Design, Synthesis, and Characterization of New δ Opioid Receptor-Selective Fluorescent Probes and Applications in Single-Molecule Microscopy of Wild-Type Receptors.

The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time.

Plasma membrane preassociation drives β-arrestin coupling to receptors and activation.

β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer.

Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus.

Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane.

The γ-aminobutyric acid type B (GABA) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABA receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABA receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments.

How Carvedilol activates β-adrenoceptors.

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β-adrenoceptors, arrestin-biased signalling via β-adrenoceptors is a molecular mechanism proposed to explain the survival benefits.

Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats.

Targeting the non-classical estrogen pathway in neurodegenerative diseases and brain injury disorders.

Estrogens can alter the biology of various tissues and organs, including the brain, and thus play an essential role in modulating homeostasis. Despite its traditional role in reproduction, it is now accepted that estrogen and its analogues can exert neuroprotective effects. Several studies have shown the beneficial effects of estrogen in ameliorating and delaying the progression of neurodegenerative diseases, including Alzheimer's and Parkinson's disease and various forms of brain injury disorders.

Detecting Transient Trapping from a Single Trajectory: A Structural Approach.

In this article, we introduce a new method to detect transient trapping events within a single particle trajectory, thus allowing the explicit accounting of changes in the particle's dynamics over time. Our method is based on new measures of a smoothed recurrence matrix. The newly introduced set of measures takes into account both the spatial and temporal structure of the trajectory.

G protein-coupled receptor-G protein interactions: a single-molecule perspective.

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling.

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

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

Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes.

Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing.

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label.

Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of μ-Opioid Receptor Dimerization.

μ-Opioid receptors (μ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity.

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis.

The subcellular dynamics of GPCR signaling.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of a multitude of extracellular cues, such as hormones, neurotransmitters, odorants and light. Because of their involvement in numerous physiological and pathological processes and their accessibility, they are extensively exploited as pharmacological targets. Biochemical and structural biology investigations have clarified the molecular basis of GPCR signaling to a high level of detail.

Endocytosis and lysosomal degradation of GluA2/3 AMPARs in response to oxygen/glucose deprivation in hippocampal but not cortical neurons.

Global cerebral ischemia results in oxygen and glucose deprivation (OGD) and consequent delayed cell death of vulnerable neurons, with hippocampal CA1 neurons more vulnerable than cortical neurons. Most AMPA receptors (AMPARs) are heteromeric complexes of subunits GluA1/GluA2 or GluA2/GluA3, and the presence of GluA2 renders AMPARs Ca-impermeable. In hippocampal CA1 neurons, OGD causes the synaptic expression of GluA2-lacking Ca-permeable AMPARs, contributing to toxic Ca influx.

Neuroprotective effects of non-classical estrogen-like signaling activators: from mechanism to potential implications.

The gonadal steroid 17β-estradiol (E2) has shown powerful cytoprotective effect on cells. In addition to classical genomic mechanisms of action, E2 also exerts non-classical effects on intracellular signal transduction. Extensive studies during the past two decades have provided evidence that the E2-induced non-classical signaling on second messenger molecules plays a critical role in the neuroprotective effect of E2.

Postlesion estradiol treatment increases cortical cholinergic innervations via estrogen receptor-α dependent nonclassical estrogen signaling in vivo.

17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex.

Action of estrogen on survival of basal forebrain cholinergic neurons: promoting amelioration.

Extensive studies during the past two decades provide compelling evidence that the gonadal steroid, estrogen, has the potential to affect the viability of basal forebrain cholinergic neurons. These observations reflect a unique ameliorative feature of estrogen as it restores and protects the cholinergic neurons against noxious stimuli or neurodegenerative processes. Hence, we first address the ameliorative function of estrogen on basal forebrain cholinergic neurons such as the actions of estrogen on neuronal plasticity of cholinergic neurons, estrogen-induced memory enhancement and the ameliorative role of estrogen on cholinergic neuron related neurodegenerative processes such as Alzheimer's disease.

PACAP-mediated neuroprotection of neurochemically identified cell types in MSG-induced retinal degeneration.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is neuroprotective in animal models of different brain pathologies and injuries, including cerebral ischemia, Parkinson's disease, and different types of retinal degenerations. We have previously shown that PACAP is protective against monosodium glutamate (MSG)-induced retinal degeneration, where PACAP-treated retinas has more retained structure and PACAP induces anti-apoptotic while it inhibits pro-apoptotic signaling pathways. The aim of the present study was to investigate cell-type specific effects of PACAP in MSG-induced retinal degeneration by means of immunohistochemistry.

Microiontophoretically applied PACAP blocks excitatory effects of kainic acid in vivo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neuroprotective in animal models of different brain pathologies, including focal and global cerebral ischemia. The application of glutaminergic excitotoxin kainic acid (KA), similar to ischemic events, may lead to neurodegeneration. In the present article, we investigated the effects of microiontophoretic application of PACAP on the excitatory effects of KA.

The application of in vivo microiontophoresis for the investigation of mast cell-neuron interactions in the rat brain.

Although mast cells are immune cells of hematopoietic origin, they can be found in parts of the central nervous system of many mammalian species. In the rat brain they are located in the thalamic region. Their function is not defined yet, although they are mostly known to secrete several chemicals, which may influence the surrounding neurons.