Repeated exposure to psychostimulants such as methamphetamine (METH) induces neuronal adaptations in the mesocorticolimbic dopamine system, including the ventral tegmental area (VTA). These changes ...lead to persistently enhanced neuronal activity causing increased dopamine release and addictive phenotypes. A factor contributing to increased dopaminergic activity in this system appears to be reduced GABA B receptor-mediated neuronal inhibition in the VTA. Dephosphorylation of serine 783 (Ser783) of the GABA B 2 subunit by protein phosphatase 2A (PP2A) appears to trigger the downregulation GABA B receptors in psychostimulant-addicted rodents. Therefore, preventing the interaction of GABA B receptors with PP2A using an interfering peptide is a promising strategy to restore GABA B receptor-mediated neuronal inhibition. We have previously developed an interfering peptide (PP2A-Pep) that inhibits the GABA B receptors/PP2A interaction and thereby restores receptor expression under pathological conditions. Here, we tested the hypothesis that restoration of GABA B receptor expression in the VTA of METH addicted mice reduce addictive phenotypes. We found that the expression of GABA B receptors was significantly reduced in the VTA and nucleus accumbens but not in the hippocampus and somatosensory cortex of METH-addicted mice. Infusion of PP2A-Pep into the VTA of METH-addicted mice restored GABA B receptor expression in the VTA and inhibited METH-induced locomotor sensitization as assessed in the open field test. Moreover, administration of PP2A-Pep into the VTA also reduced drug seeking behavior in the conditioned place preference test. These observations underscore the importance of VTA GABA B receptors in controlling addictive phenotypes. Furthermore, this study illustrates the value of interfering peptides targeting diseases-related protein-protein interactions as an alternative approach for a potential development of selective therapeutic interventions.
GABAB receptor-mediated inhibition is indispensable for maintaining a healthy neuronal excitation/inhibition balance. Many neurological diseases are associated with a disturbed excitation/inhibition ...balance and downregulation of GABAB receptors due to enhanced sorting of the receptors to lysosomal degradation. A key event triggering the downregulation of the receptors is the phosphorylation of S867 in the GABAB1 subunit mediated by CaMKIIβ. Interestingly, close to S867 in GABAB1 exists another phosphorylation site, T872. Therefore, the question arose as to whether phosphorylation of T872 is involved in downregulating the receptors and whether phosphorylation of this site is also mediated by CaMKIIβ or by another protein kinase. Here, we show that mutational inactivation of T872 in GABAB1 prevented the degradation of the receptors in cultured neurons. We found that, in addition to CaMKIIβ, also ERK1/2 is involved in the degradation pathway of GABAB receptors under physiological and ischemic conditions. In contrast to our previous view, CaMKIIβ does not appear to directly phosphorylate S867. Instead, the data support a mechanism in which CaMKIIβ activates ERK1/2, which then phosphorylates S867 and T872 in GABAB1. Blocking ERK activity after subjecting neurons to ischemic stress completely restored downregulated GABAB receptor expression to normal levels. Thus, preventing ERK1/2-mediated phosphorylation of S867/T872 in GABAB1 is an opportunity to inhibit the pathological downregulation of the receptors after ischemic stress and is expected to restore a healthy neuronal excitation/inhibition balance.
Potentially noxious stimuli are sensed by specialized nerve cells named nociceptors, which convey nociceptive signals from peripheral tissues to the central nervous system. The spinal dorsal horn and ...the trigeminal nucleus serve as first relay stations for incoming nociceptive signals. At these sites, nociceptor terminals contact a local neuronal network consisting of excitatory and inhibitory interneurons as well as of projection neurons. Blockade of neuronal inhibition in this network causes an increased sensitivity to noxious stimuli (hyperalgesia), painful sensations occurring after activation of non-nociceptive fibers (allodynia), and spontaneous pain felt in the absence of any sensory stimulation. It thus mimics the major characteristics of chronic pain states. Diminished inhibitory pain control in the spinal dorsal horn occurs naturally, e.g., through changes in the function of inhibitory neurotransmitter receptors or through altered chloride homeo-stasis in the course of inflammation or nerve damage. This review summarizes our current knowledge about endogenous mechanisms leading to diminished spinal pain control and discusses possible ways that could restore proper inhibition through facilitation of fast inhibitory neurotransmission.
The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs ...semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here, we introduce a novel genetically encoded sensor based on the engineering of a circularly permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F
=528%) and temporal resolution (τ
= 4.7 s). We further demonstrated that GLPLight1 shows comparable responses to glucagon-like peptide-1 (GLP-1) derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.
One major factor regulating the strength of GABA
B
receptor signaling and thereby neuronal excitability is the dynamic control of their cell surface expression. GABA
B
receptors are constitutively ...internalized and recycled back to the plasma membrane to maintain a stable number of receptors at cell surface for appropriate signaling. Protein phosphatase 2A (PP2A) dependent dephosphorylation of serine 783 (S783) in the GABA
B
2
subunit is a key event for downregulating GABA
B
receptor cell surface expression particularly under conditions associated with excitotoxicity. Here, we investigated the role of PP2A in regulating GABA
B
receptor cell surface expression under physiological and excitotoxic conditions. For this purpose, we developed an interfering peptide (PP2A-Pep) that inhibits the interaction of GABA
B
receptors with PP2A. Using cultured cortical neurons, we found that PP2A downregulates GABA
B
receptor cell surface expression by inhibiting recycling of the receptors and thereby promoting degradation of the receptors. Inhibition of the GABA
B
receptor/PP2A interaction by PP2A-Pep in cultured cortical neurons restored GABA
B
receptor cell surface expression after excitotoxic stress and inhibited progressing neuronal death even when added 48 h after the insult. To explore the therapeutic potential of PP2A-Pep, we further analyzed effect of PP2A-Pep in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. Incubation of brain slices prepared from MCAO-treated mice with PP2A-Pep restored normal GABA
B
receptor expression and GABA
B
receptor-mediated inhibition, reduced ischemic-induced overexcitability of neurons, and prevented neuronal death in the ischemic penumbra. This data illustrates the crucial role of regulating GABA
B
receptor phosphorylation by PP2A for controlling neuronal inhibition and excitability. The results further suggest that interfering with the GABA
B
receptor/PP2A interaction is a promising strategy for the development of specific therapeutic interventions to treat neurological diseases associated with a disturbed excitation/inhibition balance and downregulation of GABA
B
receptors.
One important function of GABA
B
receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, ...sustained activation of GABA
B
receptors with the selective agonist baclofen provides neuroprotection in
in vitro
and
in vivo
models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABA
B
receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABA
B
receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABA
B
receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABA
B
receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABA
B
receptor expression and reduced loss of neurons. Restoration of GABA
B
receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we verified the severe downregulation of GABA
B
receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABA
B
receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABA
B
receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABA
B
receptors regulate multiple beneficial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia.
Abstract
Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal ...horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABA
A
receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABA
A
receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABA
A
receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABA
A
receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.
GABA
B
receptors control neuronal excitability via slow and prolonged inhibition in the central nervous system. One important function of GABA
B
receptors under physiological condition is to prevent ...neurons from shifting into an overexcitation state which can lead to excitotoxic death. However, under ischemic conditions, GABA
B
receptors are downregulated, fostering over-excitation and excitotoxicity. One mechanism downregulating GABA
B
receptors is mediated via the interaction with the endoplasmic reticulum (ER) stress-induced transcription factor CHOP. In this study, we investigated the hypothesis that preventing the interaction of CHOP with GABA
B
receptors after an ischemic insult restores normal expression of GABA
B
receptors and reduces neuronal death. For this, we designed an interfering peptide (R2-Pep) that restored the CHOP-induced downregulation of cell surface GABA
B
receptors in cultured cortical neurons subjected to oxygen and glucose deprivation (OGD). Administration of R2-Pep after OGD restored normal cell surface expression of GABA
B
receptors as well as GABA
B
receptor-mediated inhibition. As a result, R2-Pep reduced enhanced neuronal activity and inhibited progressive neuronal death in OGD stressed cultures. Thus, targeting diseases relevant protein-protein interactions might be a promising strategy for developing highly specific novel therapeutics.
Abstract Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, ...and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.
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