Pharmacological chaperones restore proteostasis of epilepsy-associated GABAA receptor variants.

Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding, assembly, and trafficking and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner.

Exogenous methyl jasmonate enhances phytochemicals and delays senescence in harvested strawberries by modulating GABA shunt pathway.

In order to illustrate the relationship between methyl jasmonate (MeJA) and gamma aminobutyric acid (GABA) in maintaining the quality and improving the postharvest life of strawberry fruit, the harvested fruit were treated with MeJA for 16 h at 20 °C and stored at 3 ± 0.5 °C for 12 days. MeJA enhanced the expression levels of GABA shunt pathway-related genes, including glutamate decarboxylase, GABA transaminase, and succinic semialdehyde dehydrogenase, leading to an increase in GABA accumulation. Treated fruit showed higher levels of total acids, anthocyanins, total phenolics, antioxidants, and phenylalanine ammonia-lyase activity, and lower levels of cell membrane deterioration, total soluble solids, polyphenol oxidase activity and decay incidence rate. The results suggest that the positive effects of MeJA in extending the fruit postharvest life, enhancing phytochemical compounds, and decreasing the decay incidence rate may be due to the effects on motivating GABA shunt pathway and PAL enzyme activity.

dCA1-NAc shell glutamatergic projection mediates context-induced memory recall of morphine.

Opioid relapse is generally caused by the recurrence of context-induced memory reinstatement of reward. However, the internal mechanisms that facilitate and modify these processes remain unknown. One of the key regions of the reward is the nucleus accumbens (NAc) which receives glutamatergic projections from the dorsal hippocampus CA1 (dCA1). It is not yet known whether the dCA1 projection to the NAc shell regulates the context-induced memory recall of morphine. Here, we used a common model of addiction-related behavior conditioned place preference paradigm, combined with immunofluorescence, chemogenetics, optogenetics, and electrophysiology techniques to characterize the projection of the dCA1 to the NAc shell, in context-induced relapse memory to morphine. We found that glutamatergic neurons of the dCA1 and gamma aminobutyric acidergic (GABA) neurons of the NAc shell are the key brain areas and neurons involved in the context-induced reinstatement of morphine memory. The dCA1-NAc shell glutamatergic input pathway and the excitatory synaptic transmission of the dCA1-NAc shell were enhanced via the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) when mice were re-exposed to environmental cues previously associated with drug intake. Furthermore, chemogenetic and optogenetic inactivation of the dCA1-NAc shell pathway decreased the recurrence of long- and short-term morphine-paired context memory in mice. These results provided evidence that the dCA1-NAc shell glutamatergic projections mediated the context-induced memory recall of morphine.

Enhancement of γ-aminobutyric acid, avenanthramides, and other health-promoting metabolites in germinating oats (Avena sativa L.) treated with and without power ultrasound.

Power ultrasound as an emerging processing technology has been investigated for stimulating seeds to enhance germination and accumulation of health-promoting metabolites, such as γ-aminobutyric acid (GABA) and phenolic compounds. This work was undertaken to evaluate the effects of power ultrasound (25 kHz) on the nutritional properties of germinated oats, and the microstructure of oat groats after treatment. The changes in the external and internal microstructures of the ultrasound-treated oats kernel were investigated using Environmental Scanning Electron Microscopy (ESEM) and 3D X-ray Micro Computed Tomography (Micro-CT). Physicochemical properties of oats including GABA, free sugars, avenanthramides, total phenolic content, and antioxidant capacities were enhanced after germination. Furthermore, the power ultrasound treatment for 5 min after soaking significantly enhanced the GABA (48-96 h), alanine (24-96 h), succinic acid (48-72 h), total phenolic content (24 h), and total avenanthramides (24 h) in the germinated oats.

Effects of controlled germination on selected physicochemical and functional properties of whole-wheat flour and enhanced γ-aminobutyric acid accumulation by ultrasonication.

Using hard red spring (HRS), hard white (HW), and soft white (SW) wheat, this study examined how germination time affected the functionality of whole-wheat flour (WWF) and enhancement of γ-aminobutyric acid (GABA) content through ultrasonication. The falling number values significantly decreased and the glucose content increased by 227-357% after 15h of controlled germination. The setback value of WWF paste decreased from 654 to 6cP (HW), 690 to 9cP (SW), and 698 to 7cP (HRS), respectively, showing significant decreases of starch retrogradation in an aqueous system. The gluten quality and dough mixing performance of WWF after 5-15h of controlled germination was enhanced since gluten is less weakened during the dough heating stage of Mixolab testing. After a 72h germination, the GABA content increased by 339% of the non-sprouting counterpart. Furthermore, the GABA content in the ultrasound-treated SW sample was 30.7% higher than that without ultrasound treatment.

The dual modulatory effects of efavirenz on GABAA receptors are mediated via two distinct sites.

Efavirenz is a widely prescribed medicine used to treat type 1 human immunodeficiency virus (HIV-1), the most prevalent pathogenic strain of the virus responsible for the acquired immune deficiency syndrome (AIDS) pandemic. Under prescribed dosing conditions, either alone or in combination therapy, efavirenz-induced CNS disturbances are frequently reported. Efavirenz was recently reported to interact in a similar concentration range with a number of receptors, transporters and ion channels including recombinant rat α1ß2γ2 GABAA receptors whose actions were potentiated (Gatch et al., 2013; Dalwadi et al., 2016). Now we report on the molecular mechanism of efavirenz on GABAA receptors as a function of concentration and subunit composition via whole-cell recordings of GABA-activated currents from HEK293 cells expressing varying subunit configurations of GABAA receptors. Efavirenz elicited dual effects on the GABA response; it allosterically potentiated currents at low concentrations, whereas it inhibited currents at higher concentrations. The allosteric potentiating action on GABAA receptors was pronounced in the α1ß2γ2, α2ß2γ2 and α4ß2γ2 configurations, greatly diminished in the α6ß2γ2 configuration, and completely absent in the α3ß2γ2 or α5ß2γ2 configuration. In stark contrast, the inhibitory modulation of efavirenz at higher concentrations was evident in all subunit configurations examined. Moreover, efavirenz-induced modulatory effects were dependent on GABA concentration ([GABA]), with a pronounced impact on currents activated by low [GABA] but little effect at saturating [GABA]. Mutation of a highly-conserved threonine to phenylalanine in transmembrane domain 2 of the α1 subunit abolished the inhibitory effect of efavirenz in α1ß2 receptors. Finally, mutations of any of the three conserved extracellular residues in α1/2/4 subunits to the conserved residues at the corresponding positions in α3/5 subunits (i.e., R84P, M89L or I120L) completely eliminated the potentiating effect of efavirenz in α1ß2γ2 configuration. These findings demonstrate that efavirenz's positive allosteric modulation of the GABAA receptor is mediated via a novel allosteric site associated with the extracellular domain of the receptor.

Methylene blue inhibits GABAA receptors by interaction with GABA binding site.

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 µM, and it had an IC50 of 31 µM in human α1ß2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the ß2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1ß2 receptors to MB was similar to that observed in α1ß2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and ß2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.

Comparison of kinetic and pharmacological profiles of recombinant α1γ2L and α1ß2γ2L GABAA receptors - A clue to the role of intersubunit interactions.

The fastest inhibitory mechanism in the CNS is mediated by ionotropic GABAA receptors and it is known that subunit composition critically determines their properties. While a typical GABAA receptor consists of two α, two ß and one γ/δ subunit, there are some exceptions, e.g. αß receptors. Functional α1γ2 GABAA receptors can be expressed in recombinant model (Verdoorn et al., 1990) and although their role remains unknown, it seems appealing to extend their characterization to further explore the structure-function relationship of GABAA receptors. Intriguingly, this receptor is lacking canonical GABA binding sites but it can be activated by GABA and dose-response relationships for α1ß2γ2L and α1γ2L receptors overlap. Deactivation kinetics was similar for both receptors but the percentage of the fast component was smaller in the case of α1γ2L receptors and, consequently, the mean deactivation time constant was slower. The rate and extent of macroscopic desensitization were smaller in the case of α1γ2L receptors but they showed slower recovery. Both receptor types had a similar proton sensitivity showing only subtle but significant differences in pH effects on deactivation. Flurazepam exerted a similar effect on both receptors but the rapid deactivation components were differently affected and an opposite effect was observed on desensitization extent. Rebound currents evoked by pentobarbital were undistinguishable for both receptor types. Taking altogether, although some significant differences were found, α1ß2γ2L and α1γ2L receptors showed unforeseen similarity. We propose that functioning of GABAA receptors might rely on subunit-subunit cooperative interactions to a larger extent than believed so far.

Mutagenesis and computational docking studies support the existence of a histamine binding site at the extracellular ß3+ß3- interface of homooligomeric ß3 GABAA receptors.

Histamine is an important neurotransmitter that exerts its physiological actions through H1-4 metabotropic receptors in mammals. It also directly activates ionotropic GABAA receptor (GABAAR) ß3 homooligomers and potentiates GABA responses in αß heterooligomers in vitro, but the respective histamine binding sites in GABAARs are unknown. We hypothesized that histamine binds at the extracellular ß+ß- interface at a position homologous to the GABA binding site of heterooligomeric GABAARs. To test this, we individually mutated several residues at the putative ligand binding minus side of a rat GABAAR ß3 wild type subunit and of a ß3 subunit that was made insensitive to trace Zn(2+) inhibition [ß3(H267A); called (Z)ß3]. (Z)ß3, (Z)ß3(Y62L), (Z)ß3(Q64A), (Z)ß3(Q64E), α1(Z)ß3, or α1(Z)ß3(Y62L) receptors were studied in HEK293T cells using whole cell voltage clamp recording. ß3, ß3(Y62C), ß3(Q64C), ß3(N41C), ß3(D43C), ß3(A45C) or ß3(M115C) receptors were examined in Xenopus oocytes using two-electrode voltage clamp. Histamine directly activated (Z)ß3 and ß3 homooligomers and potentiated GABA actions in α1(Z)ß3 heterooligomers. Receptors containing (Z)ß3(Y62L), ß3(Y62C) and ß3(D43C) showed markedly reduced histamine potency, but homo- and heterooligomers with (Z)ß3(Q64E) exhibited increased potency. The GABAAR αß(γ) competitive antagonist bicuculline elicited sub-maximal agonist currents through (Z)ß3 homooligomers, the potency of which was strongly decreased by (Z)ß3(Y62L). Mutations ß3(N41C), ß3(A45C) and ß3(M115C) disturbed receptor expression or assembly. Computational docking into the crystal structure of homooligomeric ß3 receptors resulted in a histamine pose highly consistent with the experimental findings, suggesting that histamine activates ß3 receptors via a site homologous to the GABA site in αßγ receptors.

A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem.

Cerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the 'transient receptor potential (TRP)' superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors. Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.

An introduction to the roles of purinergic signalling in neurodegeneration, neuroprotection and neuroregeneration.

Purinergic signalling appears to play important roles in neurodegeneration, neuroprotection and neuroregeneration. Initially there is a brief summary of the background of purinergic signalling, including release of purines and pyrimidines from neural and non-neural cells and their ectoenzymatic degradation, and the current characterisation of P1 (adenosine), and P2X (ion channel) and P2Y (G protein-coupled) nucleotide receptor subtypes. There is also coverage of the localization and roles of purinoceptors in the healthy central nervous system. The focus is then on the roles of purinergic signalling in trauma, ischaemia, stroke and in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, as well as multiple sclerosis and amyotrophic lateral sclerosis. Neuroprotective mechanisms involving purinergic signalling are considered and its involvement in neuroregeneration, including the role of adult neural stem/progenitor cells. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Using pharmacological chaperones to restore proteostasis.

Normal organismal physiology depends on the maintenance of proteostasis in each cellular compartment to achieve a delicate balance between protein synthesis, folding, trafficking, and degradation while minimizing misfolding and aggregation. Defective proteostasis leads to numerous protein misfolding diseases. Pharmacological chaperones are cell-permeant small molecules that promote the proper folding and trafficking of a protein via direct binding to that protein. They stabilize their target protein in a protein-pharmacological chaperone state, increasing the natively folded protein population that can effectively engage trafficking machinery for transport to the final destination for function. Here, as regards the application of pharmacological chaperones, we focus on their capability to promote the folding and trafficking of lysosomal enzymes, G protein coupled receptors (GPCRs), and ion channels, each of which is presently an important drug target. Pharmacological chaperones hold great promise as potential therapeutics to ameliorate a variety of protein misfolding diseases.

Evidence for the participation of peripheral α5 subunit-containing GABAA receptors in GABAA agonists-induced nociception in rats.

The activation of GABAA receptor by γ-amino butyric acid (GABA) in primary afferent fibers produces depolarization. In normal conditions this depolarization causes a reduction in the release of neurotransmitters. Therefore, this depolarization remains inhibitory. However, previous studies have suggested that in inflammatory pain, GABA shifts its signaling from inhibition to excitation by an increased GABA-induced depolarization. The contribution of peripheral α5 subunit-containing GABAA receptors to the inflammatory pain is unknown. The purpose of this study was to investigate the possible pronociceptive role of peripheral α5 subunit-containing GABAA receptors in the formalin test. Formalin (0.5%) injection into the dorsum of the right hind paw produced flinching behavior in rats. Ipsilateral local peripheral pre-treatment (-10min) with exogenous GABA (0.003-0.03µg/paw) or common GABAA receptor agonists muscimol (0.003-0.03µg/paw), diazepam (0.017-0.056µg/paw) or phenobarbital (1-100µg/paw) significantly increased 0.5% formalin-induced nociceptive behavior. The pronociceptive effects of GABA (0.03µg/paw), muscimol (0.03µg/paw), diazepam (0.056µg/paw) and phenobarbital (100µg/paw) were prevented by either the GABAA receptor antagonist bicuculline (0.01-0.1µg/paw) or selective α5 subunit-containing GABAA receptor inverse agonist L-655,708 (0.017-0.17µg/paw). The α5 subunit-containing GABAA receptor protein was expressed in dorsal root ganglion (DRG) and dorsal spinal cord of naïve rats. The formalin injection did not modify α5 subunit-containing GABAA receptor expression. Overall, these results suggest that peripheral α5 subunit-containing GABAA receptors play a pronociceptive role in the rat formalin test.