obese MASLD had increased sarcosine and decreased lysine and asymmetric dimethylarginine. Using metabolites, sex, and BMI, MASLD versus healthy could be predicted with a median AUC of 86.5% and 85.6% in the lean and obese subgroups, respectively. Functional experiments in organotypic 3D primary human liver cultures showed propionate and isobutyrate induced lipid accumulation and altered expression of genes involved in lipid and glucose metabolism.CONCLUSION Lean MASLD is characterized by a distinct metabolite pattern related to amino acid metabolism, lipids, and SCFAs, while metabolic pathways of lipid accumulation are differentially activated by microbial metabolites. We highlight an important role of microbial metabolites in MASLD, with implications for predictive and mechanistic assessment of liver disease across weight categories.FUNDING Robert Bosch Stiftung, Swedish Research Council (2021-02801, 2023-03015, 2024-03401), ERC Consolidator Grant 3DMASH (101170408), Ruth and Richard Julin Foundation for Gastroenterology (2021-00158), SciLifeLab and Wallenberg National Program for Data-Driven Life Science (WASPDDLS22:006), Novo Nordisk Foundation (NNF23OC0085944, NNF23OC0084420), PMU-FFF (E-18/28/148-FEL).
Authors
Mathias Haag, Stefan Winter, Aurino M. Kemas, Julia Tevini, Alexandra Feldman, Sebastian K. Eder, Thomas K. Felder, Christian Datz, Bernhard Paulweber, Gerhard Liebisch, Oliver Burk, Volker M. Lauschke, Elmar Aigner, Matthias Schwab
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Systemic lupus erythematosus (SLE), an autoimmune disease, can cause psychiatric disorders, particularly depression, via immune activation. Human umbilical cord mesenchymal stromal cell (hUCMSC) transplantation (MSCT) has been shown to ameliorate immune dysfunction in SLE by inducing immune tolerance. However, whether MSCT can relieve the depressive symptoms in SLE remains incompletely understood. Here, we demonstrate that MSCT relieved early-onset depression-like behavior in both genetically lupus-prone (MRL/lpr) and pristane-induced lupus mice by rescuing impaired hippocampal synaptic connectivity. Transplanted hUCMSCs targeted Th1 cell–derived IFN-γ to inhibit neuronal JAK/STAT1 signaling and downstream CCL8 expression, reducing phagocytic microglia apposition to alleviate synaptic engulfment and neurological dysfunction in young (8-week-old) lupus mice. Systemic delivery of exogenous IFN-γ blunted MSCT-mediated alleviation of synaptic loss and depressive behavior in lupus mice, suggesting that the IFN-γ/CCL8 axis may be an effective therapeutic target and that MSCT is a potential therapy for lupus-related depression. In summary, transplanted hUCMSCs can target systemic immunity to ameliorate psychiatric disorders by rescuing synaptic loss, highlighting the active role of neurons as intermediaries between systemic immunity and microglia in this process.
Authors
Xiaojuan Han, Dandan Wang, Liang Chen, Hua Song, Xiulan Zheng, Xin Zhang, Shengnan Zhao, Jun Liang, Tianshu Xu, Zhibin Hu, Lingyun Sun
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Many risk-eligible women refuse tamoxifen for primary prevention of breast cancer due to concerns about common side effects such as vasomotor symptoms. Tamoxifen may also induce or worsen insulin resistance and hypertriglyceridemia, especially in women with obesity. The combination of bazedoxifene and conjugated estrogens (BZA/CE) reduces vasomotor symptoms and is currently undergoing evaluation for breast cancer risk reduction. However, the impact of BZA/CE on insulin resistance and metabolic health, particularly in those with excess adiposity, is understudied. Here, we examined the effects of obesity on response to BZA/CE in a rat model of breast cancer risk using older ovary-intact rats. Female Wistar rats received carcinogen to increase mammary cancer risk and were fed a high-fat diet to promote obesity. Lean and obese rats were selected based on adiposity, and then randomized to BZA/CE or vehicle for 8 weeks. BZA/CE reduced adiposity, enriched small (insulin-sensitive) mammary adipocytes, increased the abundance of beneficial metabolic gut microbes (Faecalbaculum rodentium and Odoribacter laneus), and reversed obesity-associated changes in lipids and adipokines. BZA/CE also reversed obesity-induced mammary enrichment of cell proliferation pathways, consistent with risk-reducing effects. Together, these data support the use of BZA/CE to improve metabolic health and reduce breast cancer risk in individuals with obesity.
Authors
Erin D. Giles, Katherine L. Cook, Ramsey M. Jenschke, Karen A. Corleto, Danilo Landrock, Tara N. Mahmood, Katherine E. Sanchez, Alina Levin, Stephen D. Hursting, Bruce F. Kimler, Barry S. Komm, Carol J. Fabian
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BACKGROUND The high-dose quadrivalent influenza vaccine (QIV-HD) showed superior efficacy against laboratory-confirmed illness compared with the standard-dose quadrivalent influenza vaccine (QIV-SD) in randomized controlled trials with the elderly. However, specific underlying mechanism remains unclear.METHODS This phase IV randomized controlled trial compared early innate responses induced by QIV-HD and QIV-SD in 59 individuals aged > 65 years. Systemic innate cells and gene signatures at day 0 (D0) and D1 as well as hemagglutinin inhibition antibody (HIA) titers at D0 and D21 after vaccination were assessed.RESULTS QIV-HD elicited robust humoral response with significantly higher antibody titers and seroconversion rates than QIV-SD. At D1 after vaccination, QIV-HD recipients showed significant reduction in innate cells, including conventional DCs and NK cells, compared with QIV-SD, correlating with significantly increased HIA titers at D21. Blood transcriptomic analysis revealed greater amplitude of gene expression in the QIV-HD arm, encompassing genes related to innate immune response, IFNs, and antigen processing and presentation, and correlated with humoral responses. Interestingly, comparative analysis with a literature dataset from young adults vaccinated with influenza standard-dose vaccine highlighted strong similarities in gene expression patterns and biological pathways with the elderly vaccinated with QIV-HD.CONCLUSION QIV-HD induces higher HIA titers than QIV-SD, a youthful boost of the innate gene expression significantly associated with high HIA titers.TRIAL REGISTRATION EudraCT no. 2021-004573-32.
Authors
Olivia Bonduelle, Tristan Delory, Isabelle Franco-Moscardini, Marion Ghidi, Selma Bennacer, Michele Wokam, Mathieu Lenormand, Melissa Petrier, Olivier Rogeaux, Simon de Bernard, Karine Alves, Julien Nourikyan, Bruno Lina, INFLUOMICS Study group, Behazine Combadiere, Cécile Janssen
×Abstract
Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases with strong female predominance. They are caused by T cell–mediated injury of hepatic parenchymal cells, but the mechanisms underlying this sex bias are unknown. Here, we investigated whether testosterone contributes to T cell activation in women with PBC. Compared with sex- and age-matched healthy controls (n = 23), cisgender (cis) women with PBC (n = 24) demonstrated decreased testosterone serum levels and proinflammatory CD4+ T cell profile in peripheral blood. Testosterone suppressed the expression of TNF and IFN-γ by human CD4+ T cells in vitro. In trans men receiving gender-affirming hormone therapy (GAHT) (n = 25), testosterone affected CD4+ T cell function by inhibiting Th1 and Th17 differentiation and by supporting the differentiation into regulatory Treg. Mechanistically, we provide evidence for a direct effect of testosterone on T cells using mice with T cell–specific deletion of the cytosolic androgen receptor. Supporting a role for testosterone in autoimmune liver disease, we observed an improved disease course and profound changes in T cell states in a trans man with AIH/primary sclerosing cholangitis (PSC) variant syndrome receiving GAHT. We here report a direct effect of testosterone on CD4+ T cells that may contribute to future personalized treatment strategies.
Authors
Lara Henze, Nico Will, Dakyung Lee, Victor Haas, Christian Casar, Jasper Meyer, Stephanie Stein, Franziska Mangler, Silja Steinmann, Tobias Poch, Jenny Krause, Johannes Fuss, Johanna Schröder, Alexandra E. Kulle, Paul-Martin Holterhus, Stefan Bonn, Marcus Altfeld, Samuel Huber, Ansgar W. Lohse, Dorothee Schwinge, Christoph Schramm
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Aberrant immune response is a hallmark of asthma, with 5%–10% of patients suffering from severe disease exhibiting poor response to standard treatment. A better understanding of the immune responses contributing to disease heterogeneity is critical for improving asthma management. T cells are major players in the orchestration of asthma, in both mild and severe disease, but it is unclear whether specific T cell subsets influence asthma symptom duration. Here we show a significant association of airway CD8+ effector memory T cells re-expressing CD45RA (TEMRAs), but not CD8+CD45RO+ or tissue-resident memory T cells, with asthma duration in patients with severe asthma (SA) but not mild to moderate asthma (MMA). Higher frequencies of IFN-γ+CD8+ TEMRAs compared with IFN-γ+CD45RO+ T cells were detected in SA airways, and the TEMRAs from patients with SA but not MMA proliferated ex vivo, although both expressed cellular senescence-associated biomarkers. Prompted by the transcriptomic profile of SA CD8+ TEMRAs and proliferative response to IL-15, airway IL15 expression was higher in patients with SA compared with MMA. Additionally, IL15 expression in asthmatic airways negatively correlated with lung function. Our findings add what we believe is a new dimension to understanding asthma heterogeneity, identifying IL-15 as a potential target for treatment.
Authors
Richard P. Ramonell, Timothy B. Oriss, Jessica C. McCreary-Partyka, Sagar L. Kale, Nicole R. Brandon, Mark A. Ross, Marc C. Gauthier, Molin Yue, Taylor J. Nee, Sudipta Das, Wei Chen, Alok V. Joglekar, Prabir Ray, Claudette M. St Croix, Dhivyaa Rajasundaram, Sally E. Wenzel, Anuradha Ray
×Abstract
Prader-Willi syndrome (PWS) is a multigenic disorder caused by the loss of 7 contiguous paternally expressed genes. Mouse models with inactivation of all PWS genes are lethal. KO mouse models for each candidate gene have been generated, but they lack the functional interactions between PWS genes. Here, we revealed an interplay between Necdin and Magel2 PWS genes and generated a mouse model (named Del Ndn-Magel2 mice) with a deletion including both genes. A subset of Del Ndn-Magel2 mice showed neonatal lethality. Behaviorally, surviving mutant mice exhibited sensory delays during infancy and alterations in social exploration at adulthood. Del Ndn-Magel2 mice had a lower body weight before weaning, persisting after weaning in males only, with reduced fat mass and improved glucose tolerance as well as altered puberty. Adult mutant mice displayed increased ventilation and a persistent increase in apneas following a hypercapnic challenge. Transcriptomics analyses revealed a dysregulation of key circadian genes and alterations of genes associated with axonal function similar to patients with PWS. At neuroanatomical levels, Del Ndn-Magel2 mice had an impaired maturation of oxytocin neurons and a disrupted development of melanocortin circuits. Together, these data indicate that the Del Ndn-Magel2 mouse is a pertinent and genetically relevant model of PWS.
Authors
Pierre-Yves Barelle, Alicia Sicardi, Fabienne Schaller, Julie Buron, Denis Becquet, Felix Omnes, Françoise Watrin, Marie-Sophie Alifrangis, Catarina Santos, Clément Menuet, Anne-Marie François-Bellan, Emilie Caron, Jessica Klucznik, Vincent Prevot, Sebastien G. Bouret, Françoise Muscatelli
×Abstract
Transplant recipients require lifelong, multimodal immunosuppression to prevent rejection by reducing alloreactive immunity. Rapamycin is known to modulate adaptive and innate immunity, but its full mechanism remains incompletely understood. We investigated the understudied effects of rapamycin on lymph node (LN) architecture, leukocyte trafficking, and gut microbiome and metabolism after 3 (early), 7 (intermediate), and 30 (late) days of rapamycin treatment. Rapamycin significantly reduced CD4+ T cells, CD8+ T cells, and Tregs in peripheral LNs, mesenteric LNs, and spleen. Rapamycin induced early proinflammation transition to protolerogenic status by modulating the LN laminin α4/α5 expression ratios (La4/La5) through LN stromal cells, laminin α5 expression, and adjustment of Treg numbers and distribution. Additionally, rapamycin shifted the Bacteroides/Firmicutes ratio and increased amino acid bioavailability in the gut lumen. These effects were evident by 7 days and became most pronounced by 30 days in naive mice, with changes as early as 3 days in allogeneic splenocyte-stimulated mice. These findings reveal what we believe to be a novel mechanism of rapamycin action through time-dependent modulation of LN architecture and gut microbiome, which orchestrates changes in immune cell trafficking, providing a framework for understanding and optimizing immunosuppressive therapies.
Authors
Long Wu, Allison Kensiski, Samuel J. Gavzy, Hnin Wai Lwin, Yang Song, Michael T. France, Ram Lakhan, Dejun Kong, Lushen Li, Vikas Saxena, Wenji Piao, Marina W. Shirkey, Valeria R. Mas, Bing Ma, Jonathan S. Bromberg
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The enzyme protein kinase C ε (PKCε) plays an important role in pain signaling and represents a promising therapeutic target for the treatment of chronic pain. We designed and generated a small molecule inhibitor of PKCε, CP612, and examined its effect in a rodent model of chemotherapy-induced neuropathic pain produced by paclitaxel, which does not respond well to current therapeutics. In addition, many patients with chronic pain use opiates, which over time can become ineffective, and attempts to discontinue them can increase pain thereby promoting sustained opioid use. Therefore, we also investigated if CP612 alters pain due to opioid withdrawal. We found that CP612 attenuated hyperalgesia produced by paclitaxel, and it both prevented and reversed hyperalgesia induced by opioid withdrawal. It was not self-administered and did not affect morphine self-administration. These findings suggest that inhibition of PKCε is an effective, nonaddictive strategy to treat chemotherapy-induced neuropathic pain, with the added benefit of preventing increases in pain that occur as opioid treatment is discontinued. This latter property could benefit individuals with chronic pain who find it difficult to discontinue opioids.
Authors
Adriana Gregory-Flores, Ivan J.M. Bonet, Stève Desaivre, Jon D. Levine, Stanton F. McHardy, Harmannus C. de Kraker, Nicholas A. Clanton, Peter M. LoCoco, Nicholas M. Russell, Caleb Fleischer, Robert O. Messing, Michela Marinelli
×Abstract
Small for gestational age (SGA), with increased risk of adult-onset cardiovascular diseases and metabolic syndromes, is known to associate with endothelial dysfunction, but the pathogenic mechanisms remain unclear. In this study, the pathological state of human umbilical vein endothelial cells (HUVECs) from SGA individuals was characterized by presenting increased angiogenesis, migration, proliferation, and wound healing ability relative to their normal counterparts. Genome-wide mapping of transcriptomes and open chromatins unveiled global gene expression alterations and chromatin remodeling in SGA-HUVECs. Specifically, we revealed increased chromatin accessibility at active enhancers, along with dysregulation of genes associated with angiogenesis, and further identified CD44 as the key gene driving HUVECs’ dysfunction by regulating pro-angiogenic genes’ expression and activating phosphorylated ERK1/2 and phosphorylated endothelial NOS expression in SGA. In SGA-HUVECs, CD44 was abnormally upregulated by 3 active enhancers that displayed increased chromatin accessibility and interacted with CD44 promoter. Subsequent motif analysis uncovered activating protein-1 (AP-1) as a crucial transcription factor regulating CD44 expression by binding to CD44 promoter and associated enhancers. Enhancers CRISPR interference and AP-1 inhibition restored CD44 expression and alleviated the hyperangiogenesis of SGA-HUVECs. Together, our study provides a foundational understanding of the epigenetic alterations driving pathological angiogenesis and offers potential therapeutic insights into addressing endothelial dysfunction in SGA.
Authors
Lingling Yan, Zhimin Zhou, Shengcai Chen, Xin Feng, Junwen Mao, Fang Luo, Jianfang Zhu, Xiuying Chen, Yingying Hu, Yuan Wang, Bingbing Wu, Lizhong Du, Chunlin Wang, Liang Gong, Yanfen Zhu
×Abstract
It is well documented that impaired DNA damage repair (DDR) induces genomic instability that can efficiently increase the sensitivity of prostate cancer (PCa) cells to PARP inhibitors; however, the underlying mechanism remains elusive. Here, we found profound genomic instability in PCa cells with SPOP gene mutations and confirmed the sensitivity of SPOP-mutated PCa cells to olaparib-induced apoptosis. Mechanistically, we identified olaparib-induced CK2-mediated phosphorylation of PIAS1-S468, which in turn mediated SUMOylation of SPOP, thus promoting its E3 ligase activity in the DDR. Moreover, an abnormal CK2/PIAS1/SPOP axis due to SPOP mutations or defects in CK2-mediated phosphorylation of PIAS1, as well as SPOP inhibitor treatment, led to impaired DDR, thus increasing olaparib-induced apoptosis of PCa cells and enhancing olaparib sensitivity in animal models and patient-derived organoids. This suggested that disruption of the CK2/PIAS1/SPOP signaling axis could serve as an indicator for targeted therapy of PCa using a PARP inhibitor.
Authors
Hui Zhang, Lili Kong, Jinhui Li, Zhihan Liu, Yiting Zhao, Xiuyi Lv, Liangpei Wu, Lin Chai, Hongjie You, Jiabei Jin, Xinyi Cao, Zhong Zheng, Yadong Liu, Zejun Yan, Xiaofeng Jin
×Abstract
Mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3) gene encoding a protein called JIP3 is an adaption protein of the kinesin-1 complex known to play a role in axonal transport of cargo. Mutations in the gene have been linked to severe neurodevelopmental disorders, resulting in developmental delay, intellectual disability, ataxia, tremor, autism, seizures, and visual impairment. A patient who has a missense mutation in the MAPK8IP3 gene (c. 1714 C>T, Arg578Cys) (R578C) manifests dystonia, gross motor delay, and developmental delay. Here, we showed that the mutation was a toxic gain-of-function mutation that altered the interactome of JIP3; disrupted axonal transport of late endosomes; increased signaling via c-Jun N-terminal kinase, resulting in apoptosis; and disrupted dopamine receptor 1 signaling while not affecting dopamine receptor 2 signaling. Furthermore, in the presence of the mutant protein, we showed that an 80% reduction of mutant JIP3 and a 60% reduction of WT JIP3 by non-allele-selective phosphorothioate-modified antisense oligonucleotides was well tolerated by several types of cells in vitro. Our study identifies what we believe to be several important new roles for JIP3 and provides important insights for therapeutic approaches, including antisense oligonucleotide reduction of JIP3.
Authors
Wei Zhang, Swapnil Mittal, Ria Thomas, Anahid Foroughishafiei, Ricardo Nunes Bastos, Wendy K. Chung, Konstantina Skourti-Stathaki, Stanley T. Crooke
×Abstract
Humoral immunity is orchestrated by follicular helper T (Tfh) cells, which promote cognate B cells to produce high-affinity, protective antibodies. In aged individuals, humoral immunity after vaccination is diminished despite the presence of Tfh cells, suggesting defects after initial Tfh cell formation. In this study, we utilized both murine and human systems to investigate how aging alters Tfh cell differentiation after influenza vaccination. We found that young Tfh cells underwent progressive differentiation after influenza vaccination, culminating in clonal expansion of effector-like cells in both draining lymph nodes and blood. In aging, early stages of Tfh cell development occurred normally. However, aging rewired the later stages of development in Tfh cells, resulting in a transcriptional program reflective of cellular senescence, sustained pro-inflammatory cytokine production, and metabolic reprogramming. We investigated the extent to which this rewiring of aged Tfh cells is due to the age-associated inflammatory (“inflammaging”) microenvironment and found that this setting was sufficient to both block the transition of Tfh cells to a post-effector resting state and skew Tfh cells toward the age-rewired state. Together, these data suggest that aging dampens humoral immunity by cytokine-mediated rewiring of late effector Tfh cell differentiation into an activated, yet less functional, cellular state.
Authors
Cody S. Nelson, Manuel A. Podestà, Maya G. Gempler, Jeong-Mi Lee, Cole J. Batty, Peterson G. Mathenge, Asra Sainju, Matthew R. Chang, Hanzhong Ke, Pragya Chandrakar, Elsa Bechu, Sierra Richardson, Cecilia B. Cavazzoni, Stefan G. Tullius, Reza Abdi, Musie Ghebremichael, Marcia C. Haigis, Wayne A. Marasco, Peter T. Sage
×Abstract
The adrenocorticotropic hormone (ACTH) test diagnoses relative adrenal insufficiency (RAI) or critical illness–related corticosteroid insufficiency (CIRCI). Initially, guidelines recommended corticosteroid/glucocorticoid (GC) therapy for septic patients with RAI, but later trials did not show a survival benefit, leading to updated guidelines that abandon targeting RAI or CIRCI. Recent studies with an RAI mouse model showed a clear survival benefit from GC therapy in mice with RAI, suggesting that inconclusive GC clinical trials might be due to issues with the ACTH test rather than targeting RAI. To investigate, we performed the ACTH test in septic mice. Interestingly, the ACTH test identified most mice as having adrenal insufficiency in early and middle stages of sepsis, even those with a normal adrenal stress response. Surprisingly, the ACTH test increased inflammatory cytokines to lethal levels, moderately increasing mortality in septic mice. This study revealed significant flaws in the ACTH test for diagnosing RAI/CIRCI. It not only fails to correctly identify these conditions, leading to misguided use of GCs, but also induces a lethal inflammatory response in sepsis. These findings suggest that inconclusive GC therapy trials may be due to the problematic nature of the ACTH test rather than ineffectiveness of targeting RAI/CIRCI.
Authors
Dan Hao, Qian Wang, Misa Ito, Jianyao Xue, Ling Guo, Bin Huang, Chieko Mineo, Philip W. Shaul, Xiang-An Li
×Abstract
In vitro fertilization (IVF) is a noncoital method of conception used to treat human infertility. Although IVF is viewed as largely safe, it is associated with adverse outcomes in the fetus, placenta, and adult offspring. Because studies focusing on the effect of IVF on the male reproductive system are limited, we used a mouse model to assess the morphological and molecular effects of IVF on male offspring. We evaluated 3 developmental stages: 18.5-day fetuses and 12- and 39-week-old adults. Regardless of age, we observed changes in testicular-to-body weight ratios, serum testosterone levels, testicular morphology, gene expression, and DNA methylation. Also, sperm showed changes in morphology and DNA methylation. To assess multigenerational phenotypes, we mated IVF-conceived and naturally conceived males with wild-type females. Offspring from IVF males exhibited decreased fetal-to-placental weight ratios and changes in placenta gene expression and morphology regardless of sex. At 12 weeks of age, offspring showed higher body weights and differences in glucose, triglyceride, insulin, total cholesterol, HDL-C, and LDL/VLDL-C levels. Both sexes showed changes in gene expression in liver, testes, and ovaries and decreased global DNA methylation. Collectively, our findings demonstrate that male IVF offspring exhibit abnormal testicular and sperm morphology and molecular alterations with a multigenerational impact.
Authors
Eric A. Rhon-Calderon, Cassidy N. Hemphill, Alexandra J. Savage, Laren Riesche, Richard M. Schultz, Marisa S. Bartolomei
×Abstract
Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell–specific effects of LHON-linked mtDNA mutations remains poorly understood, and there has been no effective treatment or cure for this disorder. Using a mouse model bearing an LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell–specific deficiencies, especially in retinal ganglion cells (RGCs), rods, and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation–induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness and increased numbers of RGCs, photoreceptors, and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide insight into retina-specific pathophysiology of mitochondrial retinopathy arising from VA deficiency and mitochondrial dysfunction induced by mtDNA mutation and a step toward therapeutic intervention for LHON and other mitochondrial retinopathies.
Authors
Cheng Ai, Huiying Li, Chunyan Wang, Yanchun Ji, Douglas C. Wallace, Junbin Qian, Yimin Zhu, Min-Xin Guan
×Abstract
The FDA-approved phosphodiesterase type 4 (PDE4) inhibitor, apremilast, has been recently investigated as a pharmacotherapy for alcohol use disorder (AUD) with promising efficacy in rodent models and humans. However, apremilast’s effects on mechanical allodynia associated with AUD as well as distinct responses of this drug between males and females are understudied. The present study examined the behavioral and electrophysiological effects of apremilast in Marchigian Sardinian alcohol-preferring (msP) rats and their Wistar counterparts. We used a 2–bottle choice (2-BC) alcohol drinking procedure and tested mechanical sensitivity across our drinking regimen. Spontaneous inhibitory GABA-mediated postsynaptic currents from the central nucleus of the amygdala (CeA) following apremilast application were tested in a subset of rats using ex vivo electrophysiology. Transcript levels for Pde4a or -4b subtypes were assessed for their modulation by alcohol. Apremilast reduced alcohol drinking in both strains of rats. Apremilast reduced mechanical allodynia immediately after drinking, persisting into early and late abstinence. Apremilast increased GABAergic transmission in CeA slices of alcohol-exposed Wistars but not msP rats, suggesting neuroadaptations in msPs by excessive drinking and mechanical allodynia. Pde4 subtype transcript levels were increased in CeA by alcohol. These results suggest that apremilast alleviates co-occurring excessive drinking and pain sensitivity, and they further confirm PDE4’s role in pain-associated AUD.
Authors
Valentina Vozella, Vittoria Borgonetti, Bryan Cruz, Celsey M. St. Onge, Ryan Bullard, Roman Vlkolinsky, Diego Gomez Ceballos, Angela R. Ozburn, Amanda J. Roberts, Roberto Ciccocioppo, Michal Bajo, Marisa Roberto
×Abstract
Mutations in the anoctamin-5 (ANO5) gene can lead to musculoskeletal disorders, with monoallelic (autosomal dominant) mutations typically presenting as skeletal abnormalities known as gnathodiaphyseal dysplasia (GDD). Clinically, GDD is characterized by thickened cortices of long bones and mandibles, narrowed medullary cavities, and increased bone fragility. While autophagy is necessary in regulating bone formation, the specific relationship between ANO5 and autophagy remains poorly understood. In this study, we demonstrated that Ano5 deficiency activates autophagy in mouse cranial osteoblasts (mCOBs), leading to enhanced osteogenic capacity in Ano5–/– mCOBs. The application of 3-methyladenine (3-MA) and chloroquine (CQ) reversed the excessive osteogenesis observed in Ano5–/– mCOBs. Further analysis revealed that Ano5 deficiency upregulated the expression of ATG9A, and silencing ATG9A significantly reduced both autophagy and osteogenic activity in Ano5–/– mCOBs. Additionally, AMP-activated protein kinase (AMPK) was found to positively regulate ATG9A, and inhibiting AMPK reduced ATG9A expression, which in turn mitigated excessive osteogenesis of Ano5–/– mCOBs. Moreover, in vivo experiments confirmed that treatment with 3-MA alleviated the bone phenotype abnormalities in Ano5–/– mice. These findings suggest that Ano5 negatively regulates autophagy, contributing to illuminate pathogenesis of GDD. Meanwhile, this research highlights potential therapeutic strategies targeting autophagy to pave the way for the clinical manifestations of GDD.
Authors
Shuai Zhang, Shengnan Wang, Sirui Liu, Xiu Liu, Mingyue Zhang, Huichong Xu, Xiaoyu Wang, Hongyu Li, Ying Hu
×Abstract
The use of genetically engineered tools, including combinations of Cre-LoxP and Flp-FRT systems, enables the interrogation of complex biology. Steroidogenic factor-1 (SF-1) is expressed in the ventromedial hypothalamic nucleus (VMH). Development of genetic tools, such as mice expressing Flp recombinase (Flp) in SF-1 neurons (Sf-1-Flp), will be useful for future studies that unravel the complex physiology regulated by the VMH. Here, we developed and characterized Sf-1-Flp mice and demonstrated their utility. The Flp sequence was inserted into the Sf-1 locus with P2A. This insertion did not affect Sf-1 mRNA expression levels and Sf-1-Flp mice do not have any visible phenotypes. They are fertile and metabolically comparable to wild-type littermate mice. Optogenetic stimulation using adeno-associated virus (AAV) carrying Flp-dependent channelrhodopsin-2 (ChR2) increased blood glucose and skeletal muscle PGC-1α in Sf-1-Flp mice. This was similar to SF-1 neuronal activation using Sf-1-BAC-Cre and AAV carrying Cre-dependent ChR2. Finally, we generated Sf-1-Flp mice that lack β2-adrenergic receptors (Adrb2) only in skeletal muscle with a combination of Cre/LoxP technology (Sf-1-Flp:SKMΔAdrb2). Optogenetic stimulation of SF-1 neurons failed to increase skeletal muscle PGC-1α in Sf-1-Flp:SKMΔAdrb2 mice, suggesting that Adrb2 in skeletal muscle is required for augmented skeletal muscle PGC-1α by SF-1 neuronal activation. Our data demonstrate that Sf-1-Flp mice are useful for interrogating complex physiology.
Authors
Marco Galvan, Mina Fujitani, Samuel R. Heaselgrave, Shreya Thomas, Bandy Chen, Jenny J. Lee, Steven C. Wyler, Joel K. Elmquist, Teppei Fujikawa
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Abstract
Bariatric surgery is associated with improved breast cancer (BC) outcomes, including greater immunotherapy effectiveness in a pre-clinical BC model. A potential mechanism of bariatric surgery-associated protection is the gut microbiota. Here, we demonstrate the dependency of improved immunotherapy response on the post-bariatric surgery gut microbiome via fecal microbial transplant (FMT). Response to αPD-1 immunotherapy was significantly improved following FMT from formerly obese bariatric surgery-treated mice. When stool from post-bariatric surgery patients was transplanted into recipient mice and compared to the patients’ pre-surgery transplants, post-surgery microbes significantly reduced tumor burden and doubled immunotherapy effectiveness. Microbes impact tumor burden through microbially derived metabolites, including branched chain amino acids (BCAA). Circulating BCAAs correlated significantly with natural killer T (NKT) cell content in the tumor microenvironment in donor mice after bariatric surgery and FMT recipients of donor cecal content after bariatric surgery compared to obese controls. BCAA supplementation replicated improved αPD-1 effectiveness in two BC models, supporting the role of BCAAs in increased immunotherapy effectiveness after bariatric surgery. Ex vivo exposure increased primary NKT cell expression of anti-tumor cytokines, demonstrating direct activation of NKT cells by BCAAs. Together, findings suggest that reinvigorating anti-tumor immunity may depend upon bariatric surgery-associated microbially derived metabolites, namely BCAAs.
Authors
Margaret S. Bohm, Sydney C. Joseph, Laura M. Sipe, Minjeong Kim, Cameron T. Leathem, Tahliyah S. Mims, Nathaniel B. Willis, Ubaid A. Tanveer, Joel H. Elasy, Emily W. Grey, Madeline E. Pye, Zeid T. Mustafa, Barbara Anne Harper, Logan G. McGrath, Deidre Daria, Brenda Landvoigt Schmitt, Jelissa A. Myers, Patricia Pantoja Newman, Brandt D. Pence, Marie Van der Merwe, Matthew J. Davis, Joseph F. Pierre, Liza Makowski
Abstract
Regulation of organismal homeostasis in response to nutrient availability is a vital physiological process that involves inter-organ communication. The role of the heart in controlling systemic metabolic health is not clear. Adopting a mouse model of diet-induced obesity, we found that the landscape of N6-methyladenosine (m6A) on cardiac mRNA is altered following high fat/high carbohydrate feeding (western diet). m6A is a critical post-transcriptional regulator of gene expression, the formation of which is catalyzed by methyltransferase-like 3 (METTL3). Through parallel unbiased approaches of nanopore sequencing, mass spectrometry, and protein array, we found regulation of circulating factors under the control of METTL3. Mice with cardiomyocyte-specific deletion of METTL3 show a systemic inability to respond to nutritional challenge, thereby mitigating the detrimental effects of western diet. Conversely, increasing cardiac METTL3 level exacerbates diet-induced body weight gain, adiposity, and glucose intolerance. Our findings position the heart at the center of systemic metabolism regulation and highlight an m6A-dependent pathway to be exploited for the battle against obesity.
Authors
Charles Rabolli, Jacob Z. Longenecker, Isabel S. Naarmann-de Vries, Joan Serrano, Jennifer M. Petrosino, George A. Kyriazis, Christoph Dieterich, Federica Accornero
Abstract
Vascular smooth muscle cells (VSMCs) possess significant phenotypic plasticity, shifting between a contractile phenotype and a synthetic state for vascular repair/remodelling. Dysregulated VSMC transformation, marked by excessive proliferation and migration, primarily drives intimal hyperplasia. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotes, plays a critical role in gene expression regulation; however, its impact on VSMC plasticity is not fully understood. This research investigates the alterations in m6A modification and its regulatory factors during VSMC phenotypic shifts and their influence on intimal hyperplasia. We demonstrate that METTL14, crucial for m6A deposition, significantly promotes VSMC dedifferentiation. METTL14 expression, initially negligible, is elevated in synthetic VSMC cultures, post-injury neointimal VSMCs, and human restenotic arteries. Reducing METTL14 in mouse primary VSMCs decreases pro-synthetic genes, suppressing their proliferation and migration. m6A-RIP-seq profiling shows key VSMC gene networks undergo altered m6A regulation in Mettl14-deficient cells. METTL14 enhances KLF4 and SERPINE1 expression through increased m6A deposition. Local METTL14 knockdown significantly curbs neointimal formation post-arterial injury, and reducing METTL14 in hyperplastic arteries halts further neointimal development. We found that METTL14 is a pivotal regulator of VSMC dedifferentiation, influencing KLF4- and SERPINE1-mediated phenotypic conversion. Inhibiting METTL14 is a viable strategy for preventing restenosis and halting restenotic occlusions.
Authors
Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin Jong Leong Wong, Renjing Liu
Abstract
Pharmacological rescue of F508del-CFTR by the triple combination CFTR modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) leads to unprecedented clinical benefits in patients with cystic fibrosis (CF), however, previous studies in CF primary human airway epithelial cultures demonstrated that chronic treatment with the potentiator ivacaftor can render the F508del protein unstable thus limiting restoration of CFTR chloride channel function. However, quantitative studies of this unwanted effect of ivacaftor on F508del channel function including dependency on cell culture conditions remain limited and the impact of chronic ivacaftor exposure on restoration of mucociliary clearance that is impaired in patients with CF has not been studied. In patient-derived primary nasal epithelial cultures, we found that different culture conditions (UNC-ALI medium vs. PneumaCult medium) have profound effects on ETI-mediated restoration of F508del-CFTR function. Chronic treatment with ivacaftor as part of ETI triple therapy limited the rescue of F508del-CFTR chloride channel function when CF nasal epithelial cultures were grown in UNC-ALI medium, but not in PneumaCult medium. In PneumaCult medium, both chronic and acute addition of ivacaftor as part of ETI treatment led to constitutive CFTR-mediated chloride secretion in the absence of exogenous cAMP-dependent stimulation. This constitutive CFTR-mediated chloride secretion was essential to improve viscoelastic properties of the mucus layer and to restore mucociliary transport on CF nasal epithelial cultures. Furthermore, nasal potential difference measurements in patients with CF showed that ETI restored constitutive F508del-CFTR activity in vivo. These results demonstrate that ivacaftor as a component of ETI therapy is essential to restore mucociliary clearance and suggest that this effect is facilitated by its constitutive activation of F508del channels following their folding-correction in patients with CF.
Authors
Anita Balázs, Tihomir Rubil, Christine K. Wong, Jasmin Berger, Marika Drescher, Kathrin Seidel, Mirjam Stahl, Simon Y. Graeber, Marcus A. Mall
Abstract
Mutations in genome maintenance factors drive sporadic and hereditary breast cancers. Here, we searched for potential drivers based on germline DNA analysis from a cohort consisting of early-onset breast cancer patients negative for BRCA1/BRCA2 mutations. This revealed candidate genes that subsequently were subjected to RNAi-based phenotype screens to reveal genome integrity impacts. We identified several genes with functional roles in genome maintenance, including Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3), SMC4, and CCDC108. Notably, G6PC3-deficient cells exhibited increased levels of γH2AX and micronuclei formation, along with defects in homologous recombination (HR) repair. Consistent with these observations, G6PC3 was required for the efficient recruitment of BRCA1 to sites of DNA double-strand breaks (DSBs). RNA sequencing analysis revealed that G6PC3 promotes the expression of multiple homologous recombination repair genes, including BRCA1. Through CRISPR-Select functional-genetic phenotype analysis of G6PC3 germline mutations, we identified two germline G6PC3 variants displaying partial loss-of-function. Furthermore, our study demonstrated that G6pc3 deficiency accelerates mammary tumor formation induced by Trp53 loss in mice. In conclusion, our cohort-based functional analysis has unveiled genome maintenance factors and identified G6PC3 as a potential candidate tumor suppressor in breast cancer.
Authors
Xin Li, Maria Rossing, Ana Moisés da Silva, Muthiah Bose, Thorkell Gudjónsson, Jan Benada, Jayashree Thatte, Jens Vilstrup Johansen, Judit Börcsök, Hanneke van der Gulden, Ji-Ying Song, Renée Menezes, Asma Tajik, Lucía Sena, Zoltan Szallasi, Morten Frödin, Jos Jonkers, Finn Cilius Nielsen, Claus Storgaard Sørensen
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