Research
Abstract
The composition of mitochondrial membrane lipids is crucial to cellular respiration, as seen in Barth syndrome (BTHS), a rare disease affecting skeletal muscle, heart, and neutrophils. In BTHS, mutations in the tafazzin (TAZ) gene reduce remodeling of the mitochondrial phospholipid, cardiolipin, causing mitochondrial dysfunction in skeletal muscle and heart. Here, we investigated effects of altering polyunsaturated fatty acid content in cardiolipin using preclinical models of BTHS. In vitro, the absence of TAZ did not impair omega-3 fatty acid incorporation into cardiolipin and resulted in increased turnover of these acyl chains. To examine this in a functional model, we generated a muscle-specific knockout mouse of TAZ (TAZ MKO), which recapitulated the human phenotype in skeletal muscle. Supplementing the diet of TAZ MKO with fish-oil-derived omega-3 fatty acids prevented lean mass loss, improved mitochondrial respiration, altered mitochondrial structure, and revealed moderate improvements in the stress response. Surprisingly, no diet-induced changes to cardiolipin species were observed in the TAZ MKO, but other phospholipids were altered by both genotype and diet, revealing complex regulation and potential compensation. Overall, this work provides evidence that omega-3 fatty acid supplementation is beneficial in muscle lacking TAZ to improve quality of life when added to current BTHS treatments.
Authors
Katharina B. Kuentzel, Ana Vranešević, Samuel A.J. Trammell, Fabian Finger, Jesper F. Havelund, Yvette L. Schooneveldt, Ivan Bradić, Nicoline R. Andersen, Anna S. Hassing, Katja T. Michler, Martin R. Larsen, Zachary Gerhart-Hines, Steven M. Claypool, Jonas T. Treebak, Andreas M. Fritzen, Matthew P. Gillum, Steen Larsen, Nils Færgeman, Trisha J. Grevengoed
Abstract
While Wilms tumors commonly arise from renal precursor cells and maintain features of the developing kidney, recent studies have demonstrated significant genetic, histologic, and molecular heterogeneity. To further investigate tumor variability as well as unifying features in tumor biology, we performed single nuclei RNA-sequencing (snRNA-seq) on treatment naïve, favorable histology Wilms tumors utilizing a reference atlas established from tumor-adjacent kidney samples and fetal kidney. Transcriptional profiles of blastemal, stromal, and epithelial components were correlated with tumor histology and demonstrate developmental-lineage plasticity, with PAX2 and PAX8 expression normally restricted to the nephron lineage of the fetal kidney found to be expressed in tumor stroma, as well as the stromal marker POSTN identified in tumor blastema. Further analyses of the blastema show shared transcriptional features with the differentiation trajectory of “uninduced” to “early differentiating” fetal nephron progenitor cells as well as aberrant expression of stromal signatures. A number of pathways from fetal nephron progenitors were maintained in the blastema, including regulation of stem cell maintainence and axonogenesis, whereas other pathways appear enriched in specific tumor samples, demonstrating the ability of snRNA-seq to identify both unifiying transcriptional signatures and uncover distinct molecular targets in signaling pathways and/or biological drivers of Wilms tumorigenesis.
Authors
Mike Adam, Keri A. Drake, Naomi Pode-Shakked, Katherine VandenHeuvel, Steve Potter, James Geller
Abstract
Cellular senescence is an irreversible stress response, which leads to loss of cellular function and remodelling of the cellular secretory profile. In humans, pancreatic β-cells undergo cellular senescence during the progression to type 2 diabetes (T2D). However, the mechanism linking β-cell senescence to islet dysfunction remains unknown and thus, the therapeutic potential of targeting senescent cells in T2D is not established. Herein, we identified a subpopulation of senescent β-cells expressing p21, which emerged early in the progression of T2D in humans and mice. Spatial transcriptomics, and proteomics analyses confirmed senescence and loss of cellular identity in this subpopulation in humans. Functional analysis revealed lack of glucose responsiveness, high basal insulin secretion, and transcription of senescence-associated secretory phenotype (SASP) factors. SASP factors from p21+ β-cells induced secondary senescence in neighbouring cells, characterized by dysfunction and loss of identity. Janus kinase inhibitors (JAKi) counteracted the induction of secondary senescence and restored β-cell function in islets from humans with T2D and in high-fat diet-fed mice. These findings reveal the critical role of p21+ β-cells in T2D pathogenesis and the therapeutic potential of targeting this pathophysiological process.
Authors
Kanako Iwasaki, Priscila Carapeto, Cristian Abarca, Francesko Hela, Stephanie Sanjines, Sebastian Pena, Sandra Le, Hui Pan, Maya Jackson, Christopher Cahill, Ayush Midha, Juliana Alcoforado Diniz, Dylan Baker, Sergii Domanskyi, Sara Espinoza, Alejandro Pena, Francisco G. Cigarroa, Jillian L. Woodworth, Jeffrey H. Chuang, Vesna D. Garovic, James L. Kirkland, Tamara Tchkonia, Nicolas Musi, George A. Kuchel, Paul Robson, Cristina Aguayo-Mazzucato
Abstract
Duchenne muscular dystrophy (DMD) is a lethal pediatric striated muscle disease caused by loss of dystrophin for which there is no cure. Cardiomyopathy is the leading cause of death amongst individuals with DMD, and effective therapeutics to treat DMD cardiomyopathy are a major unmet clinical need. This work investigated adeno-associated viral (AAV) gene therapy approaches to treat DMD cardiomyopathy by overexpression of the calcium binding proteins S100A1 and apoptosis repressor with caspase recruitment domains (ARC). Using the severe D2.mdx mouse model of DMD, we identified that S100A1 gene therapy improves the diastolic dysfunction associated with DMD cardiomyopathy, whereas ARC gene therapy prolongs survival. The combination of both S100A1 and ARC in a single bicistronic vector improves the long-term cardiac outcome and histopathology of D2.mdx mice, development of heart failure caused by micro-dystrophin expression, and exhibits safety via intracoronary delivery in a canine model of DMD. In addition to robust cardiac benefits, S100A1-ARC gene therapy benefits D2.mdx skeletal muscle function and histopathology when driven by a striated muscle promoter. Together, these findings indicate that S100A1-ARC gene therapy represents an effective treatment for DMD cardiomyopathy and may have therapeutic benefits in treating other forms of cardiomyopathy and muscle pathologies.
Authors
David W. Hammers, Cora C. Hart, Eli A. Zerpa, Karen I. Laurent, Young il Lee, Margaret M. Sleeper, H. Lee Sweeney
Abstract
Immune responses against transgene products can compromise AAV-mediated gene transfer. Although several factors influencing this immunogenicity have been described, the early in vivo events driving CD8+ T cell activation remain poorly defined. Here, we examined antigen presentation kinetics following intramuscular AAV administration in mice. Strikingly, viral genomes were detected in draining lymph nodes as early as one hour post-injection, and transgene-derived peptides were presented to CD8+ T cells from day 1, resulting in progressive activation and first cell divisions detected at day 4. Removal of the injection site demonstrated that AAV particles reaching draining lymph nodes within the first hour were sufficient to induce cytotoxic transgene-specific CD8+ T cells. Finally, AAV vectors incorporating different muscle-specific promoters and regulatory sequences were evaluated. Although muscle-specific, all promoters exhibited variable transgene expression in dendritic cells in vitro, correlating with early T-cell activation in vivo; notably, those associated with higher early antigen presentation induced robust T cell response, whereas reduced presentation correlated with absence of CD8+ T cells. These findings reveal an unexpectedly early onset of transgene-derived epitope presentation, modulated by promoter specificity, which critically shapes CD8+ T cell response. This provides a rationale for evaluating and mitigating AAV immunogenicity in gene therapy design.
Authors
Lindsay Jeanpierre, Coralie Pecquet, Hanadi Saliba, Pauline Finard, Stéphane Terry, Gianni Tavella, Inès Guesmia, Sylvie Boutin, Bérangère Bertin, Sofia Benkhelifa-Ziyyat, Giuseppe Ronzitti, David-Alexandre Gross
Abstract
In chronic beryllium disease (CBD), elevated levels of the inflammatory chemokines CCL3 and CCL4 in the lungs coincide with expanded populations of CD4+ T cells specific to beryllium (Be)-modified peptides derived from these chemokines. Here, we generated HLA-DP2 transgenic (Tg) CCL3-deficient mice (CCL3-/-) that also lack CCL4 to investigate their role in disease development. Be-exposed CCL3-/- mice maintained normal numbers of lung macrophages and dendritic cells (DCs) but exhibited significantly reduced total and HLA-DP2-CCL/Be tetramer-specific CD4+ T cells, IFN-γ-producing CD4+ T cells, and peribronchovascular aggregates, consistent with attenuated inflammation. CCL3 was predominantly expressed in macrophages and DCs, and bone marrow chimera studies confirmed that hematopoietic-derived DCs are the key regulators of CCL/Be-specific CD4+ T cell responses. RNA sequencing of lung-resident CCL4/Be tetramer-positive CD4+ T cells revealed a transcriptional profile enriched for inflammatory and cholesterol-metabolism pathways, with elevated expression of Ifng, Tnf, and Il17a. Moreover, Be-exposed HLA-DP2 Tg mice lacking TNF-α or treated with peptide-MHCII CAR-T cells targeting CCL4/Be-specific CD4+ T cells showed reduced T cell responses and cellular aggregates. These findings demonstrate that CCL3 and CCL4 promote CCL/Be-specific CD4+ T cell responses and highlight peptide-MHCII CAR-T cells as a novel strategy for depleting self-peptide/Be-specific CD4+ T cells in CBD.
Authors
Michael T. Falta, Masoom Raza, Caley J. Nevienski, Tonya M. Brunetti, Rui Fu, Rebecca M. Tucker, Joseph M. Gaballa, Faiz Minhajuddin, Kibrom M. Alula, Alberto Dinarello, Douglas G. Mack, Allison K. Martin, Joseph C. Onyiah, Michael Yarnell, Prashanth Francis, Terry J. Fry, Lisa A. Maier, Andrew P. Fontenot, Charles A. Dinarello, Shaikh M. Atif
Abstract
Tuberous sclerosis complex (TSC) and Lymphangioleiomyomatosis (LAM) lack well-defined cellular origins, limiting treatment options. In this report, scRNA-seq of Tsc2+/− mouse renal cystadenomas revealed an 80-fold increase in a tumor cell subpopulation with neural crest features, and expressing known cranial neural crest genes as SRY box transcription factor 9 (Sox9), transcription factor activator protein (Tfap2a), and candidate neurocristopathy markers, osteopontin (Spp1), lipocalin-2 (Lcn2), clusterin (Clu), and cytokeratin 18 (Krt18). These signatures were validated in mouse tumors, and LAM patient lesions and serum, identifying a tumor phenotype distinct from traditional VEGFD detection. Pathway analysis indicated activation of WNT/SHH signaling, nephric duct formation, and pro-tumorigenic signals, with transcription factor 7 (Tcf7) and ephrin-A ligands as key upstream regulators. Spp1 KO in cranial neural crest cells (CNCCs) significantly reduced proliferation (28–33%), migration (54-76%), and invasion (29-64%) without affecting viability, while Tsc2 KO increased viability 3 to 6-fold with minimal impact on chemotaxis. Elevated serum levels of SPP1 and KRT18 in some LAM patients, decreased LCN2 in nearly all, and distinct increases in VEGFD suggest complementary roles for these biomarkers. Overall, findings support a neurocristopathic model of tumor development in TSC and LAM and identify potential biomarkers and therapeutic targets beyond mTOR inhibition.
Authors
Uchenna J. Unachukwu, Enio B. Garcia, Nooralam Rai, Jeanine M. D'Armiento
Abstract
Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from both B- and T-cell lineages. Current therapy exploits ALL cells’ low expression of asparagine synthetase (ASNS) by using L-asparaginase, a bacterial enzyme that depletes circulating asparagine. However, resistance can emerge through induction of ASNS, mediated in part by the amino acid stress sensor GCN2. In this study, we addressed the efficacy of L-asparaginase in combination with genetic or pharmacological inhibition of GCN2 and a novel ASNS inhibitor designated ASX-173. Using a KrasG12D-driven mouse model of T-ALL, we found that GCN2 is dispensable for leukemogenesis. However, genetic inactivation or pharmacologic inhibition of GCN2 sensitized ALL cells to asparagine depletion, correlating with impaired ASNS induction. While GCN2 targeting enhanced sensitivity to asparagine depletion, a subset of Gcn2–/– T-ALL cells retained high ASNS expression and remained resistant to L-asparaginase. Likewise, some human T-ALL cells with elevated ASNS levels were refractory to GCN2 inhibition even under asparagine-depleted conditions. When combined with L-asparaginase, ASX-173 effectively eliminated ASNS-high leukemic cells in vitro and in vivo. These findings suggest that direct targeting of ASNS provides therapeutic benefit in leukemias that express high ASNS and are resistant to GCN2 inhibition under asparagine-depleted conditions.
Authors
Rodney Claude, Sankalp Srivastava, Kirk A. Staschke, Carlos A. Mellado Fritz, Shaoxiong Chen, Lei Liu, Minghua Zhong, Harish Kothandaraman, Nadia A. Lanman, Utpal P. Davé, Sandeep Batra, Jiehao Zhou, Yue Fang, Chi Zhang, Reuben Kapur, Jing Fan, Ronald C. Wek, Ji Zhang
Abstract
Small bowel transplantation (SBT) is the only curative treatment for intestinal failure due to short bowel syndrome (SBS); however, the 10-year graft survival rate after SBT remains below 50%. Therefore, alternative treatments are required. We developed a new therapeutic strategy for intestinal failure involving in vivo intestinal regeneration using a decellularized scaffold in a rat model. A 3-cm segment of decellularized small intestine was anastomosed to the jejunum for in vivo regeneration. After four weeks of regeneration, the entire native intestine was resected to induce SBS, and the regenerated intestine was transplanted into the same rat. Histological analysis revealed regeneration of mucosa, nerves, muscular layer, and crypts, consistent with autologous cell infiltration. An indocyanine green test confirmed blood flow from the adjacent mesentery into the regenerated intestine. The regenerated intestine exhibited absorption of nutrients in vivo, and ex vivo assessments confirmed peristalsis and absorptive capacity comparable to native intestine. Transplantation of the regenerated intestine significantly improved postoperative nutritional status in SBS rats. Our method, autogenic-regenerated intestinal transplantation, showed the therapeutic potential for intestinal failure. This is the first study to demonstrate a functionally integrated regenerated intestine, providing a foundation for future regenerative therapy.
Authors
Kentaro Iwaki, Takamichi Ishii, Hidenobu Kojima, Fumiaki Munekage, Hiroshi Horie, Kenta Makino, Takuma Karasuyama, Yusuke Hanabata, Elena Yukie Uebayashi, Satoshi Ogiso, Etsuro Hatano
Abstract
Immunosenescence, the biological aging of the immune system, leads to dysregulated immune responses, increasing susceptibility to infections and reducing vaccine efficacy in older adults, as seen with flu vaccines. In contrast, the AS01-adjuvanted recombinant herpes zoster vaccine (RZV) maintains high and sustained efficacy, offering 82% protection against herpes zoster at 11-years post-vaccination, in individuals over 50. To identify factors impacting age-dependent vaccine efficacy, we conducted a randomized, partially placebo-controlled clinical study. Young adults (18-35 years, n=84) were randomized 3:3:1:1 to receive either RZV, an inactivated quadrivalent seasonal influenza vaccine (IIV4), placebo for RZV or placebo for IIV4, while older adults (≥60, n=63) were randomized 1:1 to receive RZV or IIV4. RZV elicited robust antibody production, antigen-specific polyfunctional CD4+ T cell responses and IFN-γ from PBMCs in both age groups, while IIV4 increased antibody responses, but induced fewer antigen-specific CD4+ T cells and no elevation of IFN-γ from PBMCs. Interestingly, RZV reduced systemic inflammation in older adults, particularly after the second injection. Baseline inflammation negatively correlated with antibody production and IFN-γ response, especially after RZV. Our findings suggest that RZV may help overcome immunosenescence by enhancing cellular responses and potentially decreasing systemic inflammation, deserving further investigation into the underlying molecular mechanisms.
Authors
Gizem Kilic, Esther J.M. Taks, Leonie S. Helder, Elisabeth A. Dulfer, Büsra Geckin, Liesbeth van Emst, Heidi Lemmers, Stefano Berrè, Adhidev Biswas, Mumin Ozturk, Yutaka Negishi, Wivine Burny, Sofia Maria Buonocore, Jaap ten Oever, Musa M. Mhlanga, Mihai G. Netea
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