In humans, type 2 diabetes mellitus shows a higher prevalence in men compared with women, a phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β cell function also contribute is largely unknown. Here, we characterized the electrophysiological properties of β cells in intact male and female mouse islets. Elevation of glucose concentration above 5 mM triggered an electrical activity with a similar glucose dependence in β cells of both sexes. However, female β cells had a more depolarized membrane potential and increased firing frequency compared with males. The higher membrane depolarization in female β cells was caused by approximately 50% smaller Kv2.1 K+ currents compared with males but otherwise unchanged KATP, large-conductance and small-conductance Ca2+-activated K+ channels, and background TASK1/TALK1 K+ current densities. In female β cells, the higher depolarization caused a membrane potential–dependent inactivation of the voltage-gated Ca2+ channels (CaV), resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx was offset by a higher action potential firing frequency. Because exocytosis of insulin granules does not show a sex-specific difference, we conclude that the higher electrical activity promotes insulin release in females, improving glucose tolerance.
Noelia Jacobo-Piqueras, Tamara Theiner, Stefanie M. Geisler, Petronel Tuluc
Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.
Boang Han, Yu Wang, Shen Yue, Yun-hao Zhang, Lun Kuang, Bin-bin Gao, Yue Wang, Ziyu Zhang, Xiaohong Pu, Xin-fa Wang, Chi-chung Hui, Ting-ting Yu, Chen Liu, Steven Y. Cheng
CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag–/– hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.
Yuanfei Zhao, Leigh Nicholson, Hannah Wang, Yi Wen Qian, Wayne J. Hawthorne, Elvira Jimenez-Vera, Brian S. Gloss, Joey Lai, Adwin Thomas, Yi Vee Chew, Heather Burns, Geoff Y. Zhang, Yuan Min Wang, Natasha M. Rogers, Guoping Zheng, Shounan Yi, Stephen I. Alexander, Philip J. O’Connell, Min Hu
Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.
Safaa Hammoud, Alla Ivanova, Yosuke Osaki, Steven Funk, Haichun Yang, Olga Viquez, Rachel Delgado, Dongliang Lu, Melanie Phillips Mignemi, Jane Tonello, Selene Colon, Louise Lantier, David Wasserman, Benjamin D. Humphreys, Jeffrey Koenitzer, Justin Kern, Mark de Caestecker, Toren Finkel, Agnes Fogo, Nidia Messias, Irfan J. Lodhi, Leslie Gewin
Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation. HMGA2 variants are a rare cause of SRS and its functional role in human linear growth is unclear. Patients with suspected SRS negative for 11p15LOM/mUPD7 underwent whole-exome and/or targeted-genome sequencing. Mutant HMGA2 protein expression and nuclear localization were assessed. Two Hmga2-knockin mouse models were generated. Five clinical SRS patients harbored HMGA2 variants with differing functional impacts: 2 stop-gain nonsense variants (c.49G>T, c.52C>T), c.166A>G missense variant, and 2 frameshift variants (c.144delC, c.145delA) leading to an identical, extended-length protein. Phenotypic features were highly variable. Nuclear localization was reduced/absent for all variants except c.166A>G. Homozygous knockin mice recapitulating the c.166A>G variant (Hmga2K56E) exhibited a growth-restricted phenotype. An Hmga2Ter76-knockin mouse model lacked detectable full-length Hmga2 protein, similarly to patient 3 and 5 variants. These mice were infertile, with a pygmy phenotype. We report a heterogeneous group of individuals with SRS harboring variants in HMGA2 and describe the first Hmga2 missense knockin mouse model (Hmga2K56E) to our knowledge causing a growth-restricted phenotype. In patients with clinical features of SRS but negative genetic screening, HMGA2 should be included in next-generation sequencing testing approaches.
Avinaash V. Maharaj, Emily Cottrell, Thatchawan Thanasupawat, Sjoerd D. Joustra, Barbara Triggs-Raine, Masanobu Fujimoto, Sarina G. Kant, Danielle van der Kaay, Agnes Clement-de Boers, Alice S. Brooks, Gabriel Amador Aguirre, Irene Martín del Estal, María Inmaculada Castilla de Cortázar Larrea, Ahmed Massoud, Hermine A. van Duyvenvoorde, Christiaan De Bruin, Vivian Hwa, Thomas Klonisch, Sabine Hombach-Klonisch, Helen L. Storr
Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.
Dana Trompet, Anastasiia D. Kurenkova, Baoyi Zhou, Lei Li, Ostap Dregval, Anna P. Usanova, Tsz Long Chu, Alexandra Are, Andrei A. Nedorubov, Maria Kasper, Andrei S. Chagin
Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle–dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.
Geremy Clair, Hasmik Soloyan, Paolo Cravedi, Andrea Angeletti, Fadi Salem, Laith Al-Rabadi, Roger E. De Filippo, Stefano Da Sacco, Kevin V. Lemley, Sargis Sedrakyan, Laura Perin
Sodium-glucose cotransporter 2 (SGLT2) inhibitor, dapagliflozin (Dapa), exhibited nephroprotective effects in patients with chronic kidney disease (CKD). We assessed the efficacy of short-term Dapa administration following acute kidney injury (AKI) in preventing CKD. Male Wistar rats were randomly assigned to Sham surgery, bilateral ischemia for 30 minutes (abbreviated as IR), and IR + Dapa groups. Daily treatment with Dapa was initiated just 24 hours after IR and maintained for only 10 days. Initially, rats were euthanized at this point to study early renal repair. After severe AKI, Dapa promptly restored creatinine clearance (CrCl) and significantly reduced renal vascular resistance compared with the IR group. Furthermore, Dapa effectively reversed the mitochondrial abnormalities, including increased fission, altered mitophagy, metabolic dysfunction, and proapoptotic signaling. To study this earlier, another set of rats was studied just 5 days after AKI. Despite persistent renal dysfunction, our data reveal a degree of mitochondrial protection. Remarkably, a 10-day treatment with Dapa demonstrated effectiveness in preventing CKD transition in an independent cohort monitored for 5 months after AKI. This was evidenced by improvements in proteinuria, CrCl, glomerulosclerosis, and fibrosis. Our findings underscore the potential of Dapa in preventing maladaptive repair following AKI, emphasizing the crucial role of early intervention in mitigating AKI long-term consequences.
Miguel Ángel Martínez-Rojas, Hiram Balcázar, Isaac González-Soria, Jesús Manuel González-Rivera, Mauricio E. Rodríguez-Vergara, Laura A. Velazquez-Villegas, Juan Carlos León-Contreras, Rosalba Pérez-Villalva, Francisco Correa, Florencia Rosetti, Norma A. Bobadilla
Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving β cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.
Gene I. Uenishi, Marko Repic, Jennifer Y. Yam, Ashley Landuyt, Priya Saikumar-Lakshmi, Tingxi Guo, Payam Zarin, Martina Sassone-Corsi, Adam Chicoine, Hunter Kellogg, Martina Hunt, Travis Drow, Ritika Tewari, Peter J. Cook, Soo Jung Yang, Karen Cerosaletti, Darius Schweinoch, Benjamin Guiastrennec, Eddie James, Chandra Patel, Tiffany F. Chen, Jane H. Buckner, David J. Rawlings, Thomas J. Wickham, Karen T. Mueller
Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.
Roberto Silva-Rojas, Laura Pérez-Guàrdia, Alix Simon, Sarah Djeddi, Susan Treves, Agnès Ribes, Lorenzo Silva-Hernández, Céline Tard, Jocelyn Laporte, Johann Böhm
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