Research Article
Abstract
Tay-Sachs disease (TSD) and Sandhoff disease are fatal neurodegenerative diseases without an effective therapy that are caused by mutations in the HEXA and HEXB genes, respectively. Together they encode the heterodimeric isozyme of hexosaminidase, hexosaminidase A (HexA), that degrades GM2 ganglioside. This report describes a 5-year-long study using a bidirectional adeno-associated virus 9 (AAV9) vector (AAV9-Bic_HexA/HexB) encoding both HEXA and HEXB in the TSD sheep model. Bidirectional AAV9 was delivered i.v. or through various cerebrospinal fluid (CSF) delivery routes: intracerebroventricular (ICV), cisterna magna (CM), and lumbar intrathecal space (LIT). The longest survival and best distribution were achieved by multipoint CSF delivery (combined CM, ICV, and LIT) with treated animals that survived up to 5 years of age (untreated animals with TSD die after ~9 months). Extension in survival was accompanied by lasting improvement in neurological examination and maze testing. Improvement in biomarkers of efficacy, including MRI, magnetic resonance spectroscopy, diffusion tensor imaging, and CSF levels of GM2 ganglioside and HexA activity, was evident. Postmortem assessments showed broad HexA distribution, GM2 ganglioside clearance, and vector genome distribution, especially in deep brain structures. Therapeutic efficacy documented in this study supports translation of bidirectional vector and multipoint CSF delivery to a clinical trial in patients with TSD and Sandhoff disease.
Authors
Toloo Taghian, Jillian Gallagher, Stephanie Bertrand, William C. Baker, Kalajan Lopez Mercado, Hector R. Benatti, Erin Hall, Yvette Lopez, Abigail McElroy, John T. McCarthy, Sanjana Pulaparthi, Deborah Fernau, Samuel Mather, Sophia Esteves, Elise Diffie, Amanda Gross, Hannah G. Lahey, Xuntian Jiang, Elizabeth Parsley, Rachael Gately, Rachel Prestigiacomo, Siauna Johnson, Amanda Taylor, Lindsey Bierfeldt, Susan Tuominen, Jennifer Koehler, Guangping Gao, Jun Xie, Qin Su, Robert King, Matthew J. Gounis, Vania Anagnostakou, Ajit Puri, Ana Rita Batista, Miguel Sena-Esteves, Douglas R. Martin, Heather Gray-Edwards
Abstract
Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (noninflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of noninflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses—integrating computational transcriptomics, proteomics, and in vivo interventional studies—we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand muramyl dipeptide in colitis and sepsis models. Mechanistically, GIV’s C-terminus binds the terminal leucine-rich repeat 10 (LRR 10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2 variant, which lacks LRR 10, cannot bind GIV, which provides critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV•NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.
Authors
Gajanan D. Katkar, Mahitha Shree Anandachar, Stella-Rita C. Ibeawuchi, Ella G. McLaren, Megan L. Estanol, Kennith Carpio-Perkins, Shu-Ting Hsu, Celia R. Espinoza, Jane E. Coates, Yashaswat S. Malhotra, Madhubanti Mullick, Vanessa Castillo, Daniella Vo, Saptarshi Sinha, Pradipta Ghosh
Abstract
Tregs are critical for maintaining immune homeostasis, and their adoptive transfer can treat murine inflammatory disorders. In patients, Treg therapies have been variably efficacious. Therefore, new strategies to enhance Treg therapeutic efficacy are needed. Tregs predominantly depend on oxidative phosphorylation (OXPHOS) for energy and suppressive function. Fatty acid oxidation (FAO) contributes to Treg OXPHOS and can be important for Treg “effector” differentiation, but FAO activity is inhibited by coordinated activity of the isoenzymes acetyl-CoA carboxylase-1 and -2 (ACC1 and ACC2). Here, we show that small-molecule inhibition or Treg-specific genetic deletion of ACC1 significantly increases Treg suppressive function in vitro and in mice with established chronic graft-versus-host disease. ACC1 inhibition skewed Tregs toward an “effector” phenotype and enhanced FAO-mediated OXPHOS, mitochondrial function, and mitochondrial fusion. Inhibiting mitochondrial fusion diminished the effect of ACC1 inhibition. Reciprocally, promoting mitochondrial fusion, even in the absence of ACC1 modulation, resulted in a Treg functional and metabolic phenotype similar to that seen with ACC1 inhibition, indicating a key role for mitochondrial fusion in Treg-suppressive potency. Ex vivo–expanded, ACC1 inhibitor–treated human Tregs similarly augmented suppressor function, as observed with murine Tregs. Together, these data suggest that ACC1 manipulation may be exploited to modulate Treg function in patients.
Authors
Cameron McDonald-Hyman, Ethan G. Aguilar, Ewoud B. Compeer, Michael C. Zaiken, Stephanie Y. Rhee, Fathima A. Mohamed, Jemma H. Larson, Michael L. Loschi, Christopher Lees, Govindarajan Thangavelu, Margaret L. Sleeth, Kyle D. Smith, Jennifer S. Whangbo, Jerome Ritz, Tim D. Sparwasser, Roddy S. O’Connor, Peter A. Crawford, Jeffrey C. Rathmell, Leslie S. Kean, Robert Zeiser, Keli L. Hippen, Michael L. Dustin, Bruce R. Blazar
Abstract
Type 2 diabetes affects more than 38 million people in the United States, and a major complication is kidney disease. During the analysis of lipotoxicity in diabetic kidney disease, global fatty acid transport protein 2 (FATP2) gene deletion was noted to markedly reduce plasma glucose in db/db mice due to sustained insulin secretion. To identify the mechanism, we observed that islet FATP2 expression was restricted to α cells and that α cell FATP2 was functional. Basal glucagon and alanine-stimulated gluconeogenesis were reduced in FATP2-KO db/db mice compared with db/db mice. Direct evidence of FATP2-KO–induced α cell–mediated glucagon-like peptide 1 (GLP-1) secretion included increased GLP-1+ α cell mass in FATP2-KO db/db mice, small-molecule FATP2 inhibitor enhancement of GLP-1 secretion in αTC1-6 cells and human islets, and exendin[9-39]-inhibitable insulin secretion in FATP2 inhibitor–treated human islets. FATP2-dependent enteroendocrine GLP-1 secretion was excluded by demonstration of similar glucose tolerance and plasma GLP-1 concentrations in db/db FATP2-KO mice following oral versus i.p. glucose loading, nonoverlapping FATP2 and preproglucagon mRNA expression, and lack of FATP2 and GLP-1 coimmunolocalization in the intestines. We conclude that FATP2 deletion or inhibition exerts glucose-lowering effects through α cell–mediated GLP-1 secretion and paracrine β cell insulin release.
Authors
Shenaz Khan, Robert J. Gaivin, Zhiyu Liu, Vincent Li, Ivy Samuels, Jinsook Son, Patrick Osei-Owusu, Jeffrey L. Garvin, Domenico Accili, Jeffrey R. Schelling
Abstract
Genetic factors are fundamental in the etiology of thoracic aortic aneurysm and dissection (TAAD), but the genetic cause is detected in only about 30% of cases. To define unreported TAAD-associated sequence variants, exome and gene panel sequencing was performed in 323 patients. We identified heterozygous CDKL1 variants [c.427T>C p.(Cys143Arg), c.617C>T p.(Ser206Leu), and c.404C>T p.(Thr135Met)] in 6 patients from 3 families with TAAD spectrum disorders. CDKL1 encodes a protein kinase involved in ciliary biology. Amino acid substitutions were predicted to affect CDKL1 catalytic activity or protein binding properties. CDKL1 was expressed in vascular smooth muscle cells in normal and diseased human aortic wall tissue. Cdkl1 knockdown and transient knockout in zebrafish resulted in intersomitic vessel (ISV) malformations and aortic dilation. Coinjection of human CDKL1wild-type RNA, but not CDKL1Cys143Arg and CDKL1Ser206Leu RNA, rescued ISV malformations. All variants affected CDKL1 kinase function and profiling data, and altered protein-protein binding properties, particularly with ciliary transport molecules. Expression of CDKL1 variants in heterologous cells interfered with cilia formation and length, CDKL1 localization, and p38 MAPK and Vegf signaling. Our data suggest a role of CDKL1 variants in the pathogenesis of TAAD spectrum disorders. The association between primary cilia dysregulation and TAAD expands our knowledge of the underlying molecular pathophysiology.
Authors
Theresa Nauth, Melanie Philipp, Sina Renner, Martin D. Burkhalter, Helke Schüler, Ceren Saygi, Kristian Händler, Bente Siebels, Alice Busch, Thomas Mair, Verena Rickassel, Sophia Deden, Konstantin Hoffer, Jakob Olfe, Thomas S. Mir, Yskert von Kodolitsch, Evaldas Girdauskas, Meike Rybczynski, Malte Kriegs, Hannah Voß, Thomas Sauvigny, Malte Spielmann, Malik Alawi, Susanne Krasemann, Christian Kubisch, Till J. Demal, Georg Rosenberger
Abstract
Endoplasmic reticulum (ER) stress through IRE1/XBP1 is implicated in the onset and progression of graft-versus-host disease (GVHD), but the role of the ER stress sensor PERK in T cell allogeneic responses and GVHD remains unexplored. Here, we report that PERK is a key regulator in T cell allogeneic response and GVHD induction. PERK augments GVHD through increasing Th1 and Th17 population, while reducing Treg differentiation by activating the Nrf2 pathway. Genetic deletion or selective inhibition of PERK pharmacologically reduces GVHD while preserving graft-versus-leukemia (GVL) activity. At the cellular level, PERK positively regulates CD4+ T cell pathogenicity while negatively regulating CD8+ T cell pathogenicity in the induction of GVHD. At the molecular level, PERK interacts with SEL1L and regulates SEL1L expression, leading to augmented T cell allogeneic responses and GVHD development. In vivo, PERK deficiency in donor T cells alleviates GVHD through ER-associated degradation. Furthermore, pharmacological inhibition of PERK with AMG44 significantly suppresses the severity of GVHD induced by murine or human T cells. In summary, our findings validate PERK as a potential therapeutic target for the prevention of GVHD while preserving GVL responses, and uncover the mechanism by which PERK differentially regulates CD4+ versus CD8+ T cell allogeneic and antitumor responses.
Authors
Qiao Cheng, Hee-Jin Choi, Yongxia Wu, Xiaohong Yuan, Allison Pugel, Linlu Tian, Michael Hendrix, Denggang Fu, Reza Alimohammadi, Chen Liu, Xue-Zhong Yu
Abstract
Teplizumab, a humanized anti-CD3 mAb, represents a breakthrough in autoimmune type 1 diabetes (T1D) treatment, by delaying clinical onset in stage 2 and slowing progression in early stage 3 of the disease. However, therapeutic responses are heterogeneous. To better understand this variability, we applied single-cell transcriptomics to paired peripheral blood and pancreas samples from anti–mouse CD3–treated nonobese diabetic (NOD) mice and identified distinct gene signatures associated with the therapy outcome, with consistent patterns across compartments. Success-associated signatures were enriched in NK or CD8+ T cells and other immune cell types, whereas resistance signatures were predominantly expressed by neutrophils. The immune cell communities underlying these response signatures were confirmed in human whole blood sequencing data from the Autoimmunity-blocking Antibody for Tolerance (AbATE) study at 6 months, which assessed teplizumab therapy in individuals with stage 3 T1D. Furthermore, baseline expression profiling in the human TrialNet Anti-CD3 Prevention (TN10) (stage 2) and AbATE (stage 3) cohorts identified immune signatures predictive of therapy response, T cell–enriched signatures in responders, and neutrophil-enriched signatures in nonresponders, highlighting the roles of both adaptive and innate immunity in determining teplizumab treatment outcomes. Using an elastic net logistic regression model, we developed a 26-gene blood-based signature predicting the response to teplizumab (AUC = 0.97). These findings demonstrate the predictive potential of immune gene signatures and the value of transcriptomics profiling in guiding individualized treatment strategies with teplizumab in individuals with T1D.
Authors
Gabriele Sassi, Pierre Lemaitre, Laia Fernández Calvo, Francesca Lodi, Álvaro Cortés Calabuig, Samal Bissenova, Amber Wouters, Laure Degroote, Marijke Viaene, Niels van Damme, Lauren Higdon, Peter S. Linsley, S. Alice Long, Chantal Mathieu, Conny Gysemans
Abstract
Impaired glucose-stimulated insulin secretion (GSIS) is a hallmark of β cell dysfunction in diabetes. Epigenetic mechanisms govern cellular glucose sensing and GSIS by β cells, but they remain incompletely defined. Here, we found that BAF60a functions as a chromatin regulator that sustains biphasic GSIS and preserves β cell function under metabolic stress conditions. BAF60a was downregulated in β cells from obese and diabetic mice, monkeys, and humans. β cell–specific inactivation of BAF60a in adult mice impaired GSIS, leading to hyperglycemia and glucose intolerance. Conversely, restoring BAF60a expression improved β cell function and systemic glucose homeostasis. Mechanistically, BAF60a physically interacted with Nkx6.1 to selectively modulate chromatin accessibility and transcriptional activity of target genes critical for GSIS coupling in islet β cells. A BAF60a V278M mutation associated with decreased β cell GSIS function was identified in human donors. Mice carrying this mutation, which disrupted the interaction between BAF60a and Nkx6.1, displayed β cell dysfunction and impaired glucose homeostasis. In addition, GLP-1R and GIPR expression was significantly reduced in BAF60a-deficient islets, attenuating the insulinotropic effect of GLP-1R agonists. Together, these findings support a role for BAF60a as a component of the epigenetic machinery that shapes the chromatin landscape in β cells critical for glucose sensing and insulin secretion.
Authors
Xinyuan Qiu, Ruo-Ran Wang, Qing-Qian Wu, Hongxing Fu, Shuaishuai Zhu, Wei Chen, Wen Wang, Haide Chen, Xiuyu Ji, Wenjing Zhang, Dandan Yan, Jing Yan, Li Jin, Rong Zhang, Mengjie Shi, Ping Luo, Yingqing Yang, Qintao Wang, Ziyin Zhang, Wei Ding, Xiaowen Pan, Chengbin Li, Bin Liang, Guoji Guo, Hai-long Piao, Min Zheng, Sheng Yan, Lingyun Zhu, Cheng Hu, Zhuo-Xian Meng
Abstract
TMPRSS2:ERG gene fusion (T:E fusion) in prostate adenocarcinoma (PCa) puts ERG under androgen receptor–regulated (AR-regulated) TMPRSS2 expression. T:E fusion is associated with PTEN loss and is highly associated with decreased INPP4B expression, which together may compensate for ERG-mediated suppression of AKT signaling. We confirmed in PCa cells and a mouse PCa model that ERG suppresses IRS2 and AKT activation. In contrast, ERG downregulation did not increase INPP4B, suggesting its decrease is indirect and reflects selective pressure to suppress INPP4B function. Notably, INPP4B expression was decreased in PTEN-intact and PTEN-deficient T:E fusion tumors, suggesting selection for a nonredundant function. As ERG in T:E fusion tumors is AR regulated, we further assessed whether AR inhibition increases AKT activity in T:E fusion tumors. A T:E fusion–positive PDX had increased AKT activity in vivo and response to AKT inhibition in vitro after androgen deprivation. Moreover, two clinical trials of neoadjuvant AR inhibition prior to radical prostatectomy showed greater increases in AKT activation in the T:E fusion–positive versus –negative tumors. These findings indicate that AKT activation may mitigate the efficacy of AR-targeted therapy in T:E fusion PCa and that these patients may most benefit from combination therapy targeting AR and AKT.
Authors
Fen Ma, Sen Chen, Luigi Cecchi, Betul Ersoy-Fazlioglu, Joshua W. Russo, Seiji Arai, Seifeldin Awad, Carla Calagua, Fang Xie, Larysa Poluben, Olga Voznesensky, Anson T. Ku, Fatima Karzai, Changmeng Cai, David J. Einstein, Huihui Ye, Xin Yuan, Alex Toker, Mary-Ellen Taplin, Adam G. Sowalsky, Steven P. Balk
Abstract
Plasminogen activator inhibitor 1 (PAI-1), encoded by SERPINE1, contributes to age-related cardiovascular disease (CVD) and other aging-related pathologies. Humans with a heterozygous loss-of-function SERPINE1 variant exhibit protection against aging and cardiometabolic dysfunction. We engineered a mouse model mimicking the human mutation (Serpine1TA700/+) and compared cardiovascular responses with WT littermates. Serpine1TA700/+ mice lived 17% longer than did littermate control mice. Under l-NG-nitro-arginine methyl ester–induced (l-NAME–induced) vascular stress, Serpine1TA700/+ mice exhibited diminished pulse wave velocity (PWV), lower systolic blood pressure (SBP), and preserved left ventricular diastolic function compared with controls. Conversely, PAI-1–overexpressing mice had measurements indicating accelerated cardiovascular aging. Single-cell transcriptomics of Serpine1TA700/+ aortas revealed a vascular-protective mechanism with downregulation of the extracellular matrix regulators Ccn1 and Itgb1. Serpine1TA700/+ aortas were also enriched in a cluster of smooth muscle cells that exhibited plasticity. Finally, PAI-1 pharmacological inhibition normalized SBP and reversed l-NAME–induced PWV elevation. These findings demonstrate that PAI-1 reduction protects against cardiovascular aging-related phenotypes, while PAI-1 excess promotes vascular pathological changes. Taken together, PAI-1 inhibition represents a promising strategy to mitigate age-related CVD.
Authors
Alireza Khoddam, Anthony Kalousdian, Mesut Eren, Saul Soberanes, Andrew Decker, Elizabeth J. Lux, Benjamin W. Zywicki, Brian Dinh, Bedirhan Boztepe, Baljash S. Cheema, Carla M. Cuda, Hiam Abdala-Valencia, Arun Sivakumar, Toshio Miyata, Lisa D. Wilsbacher, Douglas E. Vaughan
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