Endoplasmic Reticulum Stress Delays Choroid Development in the HCAR1 Knockout Mouse.

The subretina, composed of the choroid and the retinal pigment epithelium (RPE), bears a critical role in proper vision. In addition to phagocytosis of photoreceptor debris, the RPE shuttles oxygen and nutrients to the neuroretina. For their own energy production, RPE cells mainly rely on lactate, a major by-product of glycolysis. Lactate, in turn, is believed to convey most of its biological effects via the hydroxycarboxylic acid receptor 1 (HCAR1). Here, the lactate-specific receptor, HCAR1, is found to be exclusively expressed in the RPE cells within the subretina, and Hcar1-/- mice exhibit a substantially thinner choroidal vasculature during development. Notably, the angiogenic properties of lactate on the choroid are impacted by the absence of Hcar1. HCAR1-deficient mice exhibit elevated endoplasmic reticulum stress along with eukaryotic initiation factor 2α phosphorylation, a significant decrease in the global protein translation rate, and a lower proliferation rate of choroidal vasculature. Strikingly, inhibition of the integrated stress response using an inhibitor that reverses the effect of eukaryotic initiation factor 2α phosphorylation restores protein translation and rescues choroidal thinning. These results provide evidence that lactate signalling via HCAR1 is important for choroidal development/angiogenesis and highlight the importance of this receptor in establishing mature vision.

Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

Versatile lactate signaling via HCAR1: a multifaceted GPCR involved in many biological processes.

G-coupled protein receptors (GPCRs) are the ultimate refuge of pharmacology and medicine as more than 40% of all marketed drugs are directly targeting these receptors. Through cell surface expression, they are at the forefront of cellular communication with the outside world. Metabolites among the conveyors of this communication are becoming more prominent with the recognition of them as ligands for GPCRs. HCAR1 is a GPCR conveyor of lactate. It is a class A GPCR coupled to Gαi which reduces cellular cAMP along with the downstream Gßγ signaling. It was first found to inhibit lipolysis, and lately has been implicated in diverse cellular processes, including neural activities, angiogenesis, inflammation, vision, cardiovascular function, stem cell proliferation, and involved in promoting pathogenesis for different conditions, such as cancer. Other than signaling from the plasma membrane, HCAR1 shows nuclear localization with different location-biased activities therein. Although different functions for HCAR1 are being discovered, its cell and molecular mechanisms are yet ill understood. Here, we provide a comprehensive review on HCAR1, which covers the literature on the subject, and discusses its importance and relevance in various biological phenomena.

Lack of HCAR1, the lactate GPCR, signaling promotes autistic-like behavior.

The GPCR HCAR1 is known to be the sole receptor for lactate, which modulates its metabolic effects. Despite its significant role in many processes, mice deficient in HCAR1 exhibit no visible phenotype and are healthy and fertile. We performed transcriptomic analysis on HCAR1 deficient cells, in combination with lactate, to explore pathophysiologically altered processes. Processes such as immune regulation, various cancers, and neurodegenerative diseases were significantly enriched for HCAR1 transcriptomic signature. However, the most affected process of all was autism spectrum disorder. We performed behavioral tests on HCAR1 KO mice and observed that these mice manifest autistic-like behavior. Our data opens new avenues for research on HCAR1 and lactate effect at a pathological level. Video Abstract.

Müller Cell-Localized G-Protein-Coupled Receptor 81 (Hydroxycarboxylic Acid Receptor 1) Regulates Inner Retinal Vasculature via Norrin/Wnt Pathways.

Ischemic retinopathies are characterized by a progressive microvascular degeneration followed by a postischemic aberrant neovascularization. To reinstate vascular supply and metabolic equilibrium to the ischemic tissue during ischemic retinopathies, a dysregulated production of growth factors and metabolic intermediates occurs, promoting retinal angiogenesis. Glycolysis-derived lactate, highly produced during ischemic conditions, has been associated with tumor angiogenesis and wound healing. Lactate exerts its biological effects via G-protein-coupled receptor 81 (GPR81) in several tissues; however, its physiological functions and mechanisms of action in the retina remain poorly understood. Herein, we show that GPR81, localized predominantly in Müller cells, governs deep vascular complex formation during development and in ischemic retinopathy. Lactate-stimulated GPR81 Müller cells produce numerous angiogenic factors, including Wnt ligands and particularly Norrin, which contributes significantly in triggering inner retinal blood vessel formation. Conversely, GPR81-null mice retina shows reduced inner vascular network formation associated with low levels of Norrin (and Wnt ligands). Lactate accumulation during ischemic retinopathy selectively activates GPR81-extracellular signal-regulated kinase 1/2-Norrin signaling to accelerate inner retinal vascularization in wild-type animals, but not in the retina of GPR81-null mice. Altogether, we reveal that lactate via GPR81-Norrin participates in inner vascular network development and in restoration of the vasculature in response to injury. These findings suggest a new potential therapeutic target to alleviate ischemic diseases.

The Inability of the Choroid to Revascularize in Oxygen-Induced Retinopathy Results from Increased p53/miR-Let-7b Activity.

Retinopathy of prematurity (ROP) is characterized by an initial retinal avascularization, followed by pathologic neovascularization. Recently, choroidal thinning has also been detected in children formerly diagnosed with ROP; a similar sustained choroidal thinning is observed in ROP models. But the mechanism underlying the lack of choroidal revascularization remains unclear and was investigated in an oxygen-induced retinopathy (OIR) model. In OIR, evidence of senescence was detected, preceded by oxidative stress in the choroid and the retinal pigment epithelium. This was associated with a global reduction of proangiogenic factors, including insulin-like growth factor 1 receptor (Igf1R). Coincidentally, tumor suppressor p53 was highly expressed in the OIR retinae. Curtailing p53 activity resulted in reversal of senescence, normalization of Igf1r expression, and preservation of choroidal integrity. OIR-induced down-regulation of Igf1r was mediated at least partly by miR-let-7b as i) let-7b expression was augmented throughout and beyond the period of oxygen exposure, ii) let-7b directly targeted Igf1r mRNA, and iii) p53 knock-down blunted let-7b expression, restored Igf1r expression, and elicited choroidal revascularization. Finally, restoration of Igf1r expression rescued choroid thickness. Altogether, this study uncovers a significant mechanism for defective choroidal revascularization in OIR, revealing a new role for p53/let-7b/IGF-1R axis in the retina. Future investigations on this (and connected) pathway could further our understanding of other degenerative choroidopathies, such as geographic atrophy.

PRMT5 orchestrates EGFR and AKT networks to activate NFκB and promote EMT.

Neuroblastoma remains a formidable challenge in pediatric oncology, representing 15% of cancer-related mortalities in children. Despite advancements in combinatorial and targeted treatments improving survival rates, nearly 50% of patients with high-risk neuroblastoma will ultimately succumb to their disease. Dysregulation of the epithelial-mesenchymal transition (EMT) is a key mechanism of tumor cell dissemination, resulting in metastasis and poor outcomes in many cancers. Our prior work identified PRMT5 as a key regulator of EMT via methylation of AKT at arginine 15, enhancing the expression of EMT-driving transcription factors and facilitating metastasis. Here, we identify that PRMT5 directly regulates the transcription of the epidermal growth factor receptor (EGFR). PRMT5, through independent modulation of the EGFR and AKT pathways, orchestrates the activation of NFκB, resulting in the upregulation of the pro-EMT transcription factors ZEB1, SNAIL, and TWIST1. Notably, EGFR and AKT form a compensatory feedback loop, reinforcing the expression of these EMT transcription factors. Small molecule inhibition of PRMT5 methyltransferase activity disrupts EGFR/AKT signaling, suppresses EMT transcription factor expression and ablates tumor growth in vivo . Our findings underscore the pivotal role of PRMT5 in the control of the EMT program in high-risk neuroblastoma.

Disentangling the nonmetabolic roles of metabolites: lactate as a case study.

Many metabolites possess covalent and noncovalent signaling functions. However, ongoing research considers them mostly as ligands, neglecting their potential involvement in post-translational modifications. In this forum article, we discuss the dual signaling functions of metabolites, using lactate as a case study, and advocate for the use of multiple complementary techniques to disentangle their functions.

Neuronal GPR81 regulates developmental brain angiogenesis and promotes brain recovery after a hypoxic ischemic insult.

Perinatal hypoxic/ischemic (HI) brain injury is a major clinical problem with devastating neurodevelopmental outcomes in neonates. During HI brain injury, dysregulated factor production contributes to microvascular impairment. Glycolysis-derived lactate accumulated during ischemia has been proposed to protect against ischemic injury, but its mechanism of action is poorly understood. Herein, we hypothesize that lactate via its G-protein coupled receptor (GPR81) controls postnatal brain angiogenesis and plays a protective role after HI injury. We show that GPR81 is predominantly expressed in neurons of the cerebral cortex and hippocampus. GPR81-null mice displayed a delay in cerebral microvascular development linked to reduced levels of various major angiogenic factors and augmented expression of anti-angiogenic Thrombospondin-1 (TSP-1) in comparison to their WT littermates. Coherently, lactate stimulation induced an increase in growth factors (VEGF, Ang1 and 2, PDGF) and reduced TSP-1 expression in neurons, which contributed to accelerating angiogenesis. HI injury in GPR81-null animals curtailed vascular density and consequently increased infarct size compared to changes seen in WT mice; conversely intracerebroventricular lactate injection increased vascular density and diminished infarct size in WT but not in GPR81-null mice. Collectively, we show that lactate acting via GPR81 participates in developmental brain angiogenesis, and attenuates HI injury by restoring compromised microvasculature.

The Succinate Receptor SUCNR1 Resides at the Endoplasmic Reticulum and Relocates to the Plasma Membrane in Hypoxic Conditions.

The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.

Location Bias as Emerging Paradigm in GPCR Biology and Drug Discovery.

GPCRs are the largest receptor family that are involved in virtually all biological processes. Pharmacologically, they are highly druggable targets, as they cover more than 40% of all drugs in the market. Our knowledge of biased signaling provided insight into pharmacology vastly improving drug design to avoid unwanted effects and achieve higher efficacy and selectivity. However, yet another feature of GPCR biology is left largely unexplored, location bias. Recent developments in this field show promising avenues for evolution of new class of pharmaceuticals with greater potential for higher level of precision medicine. Further consideration and understanding of this phenomenon with deep biochemical and molecular insights would pave the road to success. In this review, we critically analyze this perspective and discuss new avenues of investigation.

Author Correction: Antenatal IL-1-dependent inflammation persists postnatally and causes retinal and sub-retinal vasculopathy in progeny.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antenatal IL-1-dependent inflammation persists postnatally and causes retinal and sub-retinal vasculopathy in progeny.

Antenatal inflammation as seen with chorioamnionitis is harmful to foetal/neonatal organ development including to eyes. Although the major pro-inflammatory cytokine IL-1ß participates in retinopathy induced by hyperoxia (a predisposing factor to retinopathy of prematurity), the specific role of antenatal IL-1ß associated with preterm birth (PTB) in retinal vasculopathy (independent of hyperoxia) is unknown. Using a murine model of PTB induced with IL-1ß injection in utero, we studied consequent retinal and choroidal vascular development; in this process we evaluated the efficacy of IL-1R antagonists. Eyes of foetuses exposed only to IL-1ß displayed high levels of pro-inflammatory genes, and a persistent postnatal infiltration of inflammatory cells. This prolonged inflammatory response was associated with: (1) a marked delay in retinal vessel growth; (2) long-lasting thinning of the choroid; and (3) long-term morphological and functional alterations of the retina. Antenatal administration of IL-1R antagonists - 101.10 (a modulator of IL-1R) more so than Kineret (competitive IL-1R antagonist) - prevented all deleterious effects of inflammation. This study unveils a key role for IL-1ß, a major mediator of chorioamnionitis, in causing sustained ocular inflammation and perinatal vascular eye injury, and highlights the efficacy of antenatal 101.10 to suppress deleterious inflammation.

Comparative and Mixture Effect of Cynodon Dactylon, ElectroMagnetic Field and Insulin on Diabetic Mouse.

OBJECTIVE: New investigations are in progress to find some alternative treatments for diabetes mellitus. Herbs are some of the interesting medications in this regard. Cynodon dactylon (C.d) is a potential plant to be considered as a new medication. On the other hand, the effect of the Electromagnetic Field (EMF) on bio organisms is becoming clearer. In this study, the effect of C.d, EMF and insulin have been investigated on the diabetic mouse. MATERIAL AND METHODS: Diabetes was induced by a combination of ketamine (60 mg/Kg) and xylazine (10 mg/Kg) which induces a sustained hyperglycemia. Mice were divided into 12 groups: 1) control, 2) normal saline, 3 and 4) 50mg/Kg C.d, 5 and 6) 100 mg/Kg C.d, 7) insulin, 8) insulin and C.d, 9) EMF (110 KHz, 700±20 mG), 10) insulin and EMF, 11) EMF plus C.d and 12) insulin plus C.d and EMF. Blood glucose level was measured after 5 and 60 minutes in C.d administrated groups, and 5 minutes in the other groups by a glucometer set. The data were analyzed by ANOVA and different means were compared by Tukey and Bonferroni tests (p<0.05). RESULTS: According to results, both dosages of C.d had significant lowering effect on blood glucose level. The first dose was more effective than the second, and its impact was just like insulin. The 6(th), 9(th) and 10(th) groups were significant, also. However, they did not show a higher effect than insulin or C.d. The application of EMF had a significant effect compared to the second group, but it did not reduce the glucose level to the normal range. The effect of the 8th group was very impressive and the mean glucose levels in this group were lower than the control group. CONCLUSION: Considering the data, C.d is a good alternative medication for diabetes mellitus.