Peptide LCGA-17 Attenuates Behavioral and Neurochemical Deficits in Rodent Models of PTSD and Depression.

We have previously described the LCGA-17 peptide as a novel anxiolytic and antidepressant candidate that acts through the α2δ VGCC (voltage-gated calcium channel) subunit with putative synergism with GABA-A receptors. The current study tested the potential efficacy of acute and chronic intranasal (i.n.) LCGA-17 (0.05 mg/kg and 0.5 mg/kg) in rats on predator odor-induced conditioned place aversion (POCPA), a model of post-traumatic stress disorder (PTSD), and chronic unpredictable stress (CUS) that produce a range of behavioral and physiological changes that parallel symptoms of depression in humans. CUS and LCGA-17 treatment effects were tested in the sucrose preference (SPT) social interaction (SI), female urine sniffing (FUST), novelty-suppressed feeding (NSFT), and forced swim (FST) tests. Analysis of the catecholamines content in brain structures after CUS was carried out using HPLC. The efficacy of i.n. LCGA-17 was also assessed using the Elevated plus-maze (EPM) and FST. Acute LCGA-17 administration showed anxiolytic and antidepressant effects in EPM and FST, similar to diazepam and ketamine, respectively. In the POCPA study, LCGA-17 significantly reduced place aversion, with efficacy greater than doxazosin. After CUS, chronic LCGA-17 administration reversed stress-induced alterations in numerous behavioral tests (SI, FUST, SPT, and FST), producing significant anxiolytic and antidepressant effects. Finally, LCGA-17 restored the norepinephrine levels in the hippocampus following stress. Together, these results support the further development of the LCGA-17 peptide as a rapid-acting anxiolytic and antidepressant.

In silico Screening and Behavioral Validation of a Novel Peptide, LCGA-17, With Anxiolytic-Like Properties.

The aim of the study was to develop better anxiolytics and antidepressants. We focused on GABA A receptors and the α2δ auxiliary subunit of V-gated Ca2+ channels as putative targets because they are established as mediators of efficacious anxiolytics, antidepressants, and anticonvulsants. We further focused on short peptides as candidate ligands because of their high safety and tolerability profiles. We employed a structural bioinformatics approach to develop novel tetrapeptides with predicted affinity to GABA A receptors and α2δ. In silico docking studies of one of these peptides, LCGA-17, showed a high binding score for both GABA A receptors and α2δ, combined with anxiolytic-like properties in a Danio rerio behavioral screen. LCGA-17 showed anxiolytic-like effects in the novel tank test, the light-dark box, and the social preference test, with efficacy comparable to fluvoxamine and diazepam. In binding assays using rat brain membranes, [3H]-LCGA-17 was competed more effectively by gabapentinoid ligands of α2δ than ligands of GABA A receptors, suggesting that α2δ represents a likely target for LCGA-17. [3H]-LCGA-17 binding to brain lysates was unaffected by competition with ligands for GABAB, glutamate, dopamine, serotonin, and other receptors, suggesting specific interaction with α2δ. Dose-finding studies in mice using acute administration of LCGA-17 (i.p.) demonstrated anxiolytic-like effects in the open field test, elevated plus maze, and marble burying tests, as well as antidepressant-like properties in the forced swim test. The anxiolytic effects were effectively blocked by bicuculline. Therefore, LCGA-17 is a novel candidate anxiolytic and antidepressant that may act through α2δ, with possible synergism by GABA A receptors.

Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption.

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, serotonin, and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficacy of treatment. We applied active immunization against ADH to investigate its efficacy in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2% aluminum hydroxide-based adjuvant. The efficacy of immunization was demonstrated by the production of high titers of ADH-specific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats. Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum,while immunization neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum. The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and make available new prospects for developing innovative strategies for treatment of excessive alcohol intake.

Protective C allele of the single-nucleotide polymorphism rs1335532 is associated with strong binding of Ascl2 transcription factor and elevated CD58 expression in B-cells.

CD58 is expressed on the surface of antigen-presenting cells, including B-cells, and provides co-stimulation to regulatory T-cells (Treg) through CD2 receptor binding. Tregs appear to be essential suppressors of tissue-specific autoimmune responses. Thereby, CD58 plays protective role in multiple sclerosis (MS) and CD58 was identified among several loci associated with MS susceptibility. Minor (C) variant of the single-nucleotide polymorphism (SNP) rs1335532 is associated with lower MS risk according to genome-wide association studies (GWAS) and its presence correlates with higher CD58 mRNA levels in MS patients. We found that genomic region containing rs1335532 has enhancer properties and can significantly boost the CD58 promoter activity in lymphoblast cells. Using bioinformatics and pull-down assay we found that the protective (C) rs1335532 allele created functional binding site for ASCL2 transcription factor, a target of the Wnt signaling pathway. Both in B-lymphoblastoid cell lines and in primary B-cells, as well as in a monocytic cell line, activation of Wnt signaling resulted in an increased CD58 promoter activity in the presence of the protective but not the risk allele of rs1335532, whereas ASCL2 knockdown abrogated this effect. In summary, our results suggest that ASCL2 mediates the protective function of rs1335532 minor (C) allele in MS.

Individual roles of brain and serum alcohol dehydrogenase isoforms in regulation of alcohol consumption in SPF Wistar rats.

Alcohol dehydrogenases (ADH) are key enzymes of ethanol metabolism that mediate its oxidation to acetaldehyde. ADHs are also able to oxidize some types of neurotransmitters such as dopamine, serotonin and norepinephrine. Increased level of ADHs activity, induced by chronic alcohol consumption, is presumably associated with disturbed neurotransmitters metabolism that leads to stable alcohol craving. As earlier reported, intraperitoneal administration of 4-methilpirasole (non-specific inhibitor of ADHs) has shown to provide a short-term anti-alcoholic effect, but individual roles of ADH isoforms in this process were still unclear. The aim of this work was to study the roles of brain and serum ADH isoforms in alcohol consumption and neurotransmitter metabolism in the rats. In the study we used specific-pathogen-free (SPF) Wistar rats chronically alcoholized with 15% ethanol. 4-methilpirasole intranasal administration in small doses led to local inhibition of ADH III activity in the brain estimated by spectrophotometric assay. It correlated with dose-dependent reduction of dopamine concentration and increased level of its metabolic products in the brain but did not influence alcohol consumption. These data allowed us to propose an important role of brain ADHs (predominantly ADH III) in metabolism of dopamine in chronically alcoholized rats but not in regulation of alcohol consumption. To evaluate the role of serum ADH isoforms we immunized the rats with recombinant horse ADH that led to production of high levels of cross-reactive anti-ADH antibodies verified by ELISA assay. Immunization led to 30% decrease in alcohol consumption and recovery of general behavioral parameters such as motor activity, anxiety and depression level. At the same time active immunization did not cause any impairments in animal blood composition. We can conclude that immunization against ADHs appeared to be a safe way to decrease alcohol consumption that could be possibly associated with neurotransmitters metabolism correction.