Stem cell–associated osteogenic deficiency causes craniofacial deformities with progeroid accumulation of prelamin A
Li et al. report on nuclear and cytoskeletal defects that mediate stem cell–associated osteogenic deformities in a mouse model of a premature aging disorder caused by mutations in prelamin A. The cover shows filamentous actin (F-actin, red) outlining the cellular architecture of calvarial osteoprogenitor cells, with nuclei counterstained by DAPI (blue). Image credit: Kai Li and Wei Hsu.
-
Research Letter
×
Abstract
Authors
Alessandra Ciullo, Xaviar M. Jones, Hiroaki Komuro, Liang Li, Anh Nguyen, Eduardo Marbán, Ahmed Gamal-Eldin Ibrahim
-
Research Articles
×
Abstract
We provide evidence that human and murine Schlafen 5 (SLFN5) proteins are modulators of type I IFN responses and the immune response in pancreatic ductal adenocarcinoma (PDAC). Blocking expression of Slfn5 in PDAC enhanced IFN responses, suppressed tumor growth, and prolonged survival in immunocompetent mice. Notably, immunophenotypic analysis revealed a reduction in tumor-associated macrophages alongside an increase in tumor-infiltrating effector cells in tumors over time. These findings suggest SLFN5 acts as an intracellular immune checkpoint and identify it as a unique therapeutic target for the development of therapies for PDAC and possibly other malignancies.
Authors
Mariafausta Fischietti, Markella Zannikou, Elspeth M. Beauchamp, Diana Saleiro, Aneta H. Baran, Briana N. Hryhorysak, Jamie N. Guillen Magaña, Emely Lopez Fajardo, Gavin T. Blyth, Brandyn A. Castro, Jason M. Miska, Catalina Lee-Chang, Priyam Patel, Elizabeth T. Bartom, Masha Kocherginsky, Frank Eckerdt, Leonidas C. Platanias
×Abstract
Low nephron endowment constitutes a risk factor for hypertension and renal disease. Epigenetic regulation is crucial for nephron progenitor cell differentiation, affecting nephron number and renal function. The role of many epigenetic modulators, such as Lysine-specific histone demethylase 1a (LSD1 or KDM1A), remains unclear. We used Kdm1a-KO mice to demonstrate that Kdm1a depletion in nephron progenitor cells results in reduced kidney size in neonates and led to glomerulosclerosis, proteinuria, and renal cysts in adults. Notably, Kdm1a deletion in podocytes or tubular cells did not replicate these effects. CRISPR/Cas9-mediated KDM1A deletion in human kidney organoids caused cyst formation and altered gene expression, with snRNA-seq revealing downregulation of podocyte genes and upregulation of metabolic genes. The presence of noncoding RNAs indicated roles in cell proliferation. Our study reveals the critical role of Kdm1a function in nephron development and highlights its affect on transcriptional programming for long-term renal function and susceptibility to cyst formation.
Authors
Nicola Wanner, Julia Keller, Nastassia Liaukouskaya, Geoffroy Andrieux, Sandra D. Laufer, Manuel Rogg, Tillmann Bork, Wei Liang, Fabian Braun, Fabian Haas, Milagros N. Wong, Victor G. Puelles, Sydney E. Gies, Charlotte Meyer, Melanie Boerries, Martin Helmstädter, Oliver Kretz, Iris Hild, Eric Metzger, Roland Schüle, Wibke Bechtel-Walz, Tobias B. Huber
×Abstract
Chronic hyperglycemia changes the expression of various transcription factors and mRNA transcripts that impair cellular functionality and delay wound healing. Zinc finger E-box–binding homeobox 2 (ZEB2), a key transcription factor, maintains tissue-specific macrophage identities; however, its role in regulating macrophage polarization during wound healing under hyperglycemic conditions remains unclear. Here, we found that persistent hyperglycemia increases ZEB2 expression in wound macrophages via histone acetylation, contributing to chronic inflammation and delayed wound healing. Exposure to high glucose levels activated P300/CBP, a transcriptional coactivator involved in histone acetylation, which enhanced ZEB2 expression in wound macrophages. The forced expression of ZEB2 shifted macrophage polarity toward a proinflammatory state by upregulating myeloid lineage–directed transcription factors. Conversely, silencing Zeb2 at the wound site reduced hyperglycemia-induced macrophage inflammation. Topical application of C646, an inhibitor of P300, at the wound edges of streptozotocin-induced high-fat diet–fed diabetic mice significantly decreased ZEB2 expression, reduced inflammation, and accelerated wound healing. Therefore, targeted inhibition of P300 represents a promising therapeutic strategy for improving diabetic wound healing by modulating ZEB2-driven inflammation in wound macrophages.
Authors
Soumyajit Roy, Debarun Patra, Palla Ramprasad, Shivam Sharma, Parul Katiyar, Ashvind Bawa, Kanhaiya Singh, Kulbhushan Tikoo, Suman Dasgupta, Chandan K. Sen, Durba Pal
×Abstract
Psoriasis is a chronic inflammatory dermatosis characterized by pathological keratinocyte hyperproliferation and dysregulated immune activation. While ubiquitin-specific peptidase 16 (USP16) has been implicated in modulating multiple cellular signaling pathways, its functional role in psoriatic pathogenesis remains poorly understood. Our investigation revealed pronounced upregulation of USP16 expression in psoriatic epidermis compared with normal controls. Keratinocyte-specific USP16 knockdown demonstrated remarkable therapeutic efficacy, significantly ameliorating characteristic psoriatic phenotypes including epidermal hyperplasia and inflammatory infiltration. RNA-seq analysis showed that USP16 has substantial effects on cell cycle transition and keratinocytes proliferation. Through KEGG analysis, it was found that USP16 primarily regulates the NLRP3 signaling pathway, leading to enhanced cell proliferation and inflammation. Mechanically, USP16 directly binds to the NLRP3 protein to eliminate K48 ubiquitination modification, enhancing the stability of the NLRP3 protein, activating inflammasome activity. Further studies showed that the therapeutic effects of reducing USP16 on psoriasis progression were counteracted by an NLRP3 activator and keratinocyte-specific NLRP3 overexpression adenovirus. Collectively, these results shed light on how USP16 promotes NLRP3 signaling in keratinocytes, exacerbating psoriasis development. This positive regulation highlights the potential of USP16 as a therapeutic target for psoriasis.
Authors
Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu
×Abstract
Vaso-occlusive episodes (VOEs) or acute pain events, involving complex interactions between sickle erythrocytes and other blood cells, are a hallmark of sickle cell disease (SCD). In this study, we analyzed changes in peripheral blood transcriptomes between steady state and VOEs in individuals with SCD. We followed a cohort of 174 individuals with SCD with or without chronic pain and collected peripheral blood at clinic visits (steady state) and during hospitalizations (VOEs). We performed RNA-Seq profiling of CD45+ leukocytes and CD71+ erythroid cells. Pathways linked to complement activation, coagulation, and IL-6/JAK/STAT3 signaling were enriched during VOEs in the CD45+ cells. Contrastingly, the CD71+ cells showed an enrichment of pathways related to the cell cycle, such as mTORC1 signaling and the G2M checkpoint during VOEs. We then analyzed the expression changes of genes in patients with longitudinal data to determine potential biomarkers for VOEs. Expression of 4 genes — FAM20A, IL1B, MS4A4A, and SERPINB2 — was elevated during VOEs compared with steady state in the majority of patients. Furthermore, our results indicate that patients experiencing chronic pain exhibited 44% increased enrichment of significant pathways during VOEs when compared with patients without chronic pain.
Authors
Varsha Bhat, Justin J. Yoo, Srija Ponna, Alka A. Potdar, Ashwin P. Patel, G. Karen Yu, Greg Gibson, Vivien A. Sheehan
×Abstract
Type 2 diabetic nephropathy (T2DN) is a major complication of type 2 diabetes and a leading cause of chronic kidney disease. This study aimed to explore Myosin IC (MYO1C) as both a candidate biomarker and elucidate its role as a mechanistic mediator of podocyte injury in T2DN. Using urinary extracellular vesicle RNA biomarkers identified from a training and validation cohort of 33 type 2 diabetes and 40 patients with T2DN, we developed a machine learning diagnostic model for T2DN. The model achieved an AUC of 0.877 in validation and performed well in an independent test cohort with an AUC of 0.824. MYO1C was identified as the most influential feature in the final model. Mechanistic investigations in vitro and in vivo revealed that high glucose and high-fat conditions induced podocyte injury, inflammation, and apoptosis, with increased MYO1C expression. MYO1C knockdown in vitro and in vivo reduced podocyte damage and inflammatory responses. MYO1C overexpression enhanced p38, p-CREB, and TNF-α levels, while p38 inhibition mitigated these effects. These findings support MYO1C not only as a potential urinary biomarker for T2DN but also as a key pathogenic driver that promotes podocyte injury via p38 MAPK signaling.
Authors
Zihao Zhao, Qianqian Yan, Sijie Zhou, Fengxun Liu, Yong Liu, Jingjing Ren, Shaokang Pan, Zhenjie Liu, Dongwei Liu, Zhangsuo Liu, Jiayu Duan
×Abstract
BACKGROUND Idiopathic pulmonary arterial hypertension (IPAH) alters right ventricular size and function, curtailing life expectancy. Patients may experience angina and myocardial ischemia. However, the underlying mechanisms are poorly understood.METHODS This study had a cross-sectional, case-control design. Patients with IPAH undergoing right heart catheterization were prospectively enrolled and underwent functional testing during coronary angiography using a dual pressure/temperature-sensitive guidewire. Cardiovascular MRI measured left and right ventricular mass and function. Right ventricular tissue from individuals with end-stage PAH and control individuals were analyzed for pathophysiology.RESULTS Eleven IPAH and 15 control participants completed the protocol: 73% of IPAH patients had an elevated index of microcirculatory resistance (IMR > 25) and 55% had reduced coronary flow reserve (CFR < 2.0). Mean IMR was significantly higher in IPAH participants (39.2 ± 27.0 vs. 15.3 ± 5.0, P = 0.002), whereas mean CFR was lower (2.8 ± 2.1 vs. 4.0 ± 1.4; P = 0.077). Paired right coronary artery/ventricular measurements (n = 6) revealed IMR positively correlated with right ventricular mass (r = 0.91, P = 0.12) and negatively with CFR (r = –0.82, P = 0.046). Compared with controls (n = 5), PAH participants (n = 4) had reduced right ventricular capillary density, increased cardiomyocyte area, and increased mural area in pre-capillary arterioles.CONCLUSION Invasive coronary function testing was feasible and safe in IPAH. Coronary microvascular dysfunction was prevalent in IPAH and correlated with increased right ventricular mass. Histopathology revealed vascular rarefaction and remodeling of pre-capillary arterioles.FUNDING The British Heart Foundation (BHF) (PG/18/6134217) and the Golden Jubilee Research Foundation.
Authors
Erin Boland, Michael G. Freeman, David S. Corcoran, Thomas J. Ford, Barry Hennigan, Damien Collison, Aida Llucià-Valldeperas, Frances S. de Man, Kanarath P. Balachandran, Martin Johnson, Colin Church, Colin Berry
×Abstract
Next-generation sequencing technologies are increasingly used to diagnose genetic disorders, particularly immunological diseases with broad and overlapping immune dysregulation. Cryopyrin-associated periodic syndromes (CAPS) are caused by gain-of-function mutations in NLRP3 and include 3 autoinflammatory diseases spanning a continuum of severity: familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). Linking NLRP3 variants to protein dysfunction and clinical phenotype remains challenging because of genetic modifiers and environmental factors. We report the generation and phenotyping of 5 mouse lines expressing either the common human NLRP3 allele or 1 of 4 CAPS mutations spanning the disease spectrum from FCAS to NOMID. In these lines, the murine Nlrp3 locus is replaced by syntenic integration of the human NLRP3 locus, yielding 1 line with the common allele and 4 lines each carrying a distinct CAPS mutation. Unlike models in which a human mutation is introduced into the mouse protein, these lines recapitulate the spectrum of disease severity observed in humans. These findings support a model in which evaluation of nonsynonymous mutations in mice is optimized when introduced in the context of the human gene. This suggests that species-specific regulation and/or intramolecular epistasis may impact modeling of disease-associated variants.
Authors
John N. Snouwaert, MyTrang Nguyen, Christopher A. Gabel, Ivona Aksentijevich, Jenny P.-Y. Ting, Beverly H. Koller
×Abstract
Lung development relies on diverse cell intrinsic and extrinsic mechanisms to ensure proper cellular differentiation and compartmentalization. In addition, it requires precise integration of multiple signaling pathways to temporally regulate morphogenesis and appropriate cell specification. To accomplish this, organogenesis relies on epigenetic and transcriptional regulators to promote cell fate and inhibit alternative cell fates. Using genetic mouse and human embryonic stem cell (hESC) differentiation models, tissue explants, and single-cell transcriptomic analysis, we demonstrated that Bromodomain Containing Protein 4 (BRD4) is required for mammalian lung morphogenesis and cell fate. Endodermal deletion of BRD4 impaired epithelial-mesenchymal crosstalk, leading to disrupted proximal-distal patterning and branching morphogenesis. Moreover, temporal deletion of BRD4 revealed developmental stage–specific defects in airway and alveolar epithelial cell specification with a predominant role in proximal airway cell fate. Similarly, BRD4 promoted lung endodermal cell differentiation into airway lineages in a hESC-derived lung organoid model. Together, these data demonstrate that BRD4 orchestrates early lung morphogenesis and separately regulates cell specification, indicating a multifunctional and evolutionarily conserved role for BRD4 in mammalian lung development.
Authors
Hongbo Wen, Derek C. Liberti, Prashant Chandrasekaran, Shahana Parveen, Kwaku K. Quansah, Mijeong Kim, Ana N. Lange, Abigail T. Marquis, Sylvia N. Michki, Annabelle Jin, MinQi Lu, Ayomikun A. Fasan, Sriyaa Suresh, Shawyon P. Shirazi, Lisa R. Young, Jennifer M.S. Sucre, Maria C. Basil, Rajan Jain, David B. Frank
×Abstract
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune dysregulation and widespread inflammation. NK cells display marked functional impairment in SLE, including defective cytotoxicity and cytokine production, but the underlying mechanisms remain poorly defined. Here, we show that mitochondrial dysfunction and impaired mitophagy are key contributors to NK cell abnormalities in SLE. Using complementary structural, metabolic, and proteomic analyses, we found that SLE NK cells accumulate enlarged and dysfunctional mitochondria, exhibit impaired lysosomal acidification, and release mitochondrial DNA into the cytosol — features consistent with defective mitochondrial quality control. Transcriptional and proteomic profiling revealed downregulation of key mitophagy-related genes and pathways. These abnormalities correlated with reduced NK cell degranulation and cytokine production. We then tested whether enhancing mitochondrial quality control could restore NK cell function. The mitophagy activator Urolithin A improved mitochondrial and lysosomal parameters and rescued NK cell effector responses in vitro. Hydroxychloroquine partially restored mitochondrial recycling and reduced cytosolic mtDNA. These findings suggest that defective mitophagy and mitochondrial dysfunction are major contributors to NK cell impairment in SLE and that targeting mitochondrial quality control may represent a promising strategy for restoring immune balance in this disease.
Authors
Natalia Fluder, Morgane Humbel, Emeline Recazens, Alexis A. Jourdain, Camillo Ribi, George Tsokos, Denis Comte
×Abstract
Although renal fibrosis is predominantly driven by the accumulated inflammatory cells that secrete proinflammatory factors within the kidney, the key mechanisms underlying macrophage clearance from the kidney are not well understood. The interaction of hyaluronan with lymphatic endothelial hyaluronan receptor 1 (LYVE1) constitutes a critical initial step in macrophage adhesion and removal by lymphatic vessels. This study investigates alterations in LYVE1 during kidney disease and elucidates its role in macrophage trafficking. Three renal fibrosis models demonstrated a reduction in full-length LYVE1 and an increase in the soluble LYVE1 fragment. Immunostaining of fibrotic kidneys showed significantly reduced expression of soluble LYVE1 compared with the intracellular fragment (Cyto-LYVE1), demonstrating ectodomain shedding of LYVE1 in vivo and in vitro. Functionally, human lymphatic endothelial cells exposed to TGF-β1 exhibited a significant decrease in macrophage adhesion and transendothelial migration compared with controls. Mechanistic analyses identified increased matrix metalloproteinase 9 (MMP9) in renal injury as a key upstream regulator of LYVE1 shedding. MMP9 inhibitors reduced LYVE1 shedding, enhanced macrophage adhesion and trafficking, and mitigated macrophage accumulation and disease progression. In conclusion, MMP9-induced LYVE1 shedding is linked to progressive kidney fibrosis and macrophage accumulation. LYVE1 shedding inhibitors offer potential as therapeutic agents for mitigating immune overload and kidney fibrosis.
Authors
Jing Liu, Yuqing Liu, Wenqian Zhou, Saiya Zhu, Jianyong Zhong, Haichun Yang, Annet Kirabo, Valentina Kon, Chen Yu
×Abstract
BACKGROUND Clear cell renal cell carcinoma (ccRCC) with pancreatic metastases (PM) is paradoxically associated with prolonged overall survival (OS), but the biological basis for this observation remains unclear.METHODS We analyzed matched primary and metastatic samples from an international consortium of patients with PM (n = 108) and compared them with a previously characterized ccRCC cohort without PM (n = 273).RESULTS Primary ccRCC tumors associated with PM were dominated by indolent, angiogenic phenotypes, characterized by low-grade histology and reduced mTORC1 activation (all P < 0.001). Tumors of patients with PM were often PBRM1-deficient (80.4% vs. 54.8%, P < 0.001) and rarely harbored BAP1 loss (3.7% vs. 20.7%, P < 0.001). After metastasis diagnosis, patients with PM had significantly longer median OS compared with those without PM (110 vs. 33 months, HR 0.28 [95% CI, 0.19–0.39], P < 0.001). Survival was further prolonged among patients with PBRM1 loss (143 vs. 64 months, HR 0.41 [95% CI, 0.22–0.81], P = 0.008). Notably, PM lesions were typically low-grade and PBRM1-deficient even when more aggressive and evolved clones were present in primary tumors. Finally, PBRM1 loss was associated with preferential response to angiogenesis inhibitors over immune-oncology therapy, reflected by longer time on treatment (32.1 vs. 9.1 months, HR 0.16 [95% CI, 0.06–0.39], P < 0.001).CONCLUSION These findings illustrate selective tropism of indolent, less-evolved, PBRM1-deficient ccRCC clones for pancreatic dissemination. This biological bias likely underlies therapeutic sensitivity and favorable survival, supporting the consideration of PBRM1 status and metastatic tropism in risk stratification and treatment selection.FUNDING NIH Kidney Cancer SPORE grant (P50CA196516); The Cancer Prevention and Research Institute of Texas (RP220294); Endowment from Jan and Bob Pickens Distinguished Professorship in Medical Science and Brock Fund for Medical Science Chair in Pathology.
Authors
Haitao Xu, Payal Kapur, Alana Christie, Aleksandra W. Nielsen, Averi Perny, Olivia Brandenburg, Charlotte Small, Jeffrey Miyata, Hua Zhong, Courtney Roberts, Roy Elias, Vanina Tcheuyap, Cassandra Duarte, Adrie van Bokhoven, Justine Panian, Haoran Li, Katharine A Collier, Debra Zynger, Luis Meza, Benoit Beuselinck, Neeraj Agarwal, Amir Mortazavi, Sumanta Pal, Rana McKay, Elaine T. Lam, Satwik Rajaram, James Brugarolas
×Abstract
Loss of bone mass has a devastating effect on quality of life. Higher potassium (K+) intake is positively correlated with bone health. Here, we investigated whether kidney calcium (Ca2+) and phosphate (Pi) handling mechanisms mediate dietary K+ effects. Kidney Ca2+ and Pi handling proteins were altered in abundance in mice fed a 0% K+ diet for 2 weeks. In mice fed a 0.1% K+ diet for 4 or 8 weeks, urinary Ca2+ excretion increased, plasma Ca2+ levels were lower and plasma parathyroid hormone (PTH) levels were higher relative to control 1% K+ fed mice. The 0.1% K+ fed mice had greater excretion of the bone resorption marker deoxypyridinoline, increased osteoclast number, and decreased total femoral bone mineral density. During chronic low K+ intake, major changes in renal Ca2+ and Pi transport pathways were absent, except higher abundances of the sodium-potassium-chloride cotransporter (NKCC2) and the sodium-chloride cotransporter (NCC), in line with their role in kidney Ca2+ handling. Low dietary K+ induced hypocalcemia and changes in PTH were absent in mice with constitutively active NCC, supporting its role in mediating low K+ effects on Ca2+ homeostasis. Our study provides insights into the management of bone disorders in conditions of chronic electrolyte imbalance.
Authors
Sathish K. Murali, Mariavittoria D’Acierno, Xiang Zheng, Lena K. Rosenbaek, Louise N. Odgaard, P. Richard Grimm, Alice Ramesova, Robert Little, Judith Radloff, Paul A. Welling, Qi Wu, Reinhold G. Erben, Robert A. Fenton
×Abstract
Mutations in LMNA, encoding nuclear lamina protein Lamin A/C, cause premature aging disorders, most notably Hutchinson-Gilford progeria syndrome. Despite obvious skull abnormalities in patients with progeria, the etiology remains elusive. The L648R single–amino acid substitution blocks prelamin A maturation in mice, modeling a unique patient. Here, we identify prelamin A accumulation as a causative link to craniosynostosis in low bone density, contrasting conventional suture fusion in excessive ossification. The mutation causes skeletal stem cell deficiencies and subsequent osteogenesis. Intrasutural bones present in patients with progeria resemble synostosis caused by stem cell exhaustion. Comparative gene expression profiling further reveals cytoskeletal dynamics associated with skeletogenic cell aging and suture patency in mice and humans. Functional studies demonstrate that abnormal structures of progeric nuclei affect cytoskeleton organization and nucleoskeleton assembly essential for craniofacial skeletogenesis. Our findings provide compelling evidence for nuclear and cytoskeletal defects, causing stem cell–associated osteogenic defects in progeroid disorders.
Authors
Kai Li, Trunee Hsu, Hitoshi Uchida, Tingxi Wu, Susan Michaelis, Howard Worman, Wei Hsu
×Abstract
Angelman syndrome is a neurodevelopmental disorder caused by loss of the maternal UBE3A allele, the sole source of UBE3A in mature neurons owing to epigenetic silencing of the paternal allele. Although emerging therapies are being developed to restore UBE3A expression by activating the dormant paternal UBE3A allele, existing mouse models for such preclinical studies have limited throughput and utility, creating bottlenecks for both in vitro therapeutic screening and in vivo characterization. To address this, we developed the Ube3a-INSG dual-reporter knockin mouse, in which an IRES-Nanoluciferase-T2A-Sun1-sfGFP (INSG) cassette was inserted downstream of the endogenous Ube3a stop codon. The INSG model preserves UBE3A protein levels and function while enabling 2 complementary allele-specific readouts: Sun1-sfGFP and Nanoluciferase. We show that Sun1-sfGFP, a nuclear envelope–localized reporter, enables single-cell fluorescence analysis, whole-brain light-sheet imaging, and nuclear quantification by flow cytometry. Further, Nanoluciferase supports high-throughput luminescence assays for sensitive pharmacological profiling in cultured neurons and noninvasive in vivo bioluminescence imaging for pharmacodynamic assessment. By combining scalable screening, cellular analysis, and real-time in vivo monitoring in a single model, the Ube3a-INSG dual-reporter mouse provides a powerful platform to accelerate therapeutic development centered on UBE3A.
Authors
Hanna Vihma, Lucas M. James, Hannah C. Nourie, Audrey L. Smith, Siyuan Liang, Carlee A. Friar, Tasmai Vulli, Lei Xing, Dale O. Cowley, Alain C. Burette, Benjamin D. Philpot
×Abstract
The survival of patients with acute myelogenous leukemia (AML) carrying mutations in TP53 is dismal. We report the results of a detailed characterization of responses to treatment ex vivo with the MDM2 inhibitor MI219, a p53 protein stabilizer, in AML blasts from 165 patients focusing analyses on patients with WT TP53. In total 33% of AML were absolute resistant to MDM2 inhibitor–induced apoptosis, of which 45% carried TP53 mutation and 55% were TP53 WT. We conducted array-based expression profiling of 10 resistant and ten sensitive AML cases with WT TP53 status, respectively, at baseline and after 2 hours and 6 hours of MDM2 inhibitor treatment. While sensitive cases showed the induction of classical TP53 response genes, this was absent or attenuated in resistant cases. In addition, the sensitive and resistant AML samples at baseline profoundly differed in the expression of inflammation-related and mitochondrial genes. No patient with TP53 mutated AML survived. The 4-year survival of AML with defective MDM2 inhibitor–induced TP53-mediated apoptosis despite WT TP53 was dismal, at 19% when NPM1 was comutated and 6% when NPM1 was WT. In summary, we identified prevalent multicausal defects in TP53-mediated apoptosis in AML resulting in extremely poor patient survival.
Authors
Josephine Dubois, Anthony Palmer, Darren King, Mohamed Rizk, Karan Bedi, Kerby A. Shedden, Sami N. Malek
×Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease. Emerging evidence suggests manifestations beyond the neuromuscular system. Bone alterations are part of the ALS clinical picture; it remains unclear whether they are secondary to muscle denervation or due to an autonomous process. We investigated skeletal involvement in the SOD1(G93A) mouse model at presymptomatic (P45) and symptomatic (P110) stages through biomechanical and transcriptomic approaches. Three-point bending revealed significant reductions in femoral rigidity and maximum bending force in SOD1 mutants at P45, indicating early structural deficits. Micro-CT analysis demonstrated reduced trabecular bone mineral density and thickness at P45, with progressive trabecular loss and cortical thinning by P110. Histological examination revealed marked osteoblast loss at P45, suggesting impaired bone formation as the primary early mechanism. Transcriptomics of bulk bone and cultured osteoblasts from P45 mice identified dysregulation of bone differentiation, including downregulation of osteoblast differentiation genes and upregulation of negative regulators of ossification and increased cell senescence signatures. Unfolded protein response was upregulated in SOD1 osteoblasts. Immunohistochemistry confirmed the senescence phenotype with increased p16Ink4a level in SOD1 osteoblasts. These findings suggest that bone deterioration precedes overt motor symptoms and is linked to osteoblast premature senescence.
Authors
Burak Özkan, Jan-Moritz Ramge, Diana Wiesner, Jelena Scekic-Zahirovic, Stefano Antonucci, Sandra Nungeß, Dorothea Gebauer, Anita Ignatius, Jochen H. Weishaupt, Melanie Haffner-Luntzer, Francesco Roselli
×Abstract
Mycobacterium tuberculosis (Mtb) survives within multiple macrophage populations during infection, including alveolar macrophages (AMs) and recruited inflammatory macrophages. In mice, itaconate, produced in macrophages by ACOD1-mediated decarboxylation of aconitate, has direct antimicrobial activity, modulates inflammatory cytokines, and is required for resistance to Mtb infection. The role of itaconate in human macrophages is less clear, and it is unknown whether itaconate mediates distinct effects in macrophage subtypes. Here, we investigated the role of itaconate in macrophages derived from human induced pluripotent stem cells (iPSCs), induced by either GM-CSF to resemble AMs (AM-like cells, hereafter ipAM-Ls) or M-CSF to resemble monocyte-derived macrophages (MDM-like cells, hereafter ipMDM-Ls). Both human macrophage types produced substantially less itaconate than mouse macrophages, and ipAM-Ls produced 4-fold less itaconate than ipMDM-Ls. Surprisingly, ACOD1-deficient ipAM-Ls, but not ipMDM-Ls, were permissive for Mtb growth. Moreover, itaconate functioned to dampen the Mtb-induced inflammatory response in ipMDM-Ls, but not ipAM-Ls, affecting both the type I IFN and TNF pathways. These results indicate that itaconate is involved in human macrophage responses to tuberculosis, with distinct roles in different macrophage subsets. These results also show that genetically tractable iPSC-derived macrophages are a useful model to dissect cellular host-pathogen interactions in human macrophages.
Authors
Adam S. Krebs, Tomi Lazarov, Anthony T. Reynolds, Kimberly A. Dill-McFarland, Abigail Xie, James M. Bean, Muxue Du, Olivier Levy, John A. Buglino, Aaron Zhong, Anna-Lena Neehus, Stéphanie Boisson-Dupuis, Jean-Laurent Casanova, Elouise E. Kroon, Marlo Möller, Thomas R. Hawn, Ting Zhou, Lydia W.S. Finley, Marc Antoine Jean Juste, Dan W. Fitzgerald, Frederic Geissmann, Michael S. Glickman
×Abstract
Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel disease (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosal growth and the function of the gut epithelial barrier. The levels of tissue GAS5/Gas5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with IBD. Transient and tissue-specific knockdown of Gas5 in mice using CRISPR/Cas9 enhanced the renewal of the mucosa of the small intestine, increased the levels of tight junction (TJ) proteins ZO-1, ZO-2, claudin-1, and claudin-2, and improved gut barrier function. Conversely, ectopic overexpression of GAS5 in intestinal organoids and in cultured intestinal epithelium cells decreased the levels of these TJ proteins and caused epithelial barrier dysfunction. Mechanistic studies revealed that GAS5 acted as a transcriptional enhancer of the gene encoding small noncoding vault RNAs (vtRNAs) and that GAS5 repressed TJ expression by increasing the levels of vtRNAs. Together, our results indicate that GAS5 disrupts the integrity of the intestinal epithelium by impairing mucosal growth and epithelial barrier function and that it represses TJ expression, at least in part, via vtRNAs.
Authors
Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S.G. Cunningham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
×Abstract
The lymphatic system maintains fluid homeostasis and orchestrates immune cell trafficking throughout tissues. While extensively studied in cancer and lymphedema, its role in nonlymphoid organs, particularly the kidney, remains an emerging area of investigation. Previous research established molecular connections among NF-κB, VEGFR-3, and PROX-1 in regulating lymphatic growth during inflammation, and studies using global knockout mice revealed that the NF-κB1 subunit (p50) influences lymphatic vessel density. However, the role of RelA — a key component of the canonical NF-κB heterodimer — in regulating lymphatic growth and kidney function following acute kidney injury (AKI) remains unexplored. Using an inducible, predominantly lymphatic endothelial cell-specific RelA-knockout mouse model, we demonstrated that RelA expression in VEGFR-3+ cells is essential for VEGFR-3–driven lymphangiogenesis following AKI. Knockout mice exhibited substantially worse kidney function, altered histological features, impaired VEGFR-3–dependent lymphangiogenesis, and dysregulated immune cell trafficking compared with WT mice. Compensatory upregulation of PROX-1 and podoplanin occurred despite decreased VEGFR-3 and LYVE-1 total protein expression, suggesting complex regulatory mechanisms. Our findings suggest that RelA is a critical sensor for inflammation and regulator of protective lymphangiogenesis following kidney injury and provide insights into potential therapeutic targets for improved kidney injury outcomes.
Authors
Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal
×Abstract
Patients with estrogen receptor+ (ER+, ESR1+) breast cancer are most at risk of relapse, where activating mutations in ESR1 promote metastasis and therapeutic resistance. These patients are also disadvantaged in responding to immunotherapies, the mechanisms of which remain to be elucidated. Here, we engineered a transgenic mouse model carrying either Y541S or D542G mutation in ESR1, mirroring the 2 most common mutations seen in patients. ESR1mut tumors do not differ in the total number of immune cells yet display downregulation in immune pathways and decreased immune-modulatory cytokines, including IL-17a and IL-1β. T cells and macrophages have lower IFN-γ and antigen presentation, respectively. Mechanistically, ESR1mut negatively regulates immune modulator expression and upregulates Stat5 to dampen cytokine expression. In concordance, validation on ESR1mut patient tumors shows decreased IL-17a and IL-1β. Collectively, our findings reveal that ESR1 mutations contribute to an immunosuppressive tumor microenvironment by dampening cytokine secretion and immune cell activity.
Authors
Yu Gu, Dongmei Zuo, Qi-Xin Hu, Virginie Sanguin-Gendreau, Alain Pacis, Marie-Christine Guiot, Alexander Chih-Chieh Chang, Tarek Taifour, Chen Ling, Adrian V. Lee, Steffi Oesterreich, William J. Muller
×Abstract
Next-generation sequencing can identify previously uncharacterized gene expression patterns in disease. Beyond differentially expressed gene (DEG) analysis, we investigated the ability of within-population diversity (α-diversity) of the transcriptome to reveal additional biological information in alcohol-associated liver disease (ALD), comparing differential Shannon diversity (DSD) to transcriptome heterogeneity changes. RNA sequencing data from normal livers and patients with early ALD and severe AH were analyzed. α-Diversity indices and percentage Shannon diversity of a gene, which refers to this gene’s contribution to total Shannon entropy, were calculated. Ingenuity pathway analysis identified canonical pathways determined by DEG and DSD approaches. ALD significantly decreased hepatic transcriptome α-diversity, correlating with increased relative contribution of select genes. These changes were driven by lower-abundance gene expression loss. DEG and DSD analyses showed overlapping genes and canonical pathways, but DSD also identified additional genes and pathways not highlighted by DEGs, including fatty acid oxidation, extracellular matrix degradation, and cholesterol metabolism pathways that may represent additional therapeutic targets. Importantly, DSD more effectively identified differences between ASH and AH. Overall, α-diversity analysis revealed that ALD progressively reduces transcriptome heterogeneity, and that DSD provides complementary insights into disease mechanisms missed by standard approaches.
Authors
Sudrishti Chaudhary, Jia-Jun Liu, Silvia Liu, Marissa Di, Juliane I. Beier, Ramon Bataller, Josepmaria Argemi, Panayiotis V. Benos, Gavin E. Arteel
×Abstract
Congenital long QT syndrome (LQTS) promotes risk for life-threatening cardiac arrhythmia and sudden death in children and young adults. Pathogenic variants in the voltage-gated potassium channel KCNQ1 are the most frequently discovered genetic cause. Most LQTS-associated KCNQ1 variants cause loss of function secondary to impaired trafficking of the channel to the plasma membrane. There are currently no therapeutic approaches that address this underlying molecular defect. Using a high-throughput screening paradigm, we identified VU0494372, a small molecule that increases total and cell surface levels and trafficking efficiency of WT KCNQ1 as well as three LQTS-associated variants. Additionally, 16-hour treatment of cells with VU0494372 increased IKs (KCNQ1-KCNE1 current) for WT KCNQ1 and the LQTS-associated variant V207M in cells coexpressing KCNE1. VU0494372 had no impact on KCNQ1 transcription, degradation, or thermal stability, and increased the rate of KCNQ1 reaching the cell surface. We identified a potential direct interaction site with KCNQ1 at or near the binding site of the KCNQ1 potentiator ML277. Together, these findings demonstrate that small molecules can increase the expression levels and cell surface trafficking efficiency of KCNQ1 and introduce a potential new pharmacological approach for treating LQTS.
Authors
Katherine R. Clowes Moster, Carlos G. Vanoye, Ana C. Chang-Gonzalez, Ian M. Romaine, Katherine M. Stefanski, Mason C. Wilkinson, Joshua A. Bauer, Thomas P. Hasaka, Emily L. Days, Reshma R. Desai, Kathryn R. Butcher, Gary A. Sulikowski, Alex G. Waterson, Jens Meiler, Kaitlyn V. Ledwitch, Alfred L. George Jr., Charles R. Sanders
In-Press Preview - More
Abstract
Cytotoxic T lymphocytes form a critical component of SARS-CoV-2 immunity by recognizing viral peptides bound to HLA class I molecules. Here, we identified the Spike-derived peptide NYNYLYRLF448-456 (NF9) as the immunodominant HLA-A*24:02-restricted epitope in both convalescent and vaccinated donors. Across cohorts, A24/NF9-specific responses were dominated by public TCR motifs featuring TRAV12-1 (or TRAV6-1) paired with TRBJ2-7 and a conserved CDR3β sequence (CASSXXXGYEQYF). Using a panel of thirteen TCRs, we mapped recognition of single amino acid substitutions within NF9 and identified residue 5 (L452) as the principal determinant of escape. The L452R substitution, characteristic of the Delta variant, abolished recognition across all tested TCRs despite preserved HLA binding. Crystallography of a representative public TCR (P1-15) revealed that mutation at position 5 reoriented the peptide within HLA-A*24:02, flipping the adjacent Y453 side chain into the peptide-binding groove and eliminating the dominant TCR contact. This position-5-driven conformational switch provided a structural mechanism for universal loss of NF9 recognition by HLA-A*24:02-restricted T-cells. Consistent with this, Delta-infected convalescents failed to mount de novo NF9-5R-specific responses while retaining responses to the conserved A24/QI9 Spike epitope. Together, these findings defined the basis of A24/NF9 recognition and showed how one mutation remodelled peptide presentation to abrogate TCR responses.
Authors
Takeshi Nakama, Aaron Wall, Garry Dolton, Li-Rong Tan, Hannah Thomas, Hiroshi Hamana, Yoshiki Aritsu, Toong Seng Tan, Mako Toyoda, Yoshihiko Goto, Huanyu Li, Mizuki Kitamatsu, Keiko Udaka, Yusuke Miyashita, Hiroyuki Oshiumi, Kimitoshi Nakamura, Yoji Nagasaki, Rumi Minami, Hirotomo Nakata, Pierre J. Rizkallah, Hiroyuki Kishi, Takamasa Ueno, Andrew K. Sewell, Chihiro Motozono
Abstract
Sleep disturbance is a prevalent yet poorly understood comorbidity in autism spectrum disorders (ASD). Here, we uncover a bidirectional regulatory axis connecting the ASD risk gene POGZ to core circadian mechanisms. We demonstrate that Pogz is widely expressed in the suprachiasmatic nucleus (SCN), the central pacemaker of the circadian rhythms and exhibits circadian oscillations in both the hypothalamus and liver with its transcription directly regulated by the circadian molecule DBP through a D-box element in its proximal enhancer. Pogz-deficient mice exhibited prolonged circadian periodicity, impaired light-induced phase shift, delayed adaption to an 8-hour advance jet-lag, and reduced SCN c-Fos activation in response to light pulses. Mechanistically, POGZ interacts with and enhances the transcription activity of CREB, a key regulator of light-induced phase resetting. Notably, Pogz deletion leads to ASD-related deficits in social novelty and cognition, with cognitive impairments influenced by both photoperiod and behavioral paradigm. Our findings thus reveal a critical, previously unrecognized intersection between an ASD risk gene and circadian clock, offering new insights into the pathogenesis of core ASD symptoms and comorbid sleep disturbances.
Authors
Ting Wu, Jiao He, Chu-Jun Xu, Chi-Yu Li, Pingchuan Zhang, Yanfeng Wang, Shanshan Zhu, Lusi Zhang, Jingtan Zhu, Jing Zhang, Jia-Da Li, Huadie Liu
Abstract
The lung alveoli are continually exposed to inhaled pathogens and environmental hazards and rely on coordinated communication between alveolar macrophages and type 2 alveolar epithelial cells (AT2s) to maintain homeostasis. Disruption of these interactions can impair immunity and repair, contributing to acute and chronic respiratory diseases. To better define these mechanisms and support therapeutic discovery, we established a human iPSC-derived air-liquid interface platform that captures key features of AT2-macrophage crosstalk. Using this system, we show that coculture enhances AT2-specific transcriptional programs including lipid synthesis, while macrophages actively phagocytose AT2-derived surfactant. iPSC-derived macrophages adopt an alveolar macrophage-like phenotype and respond to AT2-derived M-CSF. During respiratory infection, macrophages played a crucial role in modulating epithelial inflammatory responses, augmenting antiviral immunity, and limiting viral replication. We further identify a previously unrecognized role for macrophages in epithelial repair, where VEGF-mediated signaling to macrophages increases epithelial permeability during viral infection. Together, these findings reveal dimensions of AT2-macrophage cooperation in homeostasis, infection, and repair, and demonstrate how this iPSC-derived platform can be used to dissect mechanisms that may initiate or drive the progression of respiratory diseases.
Authors
Declan L. Turner, Hannah Baric, Katelyn Patatsos, Sahel Amoozadeh, Michael See, Kathleen A. Strumila, Jack T. Murphy, Jeremy J. Wiyana, Liam Gubbels, Elizabeth S. Ng, Andrew G. Elefanty, Melanie R. Neeland, Shivanthan Shanthikumar, Sarah L. Londrigan, Mirana Ramialison, Fernando J. Rossello, Edouard G. Stanley, Rhiannon B. Werder
Abstract
Maternal opioid use disorder (OUD) poses substantial risks to maternal and fetal health. These adverse outcomes are believed to be mediated, in part, by changes in placenta structure and function; however, few studies have addressed this question. Here, we utilized flow cytometry, histology, spatial and single-cell transcriptomics to uncover the impact of OUD on placental tissues. Given that half of subjects with chronic OUD contract hepatitis C (HCV), we further stratified our findings by maternal HCV status. Our results indicate that OUD leads to higher incidence of vascular malperfusion accompanied by increased levels of inflammatory markers and dysregulated secretion of placental development factors. Spatial transcriptomics revealed that OUD disrupts the communication between trophoblasts and immune cells important for placental vascular development. Additionally, CellChat analysis revealed aberrant vascular remodeling, neuropeptide, and chemotactic signaling across trophoblast, endothelial, and myeloid cells. Processes associated with tissue homeostasis and repair were also upregulated across trophoblast and leukocytes. In addition, placental leukocytes were rewired towards regulatory/tissue surveillant phenotypes. Finally, frequencies and responses to ex-vivo stimulation of decidual macrophages and cytolytic NKcells, critical for tissue remodeling and fetal tolerance, were decreased. Altogether, these results highlight substantial disruptions to placental health by maternal OUD.
Authors
Heather E. True, Brianna M. Doratt, Sheridan B. Wagner, Delphine C. Malherbe, Nathan R. Shelman, Mahdi Eskandarian Boroujeni, Cynthia Cockerham, John M. O'Brien, Ilhem Messaoudi
Abstract
Resident cardiac fibroblast (RCF)-derived cardiac myofibroblasts (CMF) contribute to myocardial repair but also drive adverse ventricular remodeling and contractile dysfunction after myocardial infarction (MI). The sodium-activated potassium channel Slick (Slo2.1) has been described in cardiomyocyte (CM) mitochondria; however, transcriptomic analyses indicate higher Slick expression in RCFs/CMFs. Here, we investigated the role of Slick in cardiac fibroblast function and post-MI remodeling. Using live-cell imaging and whole-cell patch-clamp recordings, we found that plasma membrane Slick channels in RCFs and CMFs regulated potassium (K+) efflux and modulated store-operated calcium entry (SOCE), particularly in CMFs. Global Slick KO and conditional CMF-specific KO hearts exhibited reduced fibrosis and preserved left ventricular function following ischemia/reperfusion injury. This cardioprotection was associated with diminished CMF activation and proliferation, reduced inflammation, and improved CM survival post-MI. Collectively, these findings identify fibroblast Slick channels as regulators of SOCE-dependent fibrogenesis and demonstrate that their deletion mitigates maladaptive remodeling and functional decline after MI.
Authors
Jiaqi Yang, Lin Zhu, David Spähn, Melanie Cruz Santos, Sophia Schanz, Selina Maier, Lena Birkenfeld, Helmut Bischof, Anna Roslan, Nina Wettschureck, Oliver Borst, Lucas Matt, Robert Lukowski
