Evolutionary gravitational neocognitron neural network optimized with marine predators optimization algorithm for MRI brain tumor classification.

Magnetic resonance imaging (MRI) is a powerful tool for tumor diagnosis in human brain. Here, the MRI images are considered to detect the brain tumor and classify the regions as meningioma, glioma, pituitary and normal types. Numerous existing methods regarding brain tumor detection were suggested previously, but none of the methods accurately categorizes the brain tumor and consumes more computation period. To address these problems, an Evolutionary Gravitational Neocognitron Neural Network optimized with Marine Predators Algorithm is proposed in this article for MRI Brain Tumor Classification (EGNNN-VGG16-MPA-MRI-BTC). Initially, the brain MRI pictures are collected under Brats MRI image dataset. By using Savitzky-Golay Denoising approach, these images are pre-processed. The features are extracted utilizing visual geometry group network (VGG16). By utilizing VGG16, the features, like Grey level features, Haralick Texture features are extracted. These extracted features are given to EGNNN classifier, which categorizes the brain tumor as glioma, meningioma, pituitary gland and normal. Batch Normalization (BN) layer of EGNNN is eliminated and included with VGG16 layer. Marine Predators Optimization Algorithm (MPA) optimizes the weight parameters of EGNNN. The simulation is activated in MATLAB. Finally, the EGNNN-VGG16-MPA-MRI-BTC method attains 38.98%, 46.74%, 23.27% higher accuracy, 24.24%, 37.82%, 13.92% higher precision, 26.94%, 47.04%, 38.94% higher sensitivity compared with the existing AlexNet-SVM-MRI-BTC, RESNET-SGD-MRI-BTC and MobileNet-V2-MRI-BTC models respectively.

Evolutionary Gravitational Neocognitron Neural Network optimized with Marine Predators Algorithm is proposed in this article for MRI Brain Tumor Classification (EGNNN-VGG16-MPA-MRI-BTC). Initially, the brain MRI pictures are collected under Brats MRI image dataset. By using Savitzky-Golay Denoising approach, these images are pre-processed. The features are extracted utilizing visual geometry group network (VGG16). By utilizing VGG16, the features, like Grey level features, Haralick Texture features are extracted. These extracted features are given to EGNNN classifier, which categorizes the brain tumor as glioma, meningioma, pituitary gland and normal. Batch Normalization (BN) layer of EGNNN is eliminated and included with VGG16 layer. Marine Predators Optimization Algorithm (MPA) optimizes the weight parameters of EGNNN.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward.

Opioid analgesics exert their therapeutic and adverse effects by activating µ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein-signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT: This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

The role of holistic nutritional properties of diets in the assessment of food system and dietary sustainability.

Advancing sustainable diets for nutrition security and sustainable development necessitates clear nutrition metrics for measuring nutritional quality of diets. Food composition, nutrient requirements, and dietary intake are among the most common nutrition metrics used in the current assessment of sustainable diets. Broadly, most studies in the area classify animal-source foods (ASF) as having a substantially higher environmental footprint in comparison to plant-source foods (PSF). As a result, much of the current dietary advice promulgates diets containing higher proportions of PSF. However, this generalization is misleading since most of these studies do not distinguish between the gross and bioavailable nutrient fractions in mixed human diets. The bioavailability of essential nutrients including ß-carotene, vitamin B-12, iron, zinc, calcium, and indispensable amino acids varies greatly across different diets. The failure to consider bioavailability in sustainability measurements undermines the complementary role that ASF play in achieving nutrition security in vulnerable populations. This article critically reviews the scientific evidence on the holistic nutritional quality of diets and identifies methodological problems that exist in the way the nutritional quality of diets is measured. Finally, we discuss the importance of developing nutrient bioavailability as a requisite nutrition metric to contextualize the environmental impacts of different diets.

Biased signaling by endogenous opioid peptides.

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (µ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([35S]GTPγS binding and ß-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of ß-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction.

GPR83 engages endogenous peptides from two distinct precursors to elicit differential signaling.

PEN is an abundant neuropeptide that activates GPR83, a G protein-coupled receptor that is considered a novel therapeutic target due to its roles in regulation of feeding, reward, and anxiety-related behaviors. The major form of PEN in the brain is 22 residues in length. Previous studies have identified shorter forms of PEN in mouse brain and neuroendocrine cells; these shorter forms were named PEN18, PEN19 and PEN20, with the number reflecting the length of the peptide. The C-terminal five residues of PEN20 are identical to the C-terminus of a procholecystokinin (proCCK)-derived peptide, named proCCK56-62, that is present in mouse brain. ProCCK56-62 is highly conserved across species although it has no homology to the bioactive cholecystokinin domain. ProCCK56-62 and a longer form, proCCK56-63 were tested for their ability to engage GPR83. Both peptides bind GPR83 with high affinity, activate second messenger pathways, and induce ligand-mediated receptor endocytosis. Interestingly, the shorter PEN peptides, ProCC56-62, and ProCCK56-63 differentially activate signal transduction pathways. Whereas PEN22 and PEN20 facilitate receptor coupling to Gai, PEN18, PEN19 and ProCCK peptides facilitate coupling to Gas. Furthermore, the ProCCK peptides exhibit dose dependent Ga subtype selectivity in that they faciliate coupling to Gas at low concentrations and Gai at high concentrations. These data demonstrate that peptides derived from two distinct peptide precursors can differentially activate GPR83, and that GPR83 exhibits Ga subtype preference depending on the nature and concentration of the peptide. These results are consistent with the emerging idea that endogenous neuropeptides function as biased ligands. Significance Statement We found that peptides derived from proCCK bind and activate GPR83, a G protein-coupled receptor that is known to bind peptides derived from proSAAS. Different forms of the proCCK- and proSAAS-derived peptides show biased agonism, activating Gas or Gai depending on the length of the peptide and/or its concentration.

Ameliorative effect of enhanced Fischer ratio flaxseed protein hydrolysate in combination with antioxidant micronutrients on ethanol-induced hepatic damage in a rat model.

Alcohol abuse causes severe metabolic abnormalities inducing hepatic damage and malnutrition. Since higher Fischer ratio proteins have therapeutic value in liver diseases, an investigation was undertaken to study the ameliorative effect of the enhanced Fischer ratio flaxseed protein hydrolysate (EFR-FPH) alone and in combination with antioxidant micronutrients on ethanol-induced hepatotoxicity in a rat model. The EFR-FPH was prepared by dual enzymatic hydrolysis and charcoal treatment of flaxseed protein. The ratio of the branched-chain:aromatic amino acids (Fischer ratio) was found to be 7·08. The EFR-FPH, characterised using LC-MS/MS, showed the abundance of free leucine and isoleucine compared with phenylalanine and tyrosine. The matrix-assisted laser desorption/ionisation-time of flight MS analysis revealed the larger peptides present in EFR-FPH with mass 2·3 kDa. The EFR-FPH improved the nutritional status, liver function and antioxidant defense in the ethanol hepatotoxicity-induced rat model. The hepatoprotective effect of EFR-FPH was significantly enhanced when combined with selenium or vitamin E. Ethanol-induced changes in the liver tissue were effectively suppressed in the groups receiving EFR-FPH. Flaxseed-based hepatoprotective dietary supplement was formulated incorporating an optimum level of EFR-FPH (10 %) based on sensory acceptability and was fortified with selenium and vitamin E. The hepatoprotective formulation significantly lowered aspartate transaminase, alanine transaminase, alkaline phosphatase and bilirubin by 47, 61, 55 and 78 %, respectively, and improved the antioxidant defense in the ethanol hepatotoxicity-induced rat model. The current investigation suggests that EFR-FPH in synergy with antioxidant micronutrients is potent in ameliorating ethanol-induced hepatotoxicity and has a potential to form a hepatoprotective dietary supplement.

CHA2DS2-VASc and has-BLED scores do not accurately stratify risk for stroke or bleed in fall victims with atrial fibrillation.

BACKGROUND: Falls are the leading cause of morbidity and mortality in the elderly. Non-valvular Atrial fibrillation (AF) is present in up to 9% of this group and often requires oral anticoagulation (OAC). The CHA2DS2-VASc and HAS-BLED scores are validated tools assessing risk of ischemic stroke from AF and major bleeding (MB) from OAC. It is unclear if these predictions remain accurate in post-fall patients. This study seeks to determine the stroke and major bleeding rate in atrial fibrillation patients after a ground level fall and identify if validated risk scoring systems accurately stratify risk in this cohort. METHODS: Retrospective review of patients with AF presented to the emergency department after a fall. CHA2DS2-VASc and HAS-BLED scores were calculated. Follow up information was reviewed to 1 year. Patients were grouped according to discharge thromboprophylaxis plan (DTP): no treatment, Anti-platelet (AP), OAC, and AP + OAC. Outcomes were ischemic stroke, MB, or death at 1 year. Ischemic stroke and MB rates were calculated. Kruskal-Wallis, Χ2, Fisher's exact, and multivariable logistic regression were used to evaluate for clinical associations. RESULTS: 192 patients were included. MB rate was 14.5 bleeds/100 person-years, and ischemic stroke rate was 10.9/100 person-years. There were no observed differences between DTPs. Overall, one-year mortality was 22.1%. On unadjusted analysis, CHA2DS2-VASc did associate with ischemic stroke (p = 0.03); HAS-BLED did not associate with MB (p = 0.17). After logistic regression accounting for known risk factors, neither system associated with ischemic stroke or MB. CONCLUSIONS: Fall patients are at higher risk for both ischemic stroke and MB compared to previously published reports. Current risk assessment tools should be used with caution. Further study of risk factors is warranted to guide medication decisions in these patients.

Autoantibodies Blocking M3 Muscarinic Receptors Cause Postganglionic Cholinergic Dysautonomia.

A 10-year-old girl presented with ileus, urinary retention, dry mouth, lack of tears, fixed dilated pupils, and diffuse anhidrosis 7 days after a febrile illness. We hypothesized that her syndrome was due to autoimmunity against muscarinic acetylcholine receptors, blocking their activation. Using an indirect enzyme-linked immunosorbent assay for all 5 muscarinic receptors (M1 -M5 ), we identified in the patient's serum antibodies that selectively bound to M3 receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M3 receptor activation. Thus, autoantibodies against M3 acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243.

Use of the DELTA Model to Understand the Food System and Global Nutrition.

BACKGROUND: Increasing attention is being directed at the environmental, social, and economic sustainability of the global food system. However, a key aspect of a sustainable food system should be its ability to deliver nutrition to the global population. Quantifying nutrient adequacy with current tools is challenging. OBJECTIVE: To produce a computational model illustrating the nutrient adequacy of current and proposed global food systems. METHODS: The DELTA Model was constructed using global food commodity balance sheet data, alongside demographic and nutrient requirement data from UN and European Food Safety Authority sources. It also includes nutrient bioavailability considerations for protein, the indispensable amino acids, iron, and zinc, sourced from scientific literature. RESULTS: The DELTA Model calculates global per capita nutrient availability under conditions of equal distribution and identifies areas of nutrient deficiency for various food system scenarios. Modeling the 2018 global food system showed that it supplied insufficient calcium (64% of demographically weighted target intake) and vitamin E (69%), despite supplying sufficient macronutrients. Several future scenarios were modeled, including variations in waste; scaling up current food production for the 2030 global population; plant-based food production systems; and removing sugar crops from the global food system. Each of these scenarios fell short of meeting requirements for multiple nutrients. These results emphasize the need for a balanced approach in the design of future food systems. CONCLUSIONS: Nutrient adequacy must be at the forefront of the sustainable food system debate. The DELTA Model was designed for both experts and nonexperts to inform this debate as to what may be possible, practical, and optimal for our food system. The model results strongly suggest that both plant and animal foods are necessary to achieve global nutrition. The model is freely available for public use so that anyone can explore current and simulated global food systems.

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides.

Activation of µ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, ß-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, ß-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, ß-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of ß-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of ß-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Polyphenols are potential nutritional adjuvants for targeting COVID-19.

The newly emerging severe acute respiratory syndrome, coronavirus-2 (SARS-CoV-2) is a dangerous pathogen that causes global health problems. It causes a disease called coronavirus disease 2019 (COVID-19) with high morbidity and mortality rates. In SARS-Cov-2-infected patients, elevated oxidative stress and upsurge of inflammatory cytokines are the main pathophysiological events that contribute to the severity and progression of symptoms and death. The polyphenols are natural compounds abundant in fruits and vegetables that are characterized by their high antioxidant and anti-inflammatory effects. Polyphenols have potential as an intervention for preventing respiratory virus infection. The beneficial effects of polyphenols on COVID-19 might be due to multiple mechanisms. Polyphenols can strengthen the body's anti-inflammatory and antioxidant defenses against viral infection. Targeting virus proteins and/or blocking cellular receptors are other plausible antiviral approaches to prevent the entry of the virus and its replication in the host cells. The results on the antiviral effects of various polyphenols, especially on SARS-CoV-2, are promising. The aim of this review is to clarify the role of polyphenols in strengthening antioxidant defenses and upregulating the immune systems of COVID-19 patients and to prevent replication and spreading of the virus.

Five Decades of Research on Opioid Peptides: Current Knowledge and Unanswered Questions.

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types (µ, δ, or κ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides.

Evaluation of factors influencing obesity and the effect of a 12-week home-based exercise program in people with epilepsy - Randomized control trial.

BACKGROUND: Association of obesity, quality of life (QoL), and physical fitness in people with epilepsy (PWE) is rarely reported. We evaluate the effect of a 12-week home-based exercise program on weight reduction and physical capacity in PWE. METHODS: In 173 PWE, physical fitness was assessed by using six-minute walk test (6MWT) and one-minute step test. Self-reported QoL data was collected using a 12-Item Short Form Survey (SF-12) questionnaire; further physical (PCS) and mental (MCS) component scores were derived. Effect of exercise was evaluated using randomized study of 110 PWE, divided into control and exercise groups of 55 each. RESULTS: At baseline, mean age of study population was 25.85 ±â€¯9.62 years with 77 (44.5%) women. Average body mass index (BMI) was 29.33 ±â€¯6.17 kg/m2. Mean PCS and MCS were 45.95 ±â€¯7.92 and 45.72 ±â€¯10.40 respectively. In 124 (71.7%) PWE with obesity, while high-density lipoprotein (HDL-C) (46.10 ±â€¯12.32 vs. 39.30 ±â€¯10.39 mg/dL; p < .001) was lower, low-density lipoprotein (LDL-C) (101.60 ±â€¯37.51 vs. 113.89 ±â€¯32.65 mg/dL; p = .035) was high. Both the randomized groups were comparable for type and number of antiepileptic drugs (AEDs) used. At 12-week follow-up, PWE in the exercise group reduced 7.65 ±â€¯5.62 kg while control group gained an average of 4.01 ±â€¯4.74 kg (p < .001). Distance walked in 6MWT (293.07 ±â€¯118.73 vs. 464.29 ±â€¯55.33 m; p = .007) and PCS (48.59 ±â€¯8.57 vs. 52.62 ±â€¯4.03; p = .006) were higher in exercise group whereas MCS did not differ between the groups. None of the participants reported seizure during the 12-week follow-up period. CONCLUSION: People with epilepsy have low PCS and MCS scores; PWE with obesity have altered metabolic profile when compared to PWE without obesity. A 12-week, home-based exercise program significantly reduces weight and improves physical capacity, irrespective of AEDs used. Trials with larger sample size and longer follow-up are required to validate our findings.

Milling interventions for the production of atta for Indian flat bread with low carbohydrate digestibility.

Study was aimed to produce atta for chapati, an Indian flat bread with low carbohydrate digestibility through different milling interventions; processing and formulating a functional ingredient mix (FM). Granulation, physico-chemical, rheological and chapati making characteristics of chakki atta, CA (control), roller mill atta (RA); RA replaced with 5, 10 and 15% FM (5, 10 and 15% RAFM) were evaluated. RA and RAFM samples showed lower water absorption, higher dough stability, pasting temperature and peak viscosity than CA. Evaluation of carbohydrate digestive profile showed differences in the pattern of carbohydrate digestibility and glucose release between the chapatis prepared from CA, RA and 10% RAFM. Rapidly available glucose (RAG), an indicator of glycemic response in vivo, was found to be lower in the 10% RAFM than CA. It can be concluded that milling interventions and compositional differences together determine the carbohydrate digestibility of the atta.

Regulation of an Opioid Receptor Chaperone Protein, RTP4, by Morphine.

Signaling by classic analgesics, such as morphine, is governed primarily by the relative abundance of opioid receptors at the cell surface, and this is regulated by receptor delivery to, and retrieval from, the plasma membrane. Although retrieval mechanisms, such as receptor endocytosis, have been extensively investigated, fewer studies have explored mechanisms of receptor maturation and delivery to the plasma membrane. A previous study implicated receptor transporter proteins (RTPs) in the latter process. Since not much is known about regulation of RTP expression, we initiated studies examining the effect of chronic morphine administration on the levels of RTPs in the brain. Among the four RTPs, we detected selective and region-specific changes in RTP4 expression; RTP4 mRNA is significantly upregulated in the hypothalamus compared with other brain regions. We examined whether increased RTP4 expression impacted receptor protein levels and found a significant increase in the abundance of mu opioid receptors (MOPrs) but not other related G protein-coupled receptors (GPCRs, such as delta opioid, CB1 cannabinoid, or D2 dopamine receptors) in hypothalamic membranes from animals chronically treated with morphine. Next, we used a cell culture system to show that RTP4 expression is necessary and sufficient for regulating opioid receptor abundance at the cell surface. Interestingly, selective MOPr-mediated increase in RTP4 expression leads to increases in cell surface levels of MOPr-delta opioid receptor heteromers, and this increase is significantly attenuated by RTP4 small interfering RNA. Together, these results suggest that RTP4 expression is regulated by chronic morphine administration, and this, in turn, regulates opioid receptor cell surface levels and function.

G Protein-Coupled Receptor Heteromers.

G protein-coupled receptors (GPCRs) compose one of the largest families of membrane proteins involved in intracellular signaling. They are involved in numerous physiological and pathological processes and are prime candidates for drug development. Over the past decade, an increasing number of studies have reported heteromerization between GPCRs. Many investigations in heterologous systems have provided important indications of potential novel pharmacology; however, the physiological relevance of these findings has yet to be established with endogenous receptors in native tissues. In this review, we focus on family A GPCRs and describe the techniques and criteria to assess their heteromerization. We conclude that advances in approaches to study receptor complex functionality in heterologous systems, coupled with techniques that enable specific examination of native receptor heteromers in vivo, are likely to establish GPCR heteromers as novel therapeutic targets.

Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171.

ProSAAS is one of the most widely expressed proteins throughout the brain and was recently found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor GPR171. Although BigLEN-GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is a key brain area involved in pain modulation and opioid functions. We also found that, although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces mu opioid receptor signaling and morphine-induced antinociception, whereas the GPR171 agonist enhances morphine antinociception, suggesting that GPR171 may be a novel target toward the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.