Treatment options for Alcohol Use Disorders (AUD) have minimally advanced since 2004, while the annual deaths and economic toll have increased alarmingly. Phosphodiesterase type 4 (PDE4) is associated with alcohol and nicotine dependence. PDE4 inhibitors were identified as a potential AUD treatment using a novel bioinformatics approach. We prioritized a newer PDE4 inhibitor, apremilast, as ideal for repurposing, (i.e. FDA approved for psoriasis, low incidence of adverse events, excellent safety profile), and tested it using multiple animal strains and models, as well as in a human Phase IIa study. We found that apremilast reduced binge-like alcohol intake and behavioral measures of alcohol motivation in mouse models of genetic risk for drinking to intoxication. Apremilast also reduced excessive alcohol drinking in models for stress-facilitated drinking and alcohol dependence. Using site-directed drug infusions and electrophysiology, we uncovered that apremilast may act to lessen drinking in mice by increasing neural activity in the nucleus accumbens, a key brain region in the regulation of alcohol intake. Importantly, apremilast (90 mg/d) reduced excessive drinking in non-treatment seeking individuals with AUD in a double blind, placebo-controlled study. These results demonstrate that apremilast suppresses excessive alcohol drinking across the spectrum of AUD severity.
Kolter B. Grigsby, Regina A. Mangieri, Amanda J. Roberts, Marcelo F. Lopez, Evan J. Firsick, Kayla G. Townsley, Alan Beneze, Jessica Bess, Toby K. Eisenstein, Joseph J. Meissler, John M. Light, Jenny Miller, Susan Quello, Farhad Shadan, Michael H. Skinner, Heather C. Aziz, Pamela Metten, Richard A. Morissett, John C. Crabbe, Marisa Roberto, Howard C. Becker, Barbara J. Mason, Angela R. Ozburn
Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β-cells. To identify candidates contributing to T2D pathophysiology, we studied human pancreatic islets from ~300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified islet expression changes may predispose to diabetes, as they associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β-cells based on single-cell RNA-sequencing data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D-SNPs. Mouse knock-out strains demonstrated that T2D-associated candidates regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing β-cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we identified molecular alterations in human pancreatic islets contributing to β-cell dysfunction in T2D pathophysiology.
Karl Bacos, Alexander Perfilyev, Alexandros Karagiannopoulos, Elaine Cowan, Jones K. Ofori, Ludivine Bertonnier-Brouty, Tina Rönn, Andreas Lindqvist, Cheng Luan, Sabrina Ruhrmann, Mtakai Ngara, Åsa Nilsson, Sevda Gheibi, Claire L. Lyons, Jens O. Lagerstedt, Mohammad Barghouth, Jonathan L.S. Esguerra, Petr Volkov, Malin Fex, Hindrik Mulder, Nils Wierup, Ulrika Krus, Isabella Artner, Lena Eliasson, Rashmi B. Prasad, Luis Rodrigo Cataldo, Charlotte Ling
Christian Lacks Lino Cardenas, Lauren C. Briere, David A. Sweetser, Mark E. Lindsay, Patricia L. Musolino
The alternative sigma factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic positive and negative gene regulation essential for the spirochete’s dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by TBDCapSeq to compare the transcriptomes of wild-type and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb’s enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that BosR, a non-canonical Fur family regulator, and the c-di-GMP effector PlzA reciprocally regulate RpoS function. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting RpoN-dependent transcription of rpoS. During transmission, liganded-PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.
André A. Grassmann, Rafal Tokarz, Caroline Golino, Melissa A. McLain, Ashley M. Groshong, Justin D. Radolf, Melissa J. Caimano
Anil Dangi, Irma Husain, Collin Z. Jordan, Shuangjin Yu, Xunrong Luo
BACKGROUND. To date, only limited data is available on the mechanisms of protection against colonization with Bordetella pertussis in humans. METHODS. In this study, the cellular responses to Bordetella pertussis challenge were monitored longitudinally using high-dimensional EuroFlow-based flow cytometry, allowing quantitative detection of >250 different immune cell subsets in the blood of 15 healthy donors. RESULTS. Participants who were protected against colonization showed different early cellular responses compared to colonized participants. Especially prominent for colonization-protected participants were the early expansion of (CD36-) non classical monocytes at day 1 (d1), Natural Killer cells (d3), follicular T helper cells (d1-d3) and plasma cells (d3). Plasma cell expansion at d3 correlated negatively with the CFU load at d7 and d9 post-challenge. Increased plasma cell maturation at d11-14 was found in participants with seroconversion. CONCLUSION. These early cellular immune responses following experimental infection can now be further characterized and potentially linked to an efficient mucosal immune response, preventing colonization. Ultimately, their presence may be used to evaluate whether new Bordetella pertussis vaccine candidates are protective against Bordetella pertussis colonization, e.g., by bacterial challenge post-vaccination. TRIAL REGISTRATION. NCT03751514. FUNDING. This study is part of the PERISCOPE Project, which has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115910. The flow cytometric studies were supported by the EuroFlow Consortium.
Annieck M. Diks, Hans de Graaf, Cristina Teodosio, Rick J. Groenland, Bas de Mooij, Muktar Ibrahim, Alison R. Hill, Robert C. Read, Jacques J.M. van Dongen, Magdalena A. Berkowska
Mutations of G protein coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing Mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibit a partial maxillary-to-mandibular transformation, which is rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain-of-function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increases flexibility of this portion of the helix, thus favoring G-protein binding and leading to G-protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduces the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, that may provide the basis for drug design targeting GPCRs.
Yukiko Kurihara, Toru Ekimoto, Christopher T. Gordon, Yasunobu Uchijima, Ryo Sugiyama, Taro Kitazawa, Akiyasu Iwase, Risa Kotani, Rieko Asai, Véronique Pingault, Mitsunori Ikeguchi, Jeanne Amiel, Hiroki Kurihara
The molecular mechanisms of sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) remain incompletely understood. Single-cell RNA sequencing and morphometric data were collected from research kidney biopsies donated by young persons with type 2 diabetes (T2D), aged 12-21 years, and healthy controls (HC). Participants with T2D were obese, had higher estimated glomerular filtration rates, mesangial and glomerular volumes than HC. Ten T2D participants had been prescribed SGLT2i (T2Di(+)) and 6 not (T2Di(-)). Transcriptional profiles showed SGLT2 expression exclusively in the proximal tubular (PT) cluster with highest expression in T2Di(-). However, transcriptional alterations with SGLT2i treatment were seen across nephron segments, particularly in the distal nephron. SGLT2i treatment was associated with suppression of transcripts in the glycolysis, gluconeogenesis, tricarboxylic acid cycle pathways in PT, but enhanced in thick ascending limb. Transcripts in the energy sensitive mammalian target of rapamycin complex1 (mTORC1) signaling pathway returned towards HC levels in all tubular segments in T2Di(+), consistent with a diabetes mouse model treated with SGLT2i. Decreased levels of phosphorylated S6 protein in proximal and distal tubules in T2Di(+) confirmed changes in mTORC1 pathway activity. We propose that SGLT2i treatment benefits the kidneys by mitigating diabetes-induced metabolic perturbations via suppression of mTORC1 signaling in kidney tubules.
Jennifer A. Schaub, Fadhl M. AlAkwaa, Phillip J. McCown, Abhijit S. Naik, Viji Nair, Sean Eddy, Rajasree Menon, Edgar A. Otto, Dawit Demeke, John Hartman, Damian Fermin, Christopher O'Connor, Lalita Subramanian, Markus Bitzer, Roger Harned, Patricia Ladd, Laura Pyle, Subramaniam Pennathur, Ken Inoki, Jeffrey B. Hodgin, Frank C. Brosius, Robert G. Nelson, Matthias Kretzler, Petter Bjornstad
Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome wide methylation patterns are poorly defined. While methylation is known to alter gene expression the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n=39) that was subsequently replicated in a larger validation cohort (n=296). Gene body related regions made up >60% of the methylation differences and emphasised the importance of methylation sequencing. We observe differentially methylated genes associated with DN (DDN) in three independent T1D registries originating from Denmark (n=445), Hong Kong (n=107) and Thailand (n=130). Reduced DNA methylation at CTCF and Pol2B sites were tightly connected with DN pathways that include insulin signalling, lipid metabolism and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubules. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding and the activation of insulin signalling phosphoproteins in hyperglycaemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophage and vascular endothelial cells.
Ishant Khurana, Harikrishnan Kaipananickal, Scott Maxwell, Sørine Birkelund, Anna Syreeni, Carol Forsblom, Jun Okabe, Mark Ziemann, Antony Kaspi, Haloom Rafehi, Anne Jørgensen, Keith Al-Hasani, Merlin C. Thomas, Guozhi Jiang, Andrea O.Y. Luk, Heung Man Lee, Yu Huang, Yotsapon Thewjitcharoen, Soontaree Nakasatien, Thep Himathongkam, Christopher Fogarty, Rachel Njeim, Assaad Eid, Tine Willum Hansen, Nete Tofte, Evy Connie Ottesen, Ronald C.W. Ma, Juliana C.N. Chan, Mark Emmanuel Cooper, Peter Rossing, Per-Henrik Groop, Assam El-Osta
SIPRα on macrophages binds with CD47 to resist pro-engulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here we reported that the CD47/SIRPα axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, SHP2 is constitutively neddylated on K358 and K364 sites, thus its auto-inhibited conformation is maintained. In response to CD47-liganded SIRPα, SHP2 is deneddylated by SENP8, which leads to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that the supplementation with SHP2 allosteric inhibitors sensitized the immune treatment-resistant CRC to immunotherapy. Our results emphasized that the CRC subtype which is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs, and highlighted the need to further combine the strategy of SHP2 targeting in colorectal cancer therapy.
Yiqing Li, Hui Zhou, Pan Liu, Dandan Lv, Yichun Shi, Bufu Tang, Jiaqi Xu, Tingting Zhong, Wangting Xu, Jie Zhang, Jianying Zhou, Kejing Ying, Yongchao Zhao, YI Sun, Zhinong Jiang, Hongqiang Cheng, Xue Zhang, Yue-Hai Ke
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