Ladostigil Reduces the Adenoside Triphosphate/Lipopolysaccharide-Induced Secretion of Pro-Inflammatory Cytokines from Microglia and Modulate-Immune Regulators, TNFAIP3, and EGR1.

Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1ß, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10-11 M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1ß and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats.

AN1284 attenuates steatosis, lipogenesis, and fibrosis in mice with pre-existing non-alcoholic steatohepatitis and directly affects aryl hydrocarbon receptor in a hepatic cell line.

Non-alcoholic steatohepatitis (NASH) is an aggressive form of fatty liver disease with hepatic inflammation and fibrosis for which there is currently no drug treatment. This study determined whether an indoline derivative, AN1284, which significantly reduced damage in a model of acute liver disease, can reverse steatosis and fibrosis in mice with pre-existing NASH and explore its mechanism of action. The mouse model of dietary-induced NASH reproduces most of the liver pathology seen in human subjects. This was confirmed by RNA-sequencing analysis. The Western diet, given for 4 months, caused steatosis, inflammation, and liver fibrosis. AN1284 (1 mg or 5 mg/kg/day) was administered for the last 2 months of the diet by micro-osmotic-pumps (mps). Both doses significantly decreased hepatic damage, liver weight, hepatic fat content, triglyceride, serum alanine transaminase, and fibrosis. AN1284 (1 mg/kg/day) given by mps or in the drinking fluid significantly reduced fibrosis produced by carbon tetrachloride injections. In human HUH7 hepatoma cells incubated with palmitic acid, AN1284 (2.1 and 6.3 ng/ml), concentrations compatible with those in the liver of mice treated with AN1284, decreased lipid formation by causing nuclear translocation of the aryl hydrocarbon receptor (AhR). AN1284 downregulated fatty acid synthase (FASN) and sterol regulatory element-binding protein 1c (SREBP-1c) and upregulated Acyl-CoA Oxidase 1 and Cytochrome P450-a1, genes involved in lipid metabolism. In conclusion, chronic treatment with AN1284 (1mg/kg/day) reduced pre-existing steatosis and fibrosis through AhR, which affects several contributors to the development of fatty liver disease. Additional pathways are also influenced by AN1284 treatment.

Coordinated Transcriptional Waves Define the Inflammatory Response of Primary Microglial Culture.

The primary role of microglia is to maintain homeostasis by effectively responding to various disturbances. Activation of transcriptional programs determines the microglia's response to external stimuli. In this study, we stimulated murine neonatal microglial cells with benzoyl ATP (bzATP) and lipopolysaccharide (LPS), and monitored their ability to release pro-inflammatory cytokines. When cells are exposed to bzATP, a purinergic receptor agonist, a short-lived wave of transcriptional changes, occurs. However, only combining bzATP and LPS led to a sustainable and robust response. The transcriptional profile is dominated by induced cytokines (e.g., IL-1α and IL-1ß), chemokines, and their membrane receptors. Several abundant long noncoding RNAs (lncRNAs) are induced by bzATP/LPS, including Ptgs2os2, Bc1, and Morrbid, that function in inflammation and cytokine production. Analyzing the observed changes through TNF (Tumor necrosis factor) and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathways confirmed that neonatal glial cells exhibit a distinctive expression program in which inflammatory-related genes are upregulated by orders of magnitude. The observed capacity of the microglial culture to activate a robust inflammatory response is useful for studying neurons under stress, brain injury, and aging. We propose the use of a primary neonatal microglia culture as a responsive in vitro model for testing drugs that may interact with inflammatory signaling and the lncRNA regulatory network.

Emotional states as distinct configurations of functional brain networks.

The conceptualization of emotional states as patterns of interactions between large-scale brain networks has recently gained support. Yet, few studies have directly examined the brain's network structure during emotional experiences. Here, we investigated the brain's functional network organization during experiences of sadness, amusement, and neutral states elicited by movies, in addition to a resting-state. We tested the effects of the experienced emotion on individual variability in the brain's functional connectome. Next, for each state, we defined a community structure of the brain and quantified its segregation and integration. We found that sadness, relative to amusement, was associated with higher modular integration and increased connectivity of cognitive control networks: the salience and fronto-parietal networks. Moreover, in both the functional connectome and the emotional report, the similarity between individuals was dependent on the sex. Our results suggest that the experience of emotion is linked to a reconfiguration of whole-brain distributed, not emotion-specific, functional networks and that the brain's topological structure carries information about the subjective emotional experience.

Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

Effect of ladostigil treatment of aging rats on gene expression in four brain areas associated with regulation of memory.

Episodic and spatial memory decline in aging and are controlled by the hippocampus, perirhinal, frontal and parietal cortices and the connections between them. Ladostigil, a drug with antioxidant and anti-inflammatory activity, was shown to prevent the loss of episodic and spatial memory in aging rats. To better understand the molecular effects of aging and ladostigil on these brain regions we characterized the changes in gene expression using RNA-sequencing technology in rats aged 6 and 22 months. We found that the changes induced by aging and chronic ladostigil treatment were brain region specific. In the hippocampus, frontal and perirhinal cortex, ladostigil decreased the overexpression of genes regulating calcium homeostasis, ion channels and those adversely affecting synaptic function. In the parietal cortex, ladostigil increased the expression of several genes that provide neurotrophic support, while reducing that of pro-apoptotic genes and those encoding pro-inflammatory cytokines and their receptors. Ladostigil also decreased the expression of axonal growth inhibitors and those impairing mitochondrial function. Together, these actions could explain the protection by ladostigil against age-related memory decline.

Trait-related changes in brain network topology in premenstrual dysphoric disorder.

The female predominance in the prevalence of depression is partially accounted by reactivity to hormonal fluctuations. Premenstrual dysphoric disorder (PMDD) is a reproductive subtype of depression characterized by cyclic emotional and somatic symptoms that recur before menstruation. Despite the growing understanding that most psychiatric disorders arise from dysfunctions in distributed brain circuits, the brain's functional connectome and its network properties of segregation and integration were not investigated in PMDD. To this end, we examined the brain's functional network organization in PMDD using graph theoretical analysis. 24 drug naïve women with PMDD and 27 controls without premenstrual symptoms underwent 2 resting-state fMRI scans, during the mid-follicular and late-luteal menstrual cycle phases. Functional connectivity MRI, graph theory metrics, and levels of sex hormones were computed during each menstrual phase. Altered network topology was found in PMDD across symptomatic and remitted stages in major graph metrics (characteristic path length, clustering coefficient, transitivity, local and global efficiency, centrality), indicating decreased functional network segregation and increased functional network integration. In addition, PMDD patients exhibited hypoconnectivity of the anterior temporal lobe and hyperconnectivity of the basal ganglia and thalamus, across menstrual phases. Furthermore, the relationship between difficulties in emotion regulation and PMDD was mediated by specific patterns of functional connectivity, including connections of the striatum, thalamus, and prefrontal cortex. The shifts in the functional connectome and its topology in PMDD may suggest trait vulnerability markers of the disorder.

A Novel Indoline Derivative Ameliorates Diabesity-Induced Chronic Kidney Disease by Reducing Metabolic Abnormalities.

Both diabetes and obesity (diabesity) contribute significantly to the development of chronic kidney disease (CKD). In search of new remedies to reverse or arrest the progression of CKD, we examined the therapeutic potential of a novel compound, AN1284, in a mouse model of CKD induced by type 2 diabetes with obesity. Six-week-old BKS Cg-Dock 7m+/+ Leprdb/J mice with type 2 diabetes and obesity were treated with AN1284 (2.5 or 5 mg kg-1 per day) via micro-osmotic pumps implanted subcutaneously for 3 months. Measures included renal, pancreatic, and liver assessment as well as energy utilization. AN1284 improved kidney function in BSK-db/db animals by reducing albumin and creatinine and preventing renal inflammation and morphological changes. The treatment was associated with weight loss, decreased body fat mass, increased utilization of body fat toward energy, preservation of insulin sensitivity and pancreatic ß cell mass, and reduction of dyslipidemia, hepatic steatosis, and liver injury. This indoline derivative protected the kidney from the deleterious effects of hyperglycemia by ameliorating the metabolic abnormalities of diabetes. It could have therapeutic potential for preventing CKD in human subjects with diabesity.

Low-dose ladostigil for mild cognitive impairment: A phase 2 placebo-controlled clinical trial.

OBJECTIVE: Ladostigil reduces oxidative stress and microglial activation in aging rats. We assessed its safety and potential efficacy in a 3-year, randomized, double-blind, placebo-controlled phase 2 clinical trial in patients with mild cognitive impairment (MCI) and medial temporal lobe atrophy. METHODS: Patients 55 to 85 years of age with MCI, Clinical Dementia Rating (CDR) score of 0.5, Mini-Mental State Examination (MMSE) score >24, Wechsler Memory Scale-Revised Verbal Paired Associates I score ≤18, and Medial Temporal Lobe Atrophy Scale score >1 were stratified by APOE ε4 genotype and randomly assigned (1:1) to ladostigil 10 mg/d or placebo. Primary outcomes were safety and onset of Alzheimer disease dementia. Secondary endpoints were Neuropsychological Test Battery (NTB) composite, Disability Assessment in Dementia (DAD), and Geriatric Depression Scale (GDS) scores. Exploratory outcomes were NTB component, CDR, and MMSE scores. Biomarkers included MRI-derived whole-brain, hippocampus, and entorhinal cortex volumes. RESULTS: Two hundred ten patients from 15 sites in Austria, Germany, and Israel were randomly allocated to placebo (107 patients) or ladostigil (103 patients). After 36 months, 21 of 103 patients on placebo and 14 of 99 patients receiving ladostigil progressed to Alzheimer disease (log-rank test p = 0.162). There were no significant effects on the NTB composite, DAD, or GDS score. Whole-brain and hippocampus volumes decreased more in the placebo than in the ladostigil group (whole brain, p = 0.025, Cohen d = 0.43; hippocampus, p = 0.043, d = 0.43). Serious adverse events were reported by 28 of 107 patients treated with placebo and 26 of 103 with ladostigil. CONCLUSION: Ladostigil was safe and well tolerated but did not delay progression to dementia. Its association with reduced brain and hippocampus volume loss suggests a potential effect on atrophy. CLINICALTRIALSGOV IDENTIFIER: NCT01429623. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with MCI and medial temporal lobe atrophy, ladostigil did not significantly decrease the risk of the development of Alzheimer disease.

Sex differences during emotion processing are dependent on the menstrual cycle phase.

Sex differences in the neural processing of emotion are of special interest considering that mood and anxiety disorders predominant in females. However, these sex-related differences were typically studied without considering the hormonal status of female subjects, although emotion processing in the brain was shown to differ between phases of the menstrual cycle. In this functional MRI study, we demonstrated the influence of the menstrual cycle phase on sex differences in brain activity and functional connectivity during negative and positive emotions, using two different paradigms: emotion perception and emotion experience. Twenty naturally cycling healthy women without premenstrual symptoms were scanned twice: during the mid-follicular and late-luteal menstrual phases, and compared to a matched group of twenty healthy men. During negative emotion perception, men showed increased neural activity in the right hippocampal formation relative to women in the mid-follicular phase, and increased activity in the right cerebellum relative to women in the late-luteal phase. During experience of amusement, reduced putamen-ventrolateral prefrontal cortex and putamen-dorsomedial prefrontal cortex functional connectivity were observed for women in the late-luteal phase relative to men and associated with levels of sex hormones. These neural and hormonal findings were complemented by behavioral reports of reduced amusement and increased sadness in late-luteal women. Our results demonstrate menstrual phase-dependent sex differences in emotion perception and experience and may suggest a biological tendency for a deficient experience of pleasure and reward during the late-luteal phase. These findings may further shed light on the underlying pathophysiology of premenstrual dysphoric disorder.

Age-Induced Spatial Memory Deficits in Rats Are Correlated with Specific Brain Region Alterations in Microglial Morphology and Gene Expression.

Effect of age and ladostigil treatment (1 mg/kg/day), given for 6 months to 16 month old rats, was investigated on microglial morphology in brain regions associated with control of spatial learning. This was assessed in the Morris water maze (MWM). Microglial morphology was assessed with diaminobenzidine and fluorescent staining with Iba1 and CD11b in these brain regions. Aging did not change the number of microglia in the parietal cortex (PC) or hippocampal CA1 region (CA1-HC), but decreased microglial process tips in the CA1-HC, increased the area fraction stained by CD11b and number of bulbs on processes in PC and CA1-HC and thickness of microglial processes in corpus callosum (CC) and fornix (Fx). Performance in MWM (distance swam to escape platform) was negatively correlated with number of bulbs in PC and thickness of process in CC, and positively correlated with number of process tips in CA1-HC. Aging increased expression of MHC class II genes and others associated with motility and membrane adhesion in the PC and hippocampus, but Adora2a (Adenosine A2a receptor), only in hippocampus. Age-related increase in the number of bulbs and expression of inflammatory genes was prevented by ladostigil in PC. In the CA1-HC, ladostigil increased the number of process tips and prevented the increase in expression of Adora2a and genes regulating ion channels. Ladostigil also decreased thickening of the processes in CC and Fx. The data show brain region-specific relations induced by age in spatial learning, microglial morphology and associated genes and their response to ladostigil treatment. Graphical Abstract.

Synthesis and Biological Evaluation of Derivatives of Indoline as Highly Potent Antioxidant and Anti-inflammatory Agents.

We describe the preparation and evaluation of novel indoline derivatives with potent antioxidant and anti-inflammatory activities for the treatment of pathological conditions associated with chronic inflammation. The indolines are substituted at position 1 with chains carrying amino, ester, amide, or alcohol groups, and some have additional substituents, Cl, MeO, Me, F, HO, or BnO, on the benzo ring. Concentrations of 1 pM to 1 nM of several compounds protected RAW264.7 macrophages against H2O2 induced cytotoxicity and LPS induced elevation of NO, TNF-α, and IL-6. Several derivatives had anti-inflammatory activity at 1/100th of the concentration of unsubstituted indoline. Four compounds with ester, amine, amide, or alcohol side chains injected subcutaneously in mice at a dose of 1 µmol/kg or less, like dexamethasone (5.6 µmol/kg) prevented LPS-induced cytokine elevation in the brain and peripheral tissues. Subcutaneous injection of 100 µmol/kg of these compounds caused no noticeable adverse effects in mice during 3 days of observation.

Indoline derivatives mitigate liver damage in a mouse model of acute liver injury.

BACKGROUND: Exposure of mice to D-galactosamine (GalN) and lipopolysaccharide (LPS) induces acute liver failure through elevation of TNF-α, which causes liver damage resembling that in humans. The current study evaluated in this model the effect of two indoline derivatives, which have anti-inflammatory activity in macrophages. METHODS: AN1297 and AN1284 (0.025-0.75mg/kg) or dexamethasone (3mg/kg), were injected subcutaneously, 15min before intraperitoneal injection of GalN (800mg) plus LPS (50µg) in male Balb/C mice. After 6h, their livers were evaluated histologically by staining with hematoxylin and eosin for tissue damage and by cleaved caspase 3 for apoptosis. Activity of liver enzymes, alanine transaminase (ALT) and aspartate aminotransferase (AST) and levels of TNF-α and IL-6 were measured in plasma, and those of TNF-α and IL-6, in the liver. RESULTS: AN1297 (0.075-0.75mg/kg) and AN1284 (0.25-0.75mg/kg) maximally reduced ALT by 51% and 80%, respectively. Only AN1284 (0.25 and 0.75mg/kg) reduced AST by 41% and 48%. AN1297 and AN1284 (0.25mg/kg) decreased activation of caspase 3 (a sign of apoptosis) by 80% and plasma TNF-α by 75%. AN1297 and AN1284 (0.075mg/kg) prevented the rise in TNF-α and IL-6 in the liver. AN1284 (0.25mg/kg) reduced mortality from 90% to 20% (p<0.01) and AN1297, to 60% (p=0.121). Both indoline derivatives inhibited the phosphorylation of MAPK p38 and DNA binding of the transcription factor, AP-1. CONCLUSION: While both compounds are highly potent anti-inflammatory agents, AN1284 is more effective in mitigating the underlying causes of GalN/LPS-induced acute liver failure in mice.

Cholinergic Anti-Inflammatory Pathway Does Not Contribute to Prevention of Ulcerative Colitis by Novel Indoline Carbamates.

Indoline carbamates, AN680 and AN917 decrease cytokines, TNF-α and IL-6 in peritoneal macrophages activated by lipopolysaccharide (LPS) and in mouse tissues after LPS injection. They prevent nuclear translocation of nuclear factor κB (NF-κB) and activator protein 1. Only AN917 inhibits cholinesterase (ChE) at relevant concentrations. ChE inhibitors decrease NF-κB by activating α7 nicotinic acetylcholine receptors (α7nAChR). The current study compared the effect of rivastigmine, a ChE inhibitor, AN680 and AN917 on ulcerative colitis induced in mice by ingestion of dextran sodium sulfate (4.5%) solution. Rivastigmine (1 mg/kg), AN680 (2.5-10 mg/kg) and AN917 (2-5 mg/kg) were injected subcutaneously once daily for 8 days. Disease severity was assessed by disease activity index (DAI), colonoscopy, colon length and body weight loss, colonic levels of TNF-α, IL-6, IL-1ß and myeloid peroxidase (MPO) activity. AN680 (5 mg/kg) reduced DAI, colon shrinkage, weight loss, histopathological signs of colon damage, MPO activity, TNF-α, IL-1ß and IL-6 levels without inhibiting ChE. AN917 (5 mg/kg) and rivastigmine (1 mg/kg) inhibited ChE in plasma and colon by 65%, reduced DAI, MPO activity and IL-6, but not TNF-α or IL-1ß. AN917 did not prevent weight loss or colon shrinkage. Mecamylamine abolished the reduction of DAI, MPO activity and IL-6 by AN917 and rivastigmine, indicating they were mediated by α7nAChR. CONCLUSIONS: AN680 is very effective in preventing DSS-induced UC in mice and may therefore have potential therapeutic application in humans. Addition of ChE inhibition and indirect activation of α7nAChR lessens the efficacy of AN917 in this model.

Prenatal stressors in rodents: Effects on behavior.

The current review focuses on studies in rodents published since 2008 and explores possible reasons for any differences they report in the effects of gestational stress on various types of behavior in the offspring. An abundance of experimental data shows that different maternal stressors in rodents can replicate some of the abnormalities in offspring behavior observed in humans. These include, anxiety, in juvenile and adult rats and mice, assessed in the elevated plus maze and open field tests and depression, detected in the forced swim and sucrose-preference tests. Deficits were reported in social interaction that is suggestive of pathology associated with schizophrenia, and in spatial learning and memory in adult rats in the Morris water maze test, but in most studies only males were tested. There were too few studies on the novel object recognition test at different inter-trial intervals to enable a conclusion about the effect of prenatal stress and whether any deficits are more prevalent in males. Among hippocampal glutamate receptors, NR2B was the only subtype consistently reduced in association with learning deficits. However, like in humans with schizophrenia and depression, prenatal stress lowered hippocampal levels of BDNF, which were closely correlated with decreases in hippocampal long-term potentiation. In mice, down-regulation of BDNF appeared to occur through the action of gene-methylating enzymes that are already increased above controls in prenatally-stressed neonates. In conclusion, the data obtained so far from experiments in rodents lend support to a physiological basis for the neurodevelopmental hypothesis of schizophrenia and depression.

Novel indoline derivatives prevent inflammation and ulceration in dinitro-benzene sulfonic acid-induced colitis in rats.

BACKGROUND: In search of safer treatments for inflammatory bowel disease in subjects not responding to, or showing adverse effects to TNF-α antagonists, we tested three novel indoline carbamates in the 2,4-dinitrobenzene sulfonic acid (DNBS) model of colitis in rats. The compounds have anti-inflammatory activity in other disease models in mice. METHODS: AN827 (3-(2-(methoxy carbonyl) ethyl) indolin-4-ylethyl methyl) carbamate (0.1 or 1mg/kg), AN680 (3-(2-(methoxy carbonyl) ethyl) indolin-6-ylethyl methyl) carbamate (1.25 or 2.5mg/kg) and AN917 (3-(3-amino propyl) indolin-4-ylethyl methyl) carbamate (1 or 2mg/kg), 5-aminosalycilic acid (5-ASA) (1 or 100mg/kg) or saline (1ml/kg) were administered rectally 1h after intracolonic administration of DNBS, (35mg/kg in 30% alcohol). Disease severity was assessed four days after DNBS administration by change in body weight, colon weight, area of ulceration, myeloid peroxidase (MPO) activity, colonic TNF-α, IL-6 and IL-1ß levels. Histopathological scoring was performed after staining colon sections with hematoxylin and eosin and with antibodies to CD68 and CD11b. RESULTS: AN827 (0.1 and 1mg/kg), AN680 (2.5mg/kg) and AN917 (2.0mg/kg) significantly reduced all macroscopic and microscopic parameters of colitis, colonic pro-inflammatory cytokines, TNF-α, IL-1ß and IL-6 and MPO activity by about 80%. CONCLUSIONS: The indoline derivatives largely prevented the symptoms of colitis and were 500-50 times more potent and more effective than 5-ASA. It may be worth evaluating them in models of established colitis. Since AN827 is strongly bound by plasma proteins no adverse effects are expected if compound is absorbed into the circulation after rectal administration.

Sex-Specific Effects of Prenatal Stress on Memory and Markers of Neuronal Activity in Juvenile Rats.

Stress during pregnancy can increase the incidence of emotional problems, learning and language difficulties in human infants and pre-adolescents. Most preclinical studies in rats that attempted to find experimental support for these observations were performed in adult male offspring, but the results are inconsistent. The aim of the current study was to examine the effect of prenatal stress on novel object recognition (NOR) and spatial learning and memory in the Morris water maze (MWM) of juvenile rats of both sexes. By the use of fluorescence immunohistochemistry and protein measurements by Western blot, we measured the expression of markers of neurogenesis (doublecortin, DCX) and neuronal activity that are important for synaptic plasticity and learning (c-fos, GluR1, nNOS). Since neuronal activity in the developing and adult brain can be regulated by astrocytes, we also measured the number of astrocytes and the expression of two astroglial proteins (GFAP and S100B) in the stress-responsive hippocampal dentate gyrus (DG). Experiments were performed on littermates of rats in which its effects on behavior were measured. We found for the first time that juvenile females performed better than males in the NOR and MWM tests. They also had higher densities of DCX and c-fos in the DG, together with the expression of nNOS and GluR1 in the subgranular zone (SGZ) of the DG. There were no sex differences in the expression of GFAP and S100B in astrocytes. Prenatal stress did not affect NOR in females, but improved it in males, together with an increase in DCX+ and c-fos, the number of GFAP-expressing astrocytes and the intensity of GFAP and S100B immunofluorescence in the DG. Staining intensity of GluR1 and nNOS in the hilus and SGZ of the DG, and protein expression in the whole DG, was unchanged in prenatally stressed males. Thus, prenatal stress changed the behavior and expression of key proteins in the DG to resemble that in females. A reduction in plasma testosterone, which although not attaining statistical significance was associated with that in anogenital distance, may contribute to the effect of prenatal stress in males. In females, prenatal stress had no effect on c-fos, DCX or the number of astrocytes but reduced the staining intensity of GluR1 and nNOS. Protein expression of nNOS was also significantly lower than that in prenatally stressed males. The differential effects of prenatal stress on hippocampal neuronal and glial markers may help to explain the sex-dependent effect on spatial learning in prepubertal rats.

Perinatal citalopram does not prevent the effect of prenatal stress on anxiety, depressive-like behaviour and serotonergic transmission in adult rat offspring.

It is still not clear whether the selective serotonin reuptake inhibitors frequently prescribed to depressed pregnant women improve the behavioural outcome in their children. The current study investigated whether administration of citalopram to pregnant rats could prevent anxiety and depressive-like behaviour induced by gestational stress in their offspring, and restore the expression of serotonin 1A autoreceptors in GABAergic interneurons in the medial prefrontal cortex and dorsal raphe nuclei in males, and of corticotropin-releasing factor type 2 receptors in GABAergic interneurons in the dorsal raphe nuclei in females. Activation of these receptors modulates serotonergic transmission to target areas and is reduced in a sex-dependent manner by prenatal stress. Citalopram (10 mg/kg/day), administered orally from day 7 of gestation until 21 days postpartum, prevented the increase in anxiety in stressed mothers but did not reduce anxiety and depressive-like behaviour in their offspring and even induced depressive-like behaviour in the offspring of control mothers. Citalopram failed to restore the reduction in the expression of serotonin 1A autoreceptors in the prefrontal cortex of males and in corticotropin-releasing factor type 2 receptors in the dorsal raphe nuclei of females induced by prenatal stress. Prenatal citalopram did not prevent the behavioural changes or reduction in serotonergic transmission to target areas induced by prenatal stress. It had adverse behavioural effects in the offspring of control rats, which, together with the lack of any change in prenatally-stressed rats, may be due to inhibition of the foetal serotonin transporter thereby preventing normal development of the serotonin system.

The anti-inflammatory activity of a novel fused-cyclopentenone phosphonate and its potential in the local treatment of experimental colitis.

A novel fused-cyclopentenone phosphonate compound, namely, diethyl 3-nonyl-5-oxo-3,5,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-ylphosphonate (P-5), was prepared and tested in vitro (LPS-activated macrophages) for its cytotoxicity and anti-inflammatory activity and in vivo (DNBS induced rat model) for its potential to ameliorate induced colitis. Specifically, the competence of P-5 to reduce TNFα, IL-6, INFγ, MCP-1, IL-1α, MIP-1α, and RANTES in LPS-activated macrophages was measured. Experimental colitis was quantified in the rat model, macroscopically and by measuring the activity of tissue MPO and iNOS and levels of TNFα and IL-1ß. It was found that P-5 decreased the levels of TNFα and the tested proinflammatory cytokines and chemokines in LPS-activated macrophages. In the colitis-induced rat model, P-5 was effective locally in reducing mucosal inflammation. This activity was equal to the activity of local treatment with 5-aminosalicylic acid. It is speculated that P-5 may be used for the local treatment of IBD (e.g., with the aid of colon-specific drug platforms). Its mode of action involves inhibition of the phosphorylation of MAPK ERK but not of p38 and had no effect on IκBα.

Dose-limiting inhibition of acetylcholinesterase by ladostigil results from the rapid formation and fast hydrolysis of the drug-enzyme complex formed by its major metabolite, R-MCPAI.

Ladostigil is a pseudo reversible inhibitor of acetylcholinesterase (AChE) that differs from other carbamates in that the maximal enzyme inhibition obtainable does not exceed 50-55%. This could explain the low incidence of cholinergic adverse effects induced by ladostigil in rats and human subjects. The major metabolite, R-MCPAI is believed to be responsible for AChE inhibition by ladostigil in vivo. Therefore we determined whether the ceiling in AChE inhibition resulted from a limit in the metabolism of ladostigil to R-MCPAI by liver microsomal enzymes, or from the kinetics of enzyme inhibition by R-MCPAI. Ladostigil reduces TNF-α in lipopolysaccharide-activated microglia. In vivo, it may also reduce pro-inflammatory cytokines by inhibiting AChE and increasing the action of ACh on macrophages and splenic lymphocytes. We also assessed the contribution of AChE inhibition in the spleen of LPS-injected mice to the anti-inflammatory effect of ladostigil. As in other species, AChE inhibition by ladostigil in spleen, brain and plasma did not exceed 50-55%. Since levels of R-MCPAI increased with increasing doses of ladostigil we concluded that there was no dose or rate limitation of metabolism. The kinetics of enzyme inhibition by R-MCPAI are characterized by a rapid formation of the drug-enzyme complex and fast hydrolysis which limits the attainable degree of AChE inhibition. Ladostigil and its metabolites (1-100 nM) decreased TNF-α in lipopolysaccharide-activated macrophages. Ladostigil (5 and 10mg/kg) also reduced TNF-α in the spleen after injection of lipopolysaccharide in mice. However, AChE inhibition contributed to the anti-inflammatory effect only at a dose of 10mg/kg.