Oxytocin (OT) and vasopressin (AVP) are endogenous ligands for OT and AVP receptors in the brain and in the peripheral system. Several studies demonstrate that OT and AVP have opposite roles in ...modulating stress, anxiety and social behaviours. Interestingly, both peptides and their receptors exhibit high sequence homology which could account for the biased signalling interaction of the peptides with OT and AVP receptors. However, how and under which conditions this crosstalk occurs in vivo remains unclear. In this review we shed light on the complexity of the roles of OT and AVP, by focusing on their signalling and behavioural differences and exploring the crosstalk between the receptor systems. Moreover, we discuss the potential of OT and AVP receptors as therapeutic targets to treat human disorders, such as autism, schizophrenia and drug abuse.
LINKED ARTICLES
This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc
Post-translational modifications (PTMs) are key events in signal transduction since they affect protein function by regulating their abundance and/or activity. PTMs involve the covalent attachment of ...functional groups to specific amino acids. Since they tend to be generally reversible, PTMs serve as regulators of signal transduction pathways. G-protein-coupled receptors (GPCRs) are major signaling proteins that undergo multiple types of PTMs. In this Review, we focus on the opioid receptors, members of GPCR family A, and highlight recent advances in the field that have underscored the importance of PTMs in the functional regulation of these receptors. Since opioid receptor activity plays a central role in the development of tolerance and addiction to morphine and other drugs of abuse, understanding the molecular mechanisms regulating receptor activity is of fundamental importance.
PTMs in GPCRs are responsible for fine-tuning receptor signaling. Receptor subcellular localization, membrane distribution dynamics, protein–protein interactions, and receptor signaling are all events mediated by PTMs on specific amino acid residues.Glycosylation of the opioid receptor plays a role in modulating the steady-state levels at the cell surface. In human μ-opioid receptor, asparagine 40 (N40) is one residue that has been described to be glycosylated, and N40D polymorphism has been implicated in pain sensitivity and dependence on alcohol and heroin.Palmitoylation and phosphorylation modulate the dynamics of movement of μ-opioid receptosrs at the plasma membrane and, specifically, diffusion into lipid rafts. The crosstalk between these two PTM events regulates agonist-mediated receptor distribution and internalization.Phosphorylation of the C terminus of μ-opioid receptor is associated with agonist-induced receptor internalization, recycling, desensitization, as well as analgesia and development of tolerance.Ubiquitination of the μ-opioid receptor affects receptor degradation in a process dependent on β-arrestin recruitment.
Alzheimer's disease (AD) is the most common neurodegenerative disease in the United States (US). Animal models, specifically mouse models have been developed to better elucidate disease mechanisms ...and test therapeutic strategies for AD. A large portion of effort in the field was focused on developing transgenic (Tg) mouse models through over-expression of genetic mutations associated with familial AD (FAD) patients. Newer generations of mouse models through knock-in (KI)/knock-out (KO) or CRISPR gene editing technologies, have been developed for both familial and sporadic AD risk genes with the hope to more accurately model proteinopathies without over-expression of human AD genes in mouse brains. In this review, we summarized the phenotypes of a few commonly used as well as newly developed mouse models in translational research laboratories including the presence or absence of key pathological features of AD such as amyloid and tau pathology, synaptic and neuronal degeneration as well as cognitive and behavior deficits. In addition, advantages and limitations of these AD mouse models have been elaborated along with discussions of any sex-specific features. More importantly, the omics data from available AD mouse models have been analyzed to categorize molecular signatures of each model reminiscent of human AD brain changes, with the hope to guide future selection of most suitable models for specific research questions to be addressed in the AD field.
Abstract
Neurodegenerative diseases such as Alzheimer’s disease (AD) are characterized by the progressive loss of neurons in the brain and the spinal cord. The pathophysiology of AD is multifactorial ...with heterogeneous molecular manifestations. The lack of efficacious therapies for AD reinforces the importance of exploring in depth multifaceted disease mechanisms. Recent progresses on AD have generated a large amount of RNA-sequencing data at both bulk and single cell levels and revealed thousands of genes with expression changes in AD. However, the upstream regulators of such gene expression changes are largely unknown. Non-coding RNAs (ncRNAs) represent the majority of the human transcriptome, and regulatory ncRNAs have been found to play an important role in regulating gene expression. A single miRNA usually targets a number of mRNAs and thus such ncRNAs are particular important for understanding disease mechanisms and developing novel therapeutics. This review aims to summarize the recent findings on the roles of ncRNAs in AD from ncRNA-omics studies with a focus on ncRNA signatures, interactions between ncRNAs and mRNAs, and ncRNA-regulated pathways in AD. We also review the potential of specific ncRNAs to serve as biomarkers and therapeutic targets for AD. In the end, we point out future directions for studying ncRNAs in AD.
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.
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 G
or G
depending on the length of the peptide and/or its concentration.
Alzheimer's disease (AD) is a progressive and age-associated neurodegenerative disorder that affects women disproportionally. However, the underlying mechanisms are poorly characterized. Moreover, ...while the interplay between sex and ApoE genotype in AD has been investigated, multi-omics studies to understand this interaction are limited. Therefore, we applied systems biology approaches to investigate sex-specific molecular networks of AD.
We integrated large-scale human postmortem brain transcriptomic data of AD from two cohorts (MSBB and ROSMAP) via multiscale network analysis and identified key drivers with sexually dimorphic expression patterns and/or different responses to APOE genotypes between sexes. The expression patterns and functional relevance of the top sex-specific network driver of AD were further investigated using postmortem human brain samples and gene perturbation experiments in AD mouse models.
Gene expression changes in AD versus control were identified for each sex. Gene co-expression networks were constructed for each sex to identify AD-associated co-expressed gene modules shared by males and females or specific to each sex. Key network regulators were further identified as potential drivers of sex differences in AD development. LRP10 was identified as a top driver of the sex differences in AD pathogenesis and manifestation. Changes of LRP10 expression at the mRNA and protein levels were further validated in human AD brain samples. Gene perturbation experiments in EFAD mouse models demonstrated that LRP10 differentially affected cognitive function and AD pathology in sex- and APOE genotype-specific manners. A comprehensive mapping of brain cells in LRP10 over-expressed (OE) female E4FAD mice suggested neurons and microglia as the most affected cell populations. The female-specific targets of LRP10 identified from the single cell RNA-sequencing (scRNA-seq) data of the LRP10 OE E4FAD mouse brains were significantly enriched in the LRP10-centered subnetworks in female AD subjects, validating LRP10 as a key network regulator of AD in females. Eight LRP10 binding partners were identified by the yeast two-hybrid system screening, and LRP10 over-expression reduced the association of LRP10 with one binding partner CD34.
These findings provide insights into key mechanisms mediating sex differences in AD pathogenesis and will facilitate the development of sex- and APOE genotype-specific therapies for AD.
Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. ...Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.
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•CpeNeo/Neo mice do not express carboxypeptidase E, an important neuropeptide processing enzyme.•Homozygous CpeNeo/Neo mice have greatly reduced levels of most neuropeptides and are ...overweight, subfertile, anxious, and depressed.•Heterozygous CpeNeo/+ mice have ∼ 50% reduced expression of CPE mRNA and greatly reduced enzyme activity.•Heterozygous CpeNeo/+ mice are identical to wild-type mice in terms of neuropeptide levels, physiology and behavior, indicating that CPE is not a rate-limiting enzyme.
Carboxypeptidase E (CPE) is an essential enzyme that contributes to the biosynthesis of the vast majority of neuropeptides and peptide hormones. There are several reports claiming that small decreases in CPE activity cause physiological changes in animals and/or cultured cells, but these studies did not provide evidence that neuropeptide levels were affected by decreased CPE activity. In the present study, we tested if CPE is a rate-limiting enzyme in neuropeptide production using CpeNeo mice, which contain a neomycin cassette within the Cpe gene that eliminates enzyme expression. Homozygous CpeNeo/Neo mice show defects found in Cpefat/fat and/or Cpe global knockout (KO) mice, including greatly decreased levels of most neuropeptides, severely impaired fertility, depressive-like behavior, adult-onset obesity, and anxiety-like behavior. Removal of the neomycin cassette with Flp recombinase under a germline promoter restored expression of CPE activity and resulted in normal behavioral and physiological properties, including levels of neuropeptides. Mice heterozygous for the CpeNeo allele have greatly reduced levels of Cpe mRNA and CPE-like enzymatic activity. Despite the decreased levels of Cpe expression, heterozygous CpeNeo mice are behaviorally and physiologically identical to wild-type mice, with normal levels of most neuropeptides. These results indicate that CPE is not a rate-limiting enzyme in the production of most neuropeptides, casting doubt upon studies claiming small decreases in CPE activity contribute to obesity or other physiological effects.
Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and ...the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C βI (PKCβI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCβI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.
Opiates such as morphine and fentanyl are the most widely used analgesics for the treatment of pain; however, prolonged treatment with opiates decreases the responsiveness of opioid receptors, ...leading to the development of tolerance and opiate addiction. Recent alarming reports of the widespread misuse of fentanyl leading to the ‘Opioid Epidemic” have begun to refocus scientific investigations into the mechanisms of action of opioid receptors. Despite intense research efforts, the molecular mechanisms underlying opioid addiction remain unknown contributing to a public health challenge. Activation of opioid receptors leads to the initiation of a number of signaling cascades, resulting in enhanced protein phosphorylation. Among the various kinases, protein kinase C (PKC) has been reported to play a crucial role in the desensitization of opioid receptors. To date, the majority of studies with PKC have used genetic and/or pharmacological manipulation of PKC to explore the dynamics of PKC activation. In order to investigate the activation of endogenous PKC in native tissue, we developed conformational‐sensitive antibodies that are able to detect time‐ and ligand‐mediated activation of native PKC. We also generated antibodies to several substrates of PKC including pPP2A (regulatory subunit phosphoS90 PPP2R5D), phosphoSer143 MARCKS, and phosphoS375/T376 MOR. We used a high‐throughput microscopy approach to explore the temporal dynamics of opiate‐mediated PKC signaling and track the temporal dynamics of PKC activation in cells treated with PMA, morphine or fentanyl. Interestingly, in contrast to the PKC activation induced by morphine that is rapid but transient the activation by fentanyl is slow and sustained for a longer period of time. We are able to detect rapid activation of PKC in the striatum of animals acutely treated with morphine suggesting that the conformation‐sensitive activation, state sensitive anti‐PKC antibodies serve as hitherto unavailable, critical tools to investigate the role of PKC in opioid receptor signaling in particular and PKC‐mediated signaling in general.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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