Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o G-protein-coupled-receptors by Gβγ subunit inhibition of two mechanisms: decreased calcium entry and direct interaction of Gβγ with Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) decreasing Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact, and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. SNAP25Δ3/Δ3 mice exhibit enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in SNAP25Δ3/Δ3 mice challenged with high fat diet. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in SNAP25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neuron exocytosis from SNAP25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combatting metabolic disease.
Ryan P. Ceddia, Zack Zurawski, Analisa Thompson Gray, Feyisayo Adegboye, Ainsley McDonald-Boyer, Fubiao Shi, Dianxin Liu, Jose Maldonado, Jiesi Feng, Yulong Li, Simon Alford, Julio E. Ayala, Owen P. McGuinness, Sheila Collins, Heidi E. Hamm
Biological aging can be described as accumulative, prolonged metabolic stress, and is the major risk factor for cognitive decline and Alzheimer’s disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes over-expressed with age, and is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that in human cells, genetic removal of QR2 produces a shift in the proteome opposing that found in AD brains, while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2i’s), enabling evaluation of chronic QR2 inhibition as a novel way to reduce biological-age related metabolic stress and cognitive decline. QR2i’s replicated results obtained by genetic removal of QR2 while local QR2i microinjection improved hippocampal and cortical dependent learning in rats and mice. Continuous consumption of QR2i’s in drinking-water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity- and pathway function in the healthy and neurodegenerative brain, and the great therapeutic potential of QR2i’s as first-in-class drugs.
Nathaniel L. Gould, Gila R. Scherer, Silvia Carvalho, Khriesto Shurrush, Haneen Kayyal, Efrat Edry, Alina Elkobi, Orit David, Maria Foqara, Darshit Thakar, Tommaso Pavesi, Vijendra Sharma, Matthew Walker, Matthew Maitland, Orly Dym, Shira Albeck, Yoav Peleg, Nicolas Germain, Ilana Babaev, Haleli Sharir, Maya Lalzar, Boris Shklyar, Neta Hazut, Mohammad Khamaisy, Maxime Lévesque, Gilles Lajoie, Massimo Avoli, Gabriel Amitai, Bruce Lefker, Chakrapani Subramanyam, Brian Shilton, Haim Barr, Kobi Rosenblum
Glial activation and inflammation coincide with neurofibrillary tangles (NFT) formation in neurons. However, the mechanism behind tau fibril and glia interaction is poorly understood. Here, we found that tau preformed fibrils (PFF) caused induction of inflammation in microglia by specifically activating the TLR2-MyD88, but not TLR4-MyD88, pathway. Accordingly, TLR2 interacting domain of MyD88 (wtTIDM) peptide inhibited tau PFF-induced activation of TLR2-MyD88-NF-κB pathway resulting in reduced inflammation. Nasal administration of wtTIDM in P301S tau-expressing PS19 mice was found to inhibit gliosis and inflammatory markers, along with reduction of pathogenic tau in the hippocampus, resulting in improved cognitive behavior in PS19 mice. The inhibitory effect of wtTIDM on tau pathology was absent in PS19 mice lacking TLR2, reinforcing the essential involvement of TLR2 in wtTIDM- mediated effects in vivo. While understanding the mechanism further, we found that tau promoter harboured a potential NF-κB binding site and that proinflammatory molecules increased the transcription of tau in neurons via NF-κB. These results suggest that tau-induced neuroinflammation and neuropathology require TLR2 and that neuroinflammation directly upregulates tau in neurons via NF-κB, highlighting a direct connection between inflammation and tauopathy.
Debashis Dutta, Malabendu Jana, Ramesh Kumar Paidi, Moumita Majumder, Sumita Raha, Sridevi Dasarathy, Kalipada Pahan
Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVD), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. CHIP was identified using whole exome sequencing data of blood DNA and modeled both as a composite and for common drivers (DNMT3A, TET2, ASXL1, and JAK2) separately. We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identify IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risks. We show that CRISPR-induced Asxl1 mutated murine macrophages have a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides new evidence to support gene-specific strategies to address CHIP-associated CVD risk.
Zhi Yu, Trevor P. Filder, Yunfeng Ruan, Caitlyn Vlasschaert, Tetsushi Nakao, Md Mesbah Uddin, Taralynn Mack, Abhishek Niroula, J. Brett Heimlich, Seyedeh M. Zekavat, Christopher J. Gibson, Gabriel K. Griffin, Yuxuan Wang, Gina M. Peloso, Nancy Heard-Costa, Daniel Levy, Ramachandran S. Vasan, François Aguet, Kristin G. Ardlie, Kent D. Taylor, Stephen S. Rich, Jerome I. Rotter, Peter Libby, Siddhartha Jaiswal, Benjamin L. Ebert, Alexander G. Bick, Alan R. Tall, Pradeep Natarajan
The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII is markedly upregulated in human breast cancer (BC) and is closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of EGFR/ErbB receptor tyrosine kinase signaling and show that it forms a complex with EGFR, HER2 and α6 integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies with Col18a1 mouse models crossed with the MMTV-PyMT mammary carcinogenesis model showed that ColXVIII promotes BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells, and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.
Raman Devarajan, Valerio Izzi, Hellevi Peltoketo, Gunilla Rask, Saila Kauppila, Marja-Riitta Väisänen, Heli Ruotsalainen, Guillermo A. Martinez-Nieto, Sanna-Maria Karppinen, Timo Väisänen, Inderjeet Kaur, Jussi Koivunen, Takako Sasaki, Robert Winqvist, Aki Manninen, Fredrik Wärnberg, Malin Sund, Taina Pihlajaniemi, Ritva Heljasvaara
X-linked myotubular myopathy (XLMTM) is a fatal congenital disorder caused by mutations in the MTM1 gene. Currently, there are no approved treatments, though AAV8-mediated gene transfer therapy has shown promise in animal models and preliminarily in patients. However, four patients with XLMTM treated with gene therapy have died from progressive liver failure, and hepatobiliary disease has now been recognized more broadly in association with XLMTM. In an attempt to understand whether loss of MTM1 itself is associated with liver pathology, we have characterized a novel liver phenotype in a zebrafish model of this disease. Specifically, we have found that loss-of-function mutations in mtm1 lead to severe liver abnormalities including impaired bile flux, structural abnormalities of the bile canaliculus, and improper endosomal-mediated trafficking of canalicular transporters. Using a reporter tagged Mtm1 zebrafish line, we have established localization of Mtm1 in the liver in association with Rab11 and canalicular transport proteins, and demonstrated that hepatocyte specific re-expression of Mtm1 can rescue the cholestatic phenotype. Lastly, we completed a targeted chemical screen, and found that Dynasore, a dynamin II inhibitor, is able to partially restore bile flow and transporter localization to the canalicular membrane. In summary, we demonstrate for the first time liver abnormalities that are directly caused by MTM1 mutation in a pre-clinical model, thus establishing the critical framework for better understanding and comprehensive treatment of the human disease.
Sophie Karolczak, Ashish R. Deshwar, Evangelina Aristegui, Binita M. Kamath, Michael W. Lawlor, Gaia Andreoletti, Jonathan R. Volpatti, Jillian L. Ellis, Chunyue Yin, James J. Dowling
Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with Bone Morphogenic Protein 9 (BMP9) and BMP10. Conversely, mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in vascular development and homeostasis, and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between fluid shear stress and SMAD signaling in vascular stability and its failure in HHT. We have now found that loss of Smad4 increases ECs’ sensitivity to flow by lowering the FSS set point with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling leading to cell cycle progression - mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors, CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.
Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P-selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in SCD patients, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P-selectin antibody more effectively increased PMo numbers and reduced stasis compared to single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2-CSF-1 and IFN-I-CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.
Yunfeng Liu, Shan Su, Sarah Shayo, Weili Bao, Mouli Pal, Kai Dou, Patricia A. Shi, Banu Aygun, Sally Campbell-Lee, Cheryl A. Lobo, Avital Mendelson, Xiuli An, Deepa Manwani, Hui Zhong, Karina Yazdanbakhsh
Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11β-HSD1 (encoded by Hsd11b1). Here we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth but reduced in vivo tumor progression, which corresponded with increased frequencies of tumor-infiltrating CD8+ T cells (TIL) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of Tconv activation in tumor-infiltrating Treg. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11β-HSD1 reduced tumor growth to the same degree as gene knockout, and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11β-HSD1 is well-tolerated in clinical studies, these data suggest that targeting 11β-HSD1 may be a beneficial adjunct in cancer therapy.
Matthew D. Taves, Shizuka Otsuka, Michaela A. Taylor, Kaitlynn M. Donahue, Thomas J. Meyer, Margaret C. Cam, Jonathan D. Ashwell
Protease activated receptor (PAR) 4 (gene: F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not recognized whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice where F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared to hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P-selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of non-standard anti-platelet therapies for patients expressing PAR4 Thr120.
Frederik Denorme, Nicole D. Armstrong, Michelle L. Stoller, Irina Portier, Emilia A. Tugolukova, Rikki M. Tanner, Emilie Montenont, Seema Bhatlekar, Mark Cody, John L. Rustad, Abigail Ajanel, Neal D. Tolley, Darian C. Murray, Julie L. Boyle, Marvin T. Nieman, Steven E. McKenzie, Christian Con Yost, Leslie A. Lange, Mary Cushman, Marguerite R. Irvin, Paul F. Bray, Robert A. Campbell
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