Brain Membranes Research Articles

Mast Cells, Astrocytes, Arachidonic Acid: Do They Play a Role in Depression?

Evidence support that brain membrane fatty acids play a crucial role in psychopathologies such as depression and anxiety disorders. Although the pathogenesis of depression is not still defined, drugs commonly used to reduce arachidonic turnover in the brain can control mood disorders, such as depression. Both astrocytes and mast cells release arachidonic acid during silent inflammation. Here, we hypothesize that arachidonic acid freed from lipid droplets of mast cells, as well as the one released from activated astrocytes, could contribute to characterize a depressive condition, and the fatty acids profile of mast cells, astrocytes and microglia could also vary, reflecting the pathophysiological depressive state of the subject. Finally, there is evidence that gut microbiota is deeply implicated in mood and behavioral disorders. Human gut microbiota can control nervous system diseases through neuroimmune pathways.

Characterization of binding of Varicella zoster virus vaccine strains to preparations of mouse brain membrane receptors

Purpose: characterization of vFiraVax (the causative agent of chickenpox - VZV) and vZelVax (the causative agent of shingles - HZ) vaccine strains by their ability to bind to preparations of brain membrane receptors of SPF BALB/c mice.Materials and Methods. The study was performed on cold-adapted vFiraVax VZV and vZelVax HZ vaccine strains developed by the authors on the basis of the wild-type parental pFira VZV virus (chickenpox causative agent) and the latent parental lpZel HZ virus (shingles causative agent); vOka vaccine strains isolated from vaccines against VZV infection from two manufactures (United Kingdom and USA); the HEL-3 strain of diploid cells from human embryonic lung tissue, the MC 27 strain of diploid cells from human embryonic musculocutaneous tissue, primary and diploid cells from guinea pig fetal fibroblasts. The VZV infectivity was estimated by the limiting dilution method using MC 27 cell cultures or guinea pig fetal fibroblasts. The virus titer was measured by the hemadsorption test performed with suspensions of red blood cells from guinea pig or human type 0 positive blood. Negative staining and electron microscopy were used to study the virus preparation. The immunogenicity of vFiraVax VZV and vZelVax HZ virus strains was compared with the immunogenicity of vOka VZV virus strains from different manufacturers by using a cross-neutralization test with immune sera.Results. The Russian cold-adapted vFiraVax VZV and vZelVax HZ vaccine strains, the latent parental lpZel HZ virus and the vOka VZV vaccine strain (United Kingdom) did not bind to preparations of brain neuroreceptors of SPF BALB/c mice as distinct from the wild-type parental pFira VZV variant and vOka VZV vaccine strains (USA); the absent neurotropism of Russian vFiraVax VZV and vZelVax HZ vaccine strains is not connected with the decreased immunogenicity in relation to foreign counterparts; the electron microscope study of the vFiraVax VZV virus containing liquid concentrate detected VZV nucleocapsids.Conclusion. The differences in the VZV ability to bind to preparations of brain membrane receptors of SPF BALB/c mice can be explained by the differences in the technology of vaccine manufacturing, including attenuation techniques, obtaining of the vaccine strain, specific characteristics of the latent parental lpZel HZ virus. The absence of the binding with brain neuroreceptors of SPF mice has been proved for the Russian vFiraVax VZV and vZelVax HZ vaccine strains which was is not connected with a decrease in their immunogenicity. The method of assessment of the binding ability of VZV vaccine strains can be used as a preliminary characteristic of neurotropism for newly created vaccine strains and for vaccine products.

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism.

The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.

Beyond Oncological Hyperthermia: Physically Drivable Magnetic Nanobubbles as Novel Multipurpose Theranostic Carriers in the Central Nervous System.

Magnetic Oxygen-Loaded Nanobubbles (MOLNBs), manufactured by adding Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on the surface of polymeric nanobubbles, are investigated as theranostic carriers for delivering oxygen and chemotherapy to brain tumors. Physicochemical and cyto-toxicological properties and in vitro internalization by human brain microvascular endothelial cells as well as the motion of MOLNBs in a static magnetic field were investigated. MOLNBs are safe oxygen-loaded vectors able to overcome the brain membranes and drivable through the Central Nervous System (CNS) to deliver their cargoes to specific sites of interest. In addition, MOLNBs are monitorable either via Magnetic Resonance Imaging (MRI) or Ultrasound (US) sonography. MOLNBs can find application in targeting brain tumors since they can enhance conventional radiotherapy and deliver chemotherapy being driven by ad hoc tailored magnetic fields under MRI and/or US monitoring.

Tritiated JNJ‐63514230 is a novel GluN2B‐selective radioligand with no affinity to sigma receptors

IntroductionCommonly used GluN2B radioligands exhibit limited selectivity towards GluN2B receptors as they interact with sigma and other off‐target receptors. Here we describe development and characterization of [3H] JNJ‐63514230, a novel radioligand selective for GluN2B‐containing NMDA receptors.MethodsThe selectivity JNJ 63514230 was determined using functional assays and CEREP panel. Saturation binding and competitive radioligand binding experiments were used to characterize [3H]‐JNJ 63514230 properties. We have also tested utility of [3H]‐JNJ 63514230 as a tracer for ex‐vivo autoradiography in rat brain.ResultsWe confirmed that widely used tracers [3H] Ifenprodil and [3H] Ro 25‐6981 have substantial affinity to sigma receptors as measured in radioligand competition experiments (CEREP). In contrast, new tracer [3H] JNJ‐63514230 exhibits no affinity to sigma 1 and sigma 2 receptors in CEREP assays. Saturation binding in rat brain membranes yielded a Kd value of 2.53 nM and Bmax of 2531 fmol/mg. Association constant Kon, dissociation constant Koff, and calculated equilibrium binding constant (Kon/Koff) were 0.0067±0.001 min−1 nM‐1, 0.021±0.0002 min−1, and 3.28±0.61 nM, respectively (average of 3 individual experiments each ± SD). Enhanced selectivity for GluN2B‐containing receptors translates to reduced non‐specific binding and better signal to noise ratio in both in vitro radioligand binding assays and ex vivo autoradiography.ConclusionsWe showed that new radioligand [3H] JNJ 63514230 is superior to previous previously described GluN2B tracers as it permits accurate measurement of affinity to GluN2B‐containing receptors (Ki) without using additional blockers to mask native sigma receptors.Support or Funding InformationAuthors are full time employees of Janssen Research & Development, LLC. The research was funded by the company.

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

The TMEM189 gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.

Fetal alcohol spectrum disorders model alters the functionality of glutamatergic neurotransmission in adult zebrafish

Fetal alcohol spectrum disorders (FASD) describe a wide range of ethanol-induced developmental disabilities, including craniofacial dysmorphology, and neurochemical and behavioral impairments. Zebrafish has become a popular animal model to evaluate the long-lasting effects of, both, severe and milder forms of FASD, including alterations to neurotransmission. Glutamate is one of the most affected neurotransmitter systems in ethanol-induced developmental disabilities. Therefore, the aim of the present study was to evaluate the functionality of the glutamatergic neurotransmitter system in an adult zebrafish FASD model. Zebrafish larvae (24 h post-fertilization) were exposed to ethanol (0.1 %, 0.25 %, 0.5 %, and 1%) for 2 h. After 4 months, the animals were euthanized and their brains were removed. The following variables were measured: glutamate uptake, glutamate binding, glutamine synthetase activity, Na+/K + ATPase activity, and high-resolution respirometry. Embryonic ethanol exposure reduced Na+-dependent glutamate uptake in the zebrafish brain. This reduction was positively modulated by ceftriaxone treatment, a beta-lactam antibiotic that promotes the expression of the glutamate transporter EAAT2. Moreover, the 0.5 % and 1% ethanol groups demonstrated reduced glutamate binding to brain membranes and decreased Na+/K + ATPase activity in adulthood. In addition, ethanol reduced glutamine synthetase activity in the 1% EtOH group. Embryonic ethanol exposure did not alter the immunocontent of the glutamate vesicular transporter VGLUT2 and the mitochondrial energetic metabolism of the brain in adulthood. Our results suggest that embryonic ethanol exposure may cause significant alterations in glutamatergic neurotransmission in the adult zebrafish brain.

An Assessment of the Effects of Azodicarbonamide-containing Diet on Neurobehaviour, Brain Antioxidant Status and Membrane Lipid Peroxidation Status in Rats.

Background: Azodicarbonamide is a dough-enhancer used in the process of bread-making in countries like Nigeria. While there have been suggestions that it is a sensitizer of the respiratory system, there is a dearth of information on its effects on the central nervous system. Aim: This study assessed the effects of azodicarbonamide on the central nervous system (ADA) in rats. Objective: The effects of ADA-containing diet on neurobehaviour, brain antioxidant status, and neuromorphology of selected brain regions in rats were examined. Methods: Forty adult rats were randomly-assigned into four groups of ten rats each, and were given standard diet or diet containing ADA at 1, 2 and 4% respectively. Rats were fed a standard diet or ADA-containing diet for a period of 28 days. Weekly body weight assessment and daily estimation of food intake were done. Behavioural tests {in the Open field, Y-maze, radial-arm maze, and Elevated Plus Maze (EPM)} were conducted on day 29. Twenty-four hours after the last behavioural test, animals were euthanised, whole brains were dissected, weighed, and either homogenised for assessment of lipid peroxidation and antioxidant status; or sectioned and processed for general histology. Results: Consumption of ADA-containing diet was associated with a significant decrease in weight gain/food intake, and significant suppression of horizontal locomotion and rearing behaviours; however, grooming activity increased significantly. Also, there was a significant reduction of open-arm time in the EPM and a significant increase in Y-maze alternation (at the lowest concentration of ADA). ADA-containing diet was not associated with significant changes in brain oxidative status or neuromorphology. Conclusion: The study showed that while ADA-containing diet may alter neurobehaviour in rats; this was not associated with evidence of brain oxidative stress or neuro-histomorphological alterations.

Study on the Characteristics of ATP-Sensitive Potassium Channels in Neonatal Rat Hypoxic-Ischemic Encephalopathy

KATP channels are present on the surface of cardiac internal membranes, skeletal muscle, brain, and mitochondrial membranes. KATP channels can be activated with selective drugs including diazoxide and cromakalim that may be able to limit ischemia-induced damage or brain cell death. KATP channels have no relationship with a homogenous group, while other channels do not have the same combinations. This study was to characterize the effects of the neuro-protective KATP channel openers diazoxide and cromakalim in neonatal rats with hypoxic-ischemic encephalopathy (HIE) induced by intrauterine asphyxia. Seventy-two hours-old Sprague Dawley rat pups were subjected to intrauterine asphyxia and hypoxia for 30 min, and then randomly assigned to seven groups: Vehicle, Hypoxic-ischemia (HI), Diazoxide (Dia), Cromakalim (Cro), Diazoxide+Glibenclamide (Dia + Gli), Cromakalim+Glibenclamide (Cro + Gli), and Solvent (NaOH). Brain injury was maintained by neurologic deficit or mass injury 72 hours after HI. Moreover, gene expression of KATP channels was measured by RT-PCR 72 hours after HI. Results showed that the hippocampus and cortex had the most serious injury after HI, next highest injury was in the cerebellum, and the least amount of injury was in the thalamus and brainstem. Diazoxide and cromakalim showed a neuroprotective effect to the five brain regions tested after intrauterine asphyxia. The neuroprotective effect between diazoxide and cromakalim was not statistically significant and could be inhibited by glibenclamide. Diazoxide and cromakalim had the strongest neuroprotective effect in the hippocampus and cortex, which showed the most serious injury after intrauterine asphyxia.

Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes.

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

Functional coupling of M1 muscarinic acetylcholine receptor to Gαq/11 in dorsolateral prefrontal cortex from patients with psychiatric disorders: a postmortem study.

Accumulating studies have implicated intracellular signaling through muscarinic acetylcholine receptors (mAChRs) in psychiatric illness. In the present study, carbamylcholine chloride (carbachol)-induced Gαi/o and Gαq/11 activation was identified in postmortem human prefrontal cortical membranes. The following two sample cohorts were used: subjects [1], consisting of 40 controls without neuropsychiatric disorders, and subjects [2], consisting of 20 with bipolar disorder (BP), 20 major depressive disorder (MDD), 20 schizophrenia, and 20 controls, strictly sex- and age-matched. Carbachol-stimulated [35S]GTPγS binding to human brain membranes was assessed by the two methods, i.e., conventional method using filtration techniques (Gαi/o activation coupled to M2/M4 mAChRs) applied to subjects [1], and [35S]GTPγS binding/immuno precipitation assay (Gαq/11 activation coupled to M1 mAChR) applied to subjects [1] and [2]. The concentration eliciting the half-maximal effect (EC50), maximum percent increase (%Emax), and slope factor were obtained from concentration-response curve of carbachol-induced Gαi/o and Gαq/11 activation. The pEC50 values of both carbachol-induced Gαi/o and Gαq/11 activations in subjects [1] were significantly correlated, though its implications or underlying molecular processes are unclear. The results of M1 mAChR-mediated Gαq/11 activation in subjects [2] indicated no significant disorder-specific alterations. However, the distribution patterns of the pEC50 values showed unequal variances among the groups. There was a significant inverse correlation between the %Emax values and the pEC50 values in subjects with schizophrenia, but not in those with BP or MDD, or controls. These data support the notion that schizophrenia patients consist of biologically heterogeneous subgroups with respect to M1 mAChR-mediated signaling pathways.

Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

A New Tellurium- and Selenoorganic Compound as an Inhibitor of Acetylcholinesterase in Brain.

We studied the effect of a new cyanine dye containing selenium and tellurium on acetylcholinesterase activity in synaptic membrane in rat brain. The cyanine dye dose-dependently inhibits activity of this enzyme, and the concentration of half-maximal inhibition of acetylcholinesterase activity was 20.46 μM. The cyanine dye instantly inhibits the enzyme; the degree of inhibition depends on acetylthiocholine concentration: the lower is acetylthiocholine concentration, the higher is the degree of inhibition. On the Lineweaver-Burk plot, the concentration dependence curves of acetylcholinesterase with and without cyanine dye intersect in one point on the abscissa axis. In this case, the cyanine dye reduces the maximum inhibition rate (Vmax) and does not affect Michaelis constant (Km). The calculated inhibition constant Ki for the cyanine dye is 7.74 μM. Thus, the cyanine dye is a non-competitive inhibitor of acetylcholinesterase.

Lipid class composition of membrane and raft fractions from brains of individuals with Alzheimer's disease

Perturbation of the homeostasis of brain membrane lipids has been implicated in the pathomechanism of Alzheimer's disease (AD). The ε4 allele of the apolipoprotein E gene (APOE) confers an increased risk, in a dosage-dependent manner, for brain amyloid-β accumulation and the development of sporadic AD. An effect of the APOE genotype on brain lipid homeostasis may underlie the AD risk associated with the ε4 allele. In this research, we examined an effect of APOE ε4 on the lipid class composition of crude membranes and raft-enriched fractions of brains. We applied enzymatic reaction-based methods for the quantification of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and sphingomyelin. Our results indicate that brain lipid class composition was neither significantly altered in AD subjects nor affected by the presence of the APOE ε4 allele.

Discovery of Orexant and Anorexant Agents with Indazole Scaffold Endowed with Peripheral Antiedema Activity.

The endocannabinoid system represents an integrated neuronal network involved in the control of several organisms’ functions, such as feeding behavior. A series of hybrids of 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (mimonabant), a well-known inverse agonist of the type-1 cannabinoid receptor (CB1), once used as an antiobesity drug, and the N-(2S)-substitutes of 1-[(4-fluorophenyl)methyl]indazole-3-carboxamide with 1-amino-3-methyl-1-oxobutane (AB-Fubinaca), 1-amino-3,3-dimethyl-1-oxobutane (ADB-Fubinaca), and 3-methylbutanoate (AMB-Fubinaca), endowed with potent agonistic activity towards cannabinoid receptors CB1 and CB2 were in solution as C-terminal amides, acids, methyl esters and N-methyl amides. These compounds have been studied by binding assays to cannabinoid receptors and by functional receptor assays, using rat brain membranes in vitro. The most active among them as an agonist, (S)-1-(2,4-dichlorobenzyl)-N-(3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl)-1H-indazole-3-carboxamide (LONI11), and an antagonist, (S)-2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3-methylbutanoic acid (LONI4), were tested in vivo in mic, to evaluate their ability to stimulate or suppress feeding behavior after intraperitoneal (i.p.) administration. For a LONI11 formalin test and a tail flick test after an administration by the subcutaneous (s.c.) and intracerebroventricular (i.c.v.) routes, respectively, were also carried out in vivo in mice to investigate the antinociceptive property at the central and peripheral levesl. We observed a significant orexant effect for LONI11 and an intense anorexant effect for (S)-methyl 2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (LONI2) and LONI4. In zymosan-induced edema and hyperalgesia, LONI11 reduced the percent of paw volume increase and paw latency after s.c. administration, also suggesting a possible peripheral anti-inflammatory activity.

The Presence of Calcitonin Gene-Related Peptide and Its Receptors in Rat, Pig and Human Brain: Species Differences in Calcitonin Gene-Related Peptide Pharmacology

Aim: The aim of present study is to investigate the binding characteristics of non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists (i.e., gepants) in the brain membranes of rat, pig and human. Methods: The interaction of available gepants with the CGRP receptor was studied in the brain membranes of 3 different species using a radioligand competitive binding assay. In addition, the distribution of CGRP and its receptor component receptor activity modifying protein 1 (RAMP1) in rat cerebellum and cortex was explored using immunohistochemistry. Results: All gepants, except SB268262, displaced 100% of the radioligand specific binding in the brain tissue of all 3 species and showed highest affinity for CGRP receptors in human brain as compared to rat and pig brain membranes. Furthermore, radioligand binding studies revealed the presence of higher CGRP receptor density in human cerebellum compared to human cortex. The morphology, size and density of CGRP immunoreactive cells suggest that all cerebral cortical neurons were positive for CGRP. Slender receptor immunoreactive fibres were found spanning through the entire cortex. CGRP immunoreactivity was displayed in the cell soma of cerebellar Purkinje cells and in large neurons in the medial cerebellar nucleus. RAMP1 was found on the surface of the Purkinje cells and in parallel fibres, indicating presence in the granule cell axons. Conclusion: Cerebellum and cerebral cortex are rich in CGRP and CGRP receptors, which can be antagonized by gepants. However, all gepants display higher affinity for human CGRP receptors as compared to rat and pig CGRP receptors. Furthermore, human cerebellum seems to express higher density of CGRP receptors.

The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3β/WDR7 in neuroendocrine cells

Vacuolar-type H+-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3β/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3β/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3β/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3β/WDR7, and cell assays indicated that Rbcn3β/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3β in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3β/WDR7 interactions.

Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in β-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor α. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of β-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.