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Abstract
We hypothesized that performing bone marrow transplant (BMT) using marrow extracted from the vertebral bodies (VB) of an unrelated deceased lung transplant (LTX) donor would be able to establish persistent hematopoiesis, generate immunity, and tolerance. A teenager with severe combined immunodeficiency with lung failure due to recurrent pneumonias underwent LTX in 2016 from a 1/8 HLA allele-matched unrelated donor, followed by BMT 4 months later using T-cell/B-cell-depleted, cryopreserved VB marrow. Rapid engraftment was followed by accelerating immune competence at 6 months, with independence from immunosuppression by 16 months. Donor T-cell (>95%) and myeloid chimerism (7-10%) have persisted for over nine years. At two years post-BMT, circulating T cells were hyporesponsive to host dendritic cells in vitro. T-cell receptor clonotyping revealed the disappearance of host-reactive clones, and T-cell RNA-sequencing exhibited downmodulated signaling pathways for cytotoxicity/rejection, paired with upregulated immunomodulatory pathways, suggesting active suppression. In parallel, host monocytes upregulated certain signaling pathways, indicating active interactions between post-thymic donor T cells and host monocytes. In summary, durable hematopoietic engraftment, immunity, and tolerance were demonstrable for the first time in a recipient of BMT obtained from VB graft.
Authors
Paul Szabolcs, Xiaohua Chen, Marian G. Michaels, Memphis Hill, Evelyn Garchar, Zarreen Amin, Heather M. Stanczak, Shawna McIntyre, Aleksandra Petrovic, Dhivyaa Rajasundaram, Ansuman Chattopadhyay, Jonathan E. Spahr, Peter D. Wearden, Geoffrey Kurland
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 progeroid patients, the disease-causing mechanism remains elusive. The L648R single amino acid substitution blocks prelamin A maturation in mice, modeling a unique human patient. Here, we describe skull deformities in premature aging caused by aberrant suture fusion resembling those of patients with craniosynostosis. Further examinations identify prelamin A accumulation causatively linked to multiple suture synostoses in low bone density. This etiology is distinct from conventional suture fusion mediated by excessive ossification. In addition, the mutation disrupts skeletal stem cell stemness and subsequent stem cell-mediated proliferation and differentiation in osteogenesis. Intrasutural bones present in progeroid patients are highly reminiscent of 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 caused by prelamin A accumulation affect cytoskeleton organization and nucleoskeleton assembly essential for craniofacial skeletogenesis. Pharmacogenetic analyses indicate alleviation of osteogenic defects via actin polymerization. Our findings provide compelling evidence for nuclear and cytoskeletal defects, mediating stem cell-associated osteogenic deformities in progeroid disorders.
Authors
Kai Li, Trunee Hsu, Hitoshi Uchida, Tingxi Wu, Susan Michaelis, Howard J. Worman, Wei Hsu
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 demonstrated 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. Natural killer (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 W. Fluder, Morgane Humbel, Emeline Recazens, Alexis A. Jourdain, Camillo Ribi, George C. Tsokos, Denis Comte
Abstract
Authors
Alessandra Ciullo, Xaviar M. Jones, Hiroaki Komuro, Liang Li, Anh Nguyen, Eduardo Marbán, Ahmed Gamal-Eldin Ibrahim
Abstract
Chronic hyperglycemia changes the expression of various transcription factors and mRNA transcripts, which impair the cellular functionality and delayed wound healing. ZEB2 (zinc finger E-box binding homeobox 2), a key transcription factor maintains the tissue specific macrophage identities, however, its role in regulating macrophage polarization during wound healing under hyperglycemic conditions remains unclear. Here, we have 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 activates P300/CBP, a transcriptional coactivator involved in histone acetylation, enhances ZEB2 expression in wound macrophages. The forced expression of ZEB2 shifts macrophage polarity toward a pro-inflammatory state by upregulating myeloid lineage directed transcription factors (MLDTFs). 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
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 TP53 wildtype (WT) patients. 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 ten resistant and ten sensitive AML cases with WT TP53 status, respectively, at baseline and after 2h and 6h 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 TP53 mutated AML patient 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 co-mutated and 6% when NPM1 was WT. In summary, we identified prevalent multi-causal 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
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 to normal controls. Keratinocyte-specific USP16 knockdown demonstrated remarkable therapeutic efficacy, significantly ameliorating characteristic psoriatic phenotypes including epidermal hyperplasia and inflammatory infiltration. RNA sequencing 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 effect 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
We provide evidence that human and murine SLFN5 proteins are modulators of Type I IFN responses and the immune response in pancreatic cancer. Blocking expression of Slfn5 in PDAC enhances IFN-responses, suppresses tumor growth, and prolongs survival in immunocompetent mice. Notably, immunophenotypic analysis reveals a reduction in tumor-associated macrophages (TAMs) alongside an increase in tumor infiltrating effector cells in tumors over time. These findings implicate 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
CD4 T cells are essential for immunity to M. tuberculosis (Mtb), and emerging evidence indicates that IL-17-producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that, as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in two distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoRγt and produce IL-17, in contrast to ‘classical’ Mtb antigens that induce T cells that produce IFNγ. Together with emerging evidence showing human Th17 responses are associated with prevention of progression to TB disease, our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.
Authors
Paul Ogongo, Liya Wassie, Anthony Tran, Devin Columbus, Julia Huffaker, Lisa Sharling, Gregory Ouma, Samuel Gurrion Ouma, Kidist Bobosha, Cecilia S. Lindestam Arlehamn, Neel R. Gandhi, Sara C. Auld, Jyothi Rengarajan, Cheryl L. Day, Artur Queiroz, Mariana Araújo-Pereira, Eduardo Fukutani, Bruno B. Andrade, John D. Altman, Henry M. Blumberg, Joel D. Ernst
Abstract
Alveolar hemorrhage (AH) is a life-threatening condition with high mortality, yet the immunologic mechanisms governing disease severity remain poorly defined. Here, we demonstrate a protective role for T cell–intrinsic β-catenin stabilization in AH using a transgenic mouse model (CAT-Tg) in which β-catenin is stabilized under the Lck promoter. β-Catenin stabilization induced a distinct T cell phenotype marked by expansion of central effector memory cells (CD44+CD122+Eomes+T-bet+) and suppression of proinflammatory signaling, including reduced phosphorylation of STAT1, STAT3, and JAK1. Pristane-induced AH was attenuated in CAT-Tg mice, which exhibited reduced lung injury, decreased proteinuria, and diminished pulmonary proinflammatory cytokine production compared with wild-type controls. Protection was associated with a marked expansion of FOXP3+ regulatory T cells (Tregs). Mechanistically, β-catenin stabilization enhanced lung expression of Amphiregulin and BATF, mediators of Treg stability and tissue repair. Adoptive transfer of CAT-Tg–derived Tregs into wild-type mice conferred superior protection against AH, reducing lung inflammation and proteinuria. Transcriptomic analyses revealed enrichment of tissue repair and immune homeostasis pathways, including PI3K–Akt, angiogenesis, and STAT5 signaling. Collectively, these findings identify β-catenin as a regulator of a protective Amphiregulin–BATF–Treg axis, highlighting a immunomodulatory pathway with therapeutic potential for AH and inflammatory lung disease.
Authors
Fiona Mason, Hui Xiong, Ali Mobeen, Md Saddam Hossain, Sara Mahmudlu, Rosanne Trevail, Mikyal Mobeen, Li Chen, Sunny Lee, Tuncay Delibasi, Jyoti Misra Sen, Mobin Karimi
Abstract
Cachexia is a debilitating syndrome characterized by progressive skeletal muscle wasting, commonly affecting cancer patients, particularly those with pancreatic cancer. Despite its clinical significance, the molecular mechanisms underlying cancer cachexia remain poorly understood. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq, complemented by biochemical and histological analyses, to investigate molecular alterations in the skeletal muscle of the KPC mouse model of pancreatic cancer cachexia. Our findings demonstrated that KPC tumor growth induced myofiber-specific changes in the expression of genes involved in proteolytic pathways, mitochondrial biogenesis, and angiogenesis. Notably, tumor progression enhanced the activity of specific transcription factors that regulate the mTORC1 signaling pathway, along with genes involved in translational initiation and ribosome biogenesis. Skeletal muscle-specific, inducible inhibition of mTORC1 activity further exacerbated muscle loss in tumor-bearing mice, highlighting its protective role in maintaining muscle mass. Additionally, we uncovered new intercellular signaling networks within the skeletal muscle microenvironment during pancreatic cancer-induced cachexia. Together, our study revealed previously unrecognized molecular mechanisms that regulates skeletal muscle homeostasis and identified potential therapeutic targets for the treatment of pancreatic cancer–associated cachexia.
Authors
Bowen Xu, Aniket S. Joshi, Meiricris Tomaz da Silva, Silin Liu, Ashok Kumar
Abstract
Although renal fibrosis is predominantly driven by the accumulated inflammatory cells that secrete pro-inflammatory factors within the kidney, the key mechanisms underlying macrophage clearance from the kidney are not well understood. The interaction of hyaluronan (HA) 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 intracellular fragment (Cyto-LYVE1), demonstrating ectodomain shedding of LYVE1 in vivo and in vitro. Functionally, human lymphatic endothelial cells exposed to TGF-β1 exhibited significant decrease in macrophage adhesion and transendothelial migration compared to controls. Mechanistic analyses identified increased matrix metalloproteinase (MMP)9 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
The lymphatic system maintains fluid homeostasis and orchestrates immune cell trafficking throughout tissues. While extensively studied in cancer and lymphedema, its role in non-lymphoid organs, particularly the kidney, remains an emerging area of investigation. Previous research established molecular connections between 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-specific RelA knockout mouse model, we demonstrate that RelA expression in VEGFR-3+ cells is essential for VEGFR-3 driven lymphangiogenesis following AKI. Knockout mice exhibited significantly worse kidney function, altered histological features, impaired VEGFR-3-dependent lymphangiogenesis, and dysregulated immune cell trafficking. 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
BACKGROUND. Idiopathic pulmonary arterial hypertension (IPAH) alters right ventricular size and function, curtailing life-expectancy. Patients may experience angina and myocardial ischemia. However, the mechanisms underlying these changes are poorly understood. METHODS. A cross-sectional, case-control design of coronary pathophysiology (in vivo and ex vivo) in IPAH. Patients with IPAH (Group-1.1) undergoing clinically indicated right heart catheterization were prospectively enrolled. Participants underwent functional testing during coronary angiography using a dual pressure/temperature-sensitive guidewire. Cardiovascular magnetic resonance measured left and right ventricular mass and function. Autopsy cardiac tissues from end-stage PAH (Group-1) and control individuals were analyzed for right ventricular pathophysiology. RESULTS. Eleven participants with IPAH and 15 control participants completed the protocol (IPAH: 45±15 years, 73% female; controls: 58.3±9.1 years, 73% female). 73% (n=8) of IPAH patients had an elevated index of microcirculatory resistance (IMR >25) and 55% (n=6) had reduced coronary flow reserve (CRF <2.0). The 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 to controls (n=5), PAH participants (n=4) had reduced right ventricular capillary density (111±18 vs. 167±20, p=0.032), increased cardiomyocyte area (383±118μm2 vs. 231±61μm2, p=0.0390), and increased mural area in small pre-capillary arterioles (127±10μm2 vs. 107±20μm2, p=0.041). CONCLUSIONS. Coronary microvascular dysfunction is prevalent in IPAH and correlates with increased right ventricular mass. Histopathology revealed vascular rarefaction and remodeling of pre-capillary arterioles. The clinical significance merits prospective evaluation. Invasive coronary function testing was feasible and safe in IPAH, providing a platform to assess therapeutic impacts on cardiac microvascular function.
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
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 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 T2DN patients, 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, thereby highlighting its therapeutic promise.
Authors
Zihao Zhao, Qianqian Yan, Sijie Zhou, Fengxun Liu, Yong Liu, Jingjing Ren, Shaokang Pan, Zhenjie Liu, Dongwei Liu, Zhangsuo Liu, Jiayu Duan
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 co-transporter (NKCC2) and the sodium-chloride co-transporter (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, Paul Richard Grimm, Alice Ramesova, Robert Little, Judith Radloff, Paul A. Welling, Qi Wu, Reinhold G. Erben, Robert A. Fenton
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) stage 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 precede overt motor symptoms and are 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
Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel diseases (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosa growth and the function of the gut epithelium barrier. The levels of tissue GAS5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from IBD patients. 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. Cunnigham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
Abstract
Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Co-inheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR-deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering new insights into the complex role of PKR deficiency in SCD pathology.
Authors
Xunde Wang, Meghann Smith, Sayuri Kamimura, Quan Li, Niharika Shah, Martha Quezado, Luis E.F. Almeida, Sebastian Vogel, Mickias B. Tegegn, Kevin Y. Sun, Rafael Villasmil, Chengyu Liu, William A. Eaton, Swee Lay Thein, Zenaide M.N. Quezado
