Abstract
Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector–mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X–KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector–treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector–mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.
Authors
Hayes Wong, Alexander W.M. Hooper, Hye Ri Kang, Shiron J. Lee, Jiayi Zhao, Chanchal Sadhu, Satinder Rawat, Steven J. Gray, David R. Hampson
Abstract
Previous studies implicate extracellular adenosine signaling in attenuating myocardial ischemia and reperfusion injury (IRI). This extracellular adenosine signaling is terminated by its uptake into cells by equilibrative nucleoside transporters (ENTs). Thus, we hypothesized that targeting ENTs would function to increase cardiac adenosine signaling and concomitant cardioprotection against IRI. Mice were exposed to myocardial ischemia and reperfusion injury. Myocardial injury was attenuated in mice treated with the nonspecific ENT inhibitor dipyridamole. A comparison of mice with global Ent1 or Ent2 deletion showed cardioprotection only in Ent1–/– mice. Moreover, studies with tissue-specific Ent deletion revealed that mice with myocyte-specific Ent1 deletion (Ent1loxP/loxP Myosin Cre+ mice) experienced smaller infarct sizes. Measurements of cardiac adenosine levels demonstrated that postischemic elevations of adenosine persisted during reperfusion after targeting ENTs. Finally, studies in mice with global or myeloid-specific deletion of the Adora2b adenosine receptor (Adora2bloxP/loxP LysM Cre+ mice) implied that Adora2b signaling on myeloid-inflammatory cells in cardioprotection provided by ENT inhibition. These studies reveal a previously unrecognized role for myocyte-specific ENT1 in cardioprotection by enhancing myeloid-dependent Adora2b signaling during reperfusion. Extension of these findings implicates adenosine transporter inhibitors in cardioprotection against ischemia and reperfusion injury.
Authors
Wei Ruan, Jiwen Li, Seungwon Choi, Xinxin Ma, Yafen Liang, Ragini Nair, Xiaoyi Yuan, Tingting W. Mills, Holger K. Eltzschig
Abstract
Ischemic-reperfusion injury (IRI) is a major pathogenic factor in acute kidney injury (AKI), which directly leads to the hypoxic injury of renal tubular epithelial cells (RTECs). Although emerging studies suggest repressor element 1–silencing transcription factor (REST) as a master regulator of gene repression under hypoxia, its role in AKI remains elusive. Here, we found that REST was upregulated in AKI patients, mice, and RTECs, which was positively associated with the degree of kidney injury, while renal tubule–specific knockout of Rest significantly alleviated AKI and its progression to chronic kidney disease (CKD). Subsequent mechanistic studies indicated that suppression of ferroptosis was responsible for REST-knockdown-induced amelioration of hypoxia-reoxygenation injury, during which process Cre-expressing adenovirus–mediated REST downregulation attenuated ferroptosis through upregulating glutamate-cysteine ligase modifier subunit (GCLM) in primary RTECs. Further, REST transcriptionally repressed GCLM expression via directly binding to its promoter region. In conclusion, our findings revealed the involvement of REST, a hypoxia regulatory factor, in AKI-to-CKD transition and identified the ferroptosis-inducing effect of REST, which may serve as a promising therapeutic target for ameliorating AKI and its progression to CKD.
Authors
Shuiqin Gong, Aihong Zhang, Mengying Yao, Wang Xin, Xu Guan, Shaozong Qin, Yong Liu, Jiachuan Xiong, Ke Yang, Li Xiong, Ting He, Yinghui Huang, Jinghong Zhao
Abstract
Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.
Authors
Aaron Bodansky, Chung-Yu Wang, Aditi Saxena, Anthea Mitchell, Andrew F. Kung, Saki Takahashi, Khamal Anglin, Beatrice Huang, Rebecca Hoh, Scott Lu, Sarah A. Goldberg, Justin Romero, Brandon Tran, Raushun Kirtikar, Halle Grebe, Matthew So, Bryan Greenhouse, Matthew S. Durstenfeld, Priscilla Y. Hsue, Joanna Hellmuth, J. Daniel Kelly, Jeffrey N. Martin, Mark S. Anderson, Steven G. Deeks, Timothy J. Henrich, Joseph L. DeRisi, Michael J. Peluso
Abstract
Forkhead box M1 (FOXM1) plays a critical role in development physiologically and tumorigenesis pathologically. However, insufficient efforts have been dedicated to exploring the regulation, in particular the degradation of FOXM1. Here, the ON-TARGETplus siRNA library targeting E3 ligases was used to screen potential candidates to repress FOXM1. Of note, mechanism study revealed that RNF112 directly ubiquitinates FOXM1 in gastric cancer, resulting in a decreased FOXM1 transcriptional network and suppressing the proliferation and invasion of gastric cancer. Interestingly, the well-established small-molecule compound RCM-1 significantly enhanced the interaction between RNF112 and FOXM1, which further promoted FOXM1 ubiquitination and subsequently exerted promising anticancer effects in vitro and in vivo. Altogether, we demonstrate that RNF112 suppresses gastric cancer progression by ubiquitinating FOXM1 and highlight the RNF112/FOXM1 axis serves as both prognosis biomarker and therapeutic target in gastric cancer.
Authors
Shengwei Zhang, Jing Wang, Weichao Hu, Lijiao He, Qingyun Tang, Jie Li, Mengmeng Jie, Xinzhe Li, Cheng Liu, Qin Ouyang, Shiming Yang, Changjiang Hu
Abstract
Insulin secretion from pancreatic β cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell–derived β cells (SC–β cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC–β cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC–β cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human β cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.
Authors
Eleonora de Klerk, Yini Xiao, Christopher H. Emfinger, Mark P. Keller, David I. Berrios, Valentina Loconte, Axel A. Ekman, Kate L. White, Rebecca L. Cardone, Richard G. Kibbey, Alan D. Attie, Matthias Hebrok
Abstract
Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus–based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.
Authors
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
Abstract
Viral illnesses like SARS-CoV-2 have pathologic effects on nonrespiratory organs in the absence of direct viral infection. We injected mice with cocktails of rodent equivalents of human cytokine storms resulting from SARS-CoV-2/COVID-19 or rhinovirus common cold infection. At low doses, COVID-19 cocktails induced glomerular injury and albuminuria in zinc fingers and homeoboxes 2 (Zhx2) hypomorph and Zhx2+/+ mice to mimic COVID-19–related proteinuria. Common Cold cocktail induced albuminuria selectively in Zhx2 hypomorph mice to model relapse of minimal change disease, which improved after depletion of TNF-α, soluble IL-4Rα, or IL-6. The Zhx2 hypomorph state increased cell membrane to nuclear migration of podocyte ZHX proteins in vivo (both cocktails) and lowered phosphorylated STAT6 activation (COVID-19 cocktail) in vitro. At higher doses, COVID-19 cocktails induced acute heart injury, myocarditis, pericarditis, acute liver injury, acute kidney injury, and high mortality in Zhx2+/+ mice, whereas Zhx2 hypomorph mice were relatively protected, due in part to early, asynchronous activation of STAT5 and STAT6 pathways in these organs. Dual depletion of cytokine combinations of TNF-α with IL-2, IL-13, or IL-4 in Zhx2+/+ mice reduced multiorgan injury and eliminated mortality. Using genome sequencing and CRISPR/Cas9, an insertion upstream of ZHX2 was identified as a cause of the human ZHX2 hypomorph state.
Authors
Maria Del Nogal Avila, Ranjan Das, Joubert Kharlyngdoh, Eduardo Molina-Jijon, Hector Donoro Blazquez, Stéphanie Gambut, Michael Crowley, David K. Crossman, Rasheed A. Gbadagesin, Sunveer S. Chugh, Sunjeet S. Chugh, Carmen Avila-Casado, Camille Macé, Lionel C. Clement, Sumant S. Chugh
Abstract
The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCSs) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulation. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A–exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that was upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulated some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring’s metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.
Authors
Soyoung Park, Amine M. Belfoul, Marialetizia Rastelli, Alice Jang, Magali Monnoye, Hosung Bae, Anna Kamitakahara, Patrick Giavalisco, Shan Sun, Pierre-Yves Barelle, Jasmine Plows, Cholsoon Jang, Anthony Fodor, Michael I. Goran, Sebastien G. Bouret
Abstract
Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.
Authors
Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Michael Feely, Elham Nasri, Robert P. Seifert, Xiaoping Luo, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens III, Frederic J. Kaye, Maria Zajac-Kaye
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