Docosahexaenoic Acid (DHA) Induced Morphological Differentiation of Astrocytes Is Associated with Transcriptional Upregulation and Endocytosis of ß2-AR.

Docosahexaenoic acid (DHA), an important ω-3 fatty acid, is abundantly present in the central nervous system and is important in every step of brain development. Much of this knowledge has been based on studies of the role of DHA in the function of the neurons, and reports on its effect on the glial cells are few and far between. We have previously reported that DHA facilitates astrocyte differentiation in primary culture. We have further explored the signaling mechanism associated with this event. It was observed that a sustained activation of the extracellular signal-regulated kinase (ERK) appeared to be critical for DHA-induced differentiation of the cultured astrocytes. Prior exposure to different endocytic inhibitors blocked both ERK activation and differentiation of the astrocytes during DHA treatment suggesting that the observed induction of ERK-2 was purely endosomal. Unlike the ß1-adrenergic receptor (ß1-AR) antagonist, atenolol, pre-treatment of the cells with the ß2-adrenergic receptor (ß2-AR) antagonist, ICI-118,551 inhibited the DHA-induced differentiation process, indicating a downstream involvement of ß2-AR in the differentiation process. qRT-PCR and western blot analysis demonstrated a significant induction in the mRNA and protein expression of ß2-AR at 18-24 h of DHA treatment, suggesting that the induction of ß2-AR may be due to transcriptional upregulation. Moreover, DHA caused activation of PKA at 6 h, followed by activation of downstream cAMP response element-binding protein, a known transcription factor for ß2-AR. Altogether, the observations suggest that DHA upregulates ß2-AR in astrocytes, which undergo endocytosis and signals for sustained endosomal ERK activation to drive the differentiation process.

Efficacy of Arsenicum album 30cH in preventing febrile episodes following DPT-HepB-Polio vaccination - a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Among the post-immunization adverse events, especially of Diphtheria-Pertusis-Tetanus (DPT), fever is a common systemic reaction. There is anecdotal support for the use of the homeopathic medicine Arsenicum album in preventing post-vaccination fever. The investigators intended to evaluate its efficacy in preventing febrile episodes following vaccination. METHODS: In the community medicine out-patient of Mahesh Bhattacharyya Homoeopathic Medical College and Hospital, West Bengal, India, between August 2014 and January 2017, a double-blind, randomized, placebo-controlled trial was conducted on 120 children (verum: 60, placebo: 60) who presented for the 2nd and 3rd dose of DPT-HepB-Polio vaccination and reported febrile episodes following the 1st dose. Intervention used was Arsenicum album 30cH 6 doses or placebo (indistinguishable from verum), thrice daily for two subsequent days. Parents were advised to report any event of febrile attacks within 48h of vaccination, either directly or over telephone. RESULTS: The groups were comparable at baseline. Children reporting fever after the 2nd dose was 29.8% and 30.4% respectively for the homeopathy group and control group respectively [Relative Risk (RR)=1.008] with no significant difference (P=0.951) between groups. Again after the 3rd dose, children reporting fever were 31.5% and 28.3% respectively for the homeopathy group and control group respectively (RR=0.956) with no significant difference (P=0.719) between groups. CONCLUSION: Empirically selected Arsenicum album 30cH could not produce differentiable effect from placebo in preventing febrile episodes following DPT-HepB-Polio vaccination. [Trial registration: CTRI/2017/02/007939].

Thyroid Hormone and Astrocyte Differentiation.

Thyroid hormones (THs) have important contributions to the development of the mammalian brain, targeting its actions on both neurons and glial cells. Astrocytes, which constitute about half of the glial cells, characteristically undergo dramatic changes in their morphology during development and such changes become necessary for the proper development of the brain. Interestingly, a large number of studies have suggested that THs play a profound role in such morphological maturation of the astrocytes. This review discusses the present knowledge on the mechanisms by which THs elicit progressive differentiation and maturation of the astrocytes. As a prelude, information on astrocyte morphology during development and its regulations, the role of THs in the various functions of astrocyte shall be dealt with for a thorough understanding of the subject of this review.

Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells.

Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione-S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.

Thyroid Hormone-Induced Differentiation of Astrocytes is Associated with Transcriptional Upregulation of ß-arrestin-1 and ß-adrenergic Receptor-Mediated Endosomal Signaling.

Thyroid hormones (TH) promote differentiation of astrocytes. We have previously reported that a downstream role ß-adrenergic receptor (ß-AR) system in such effects of TH. Although evidences indicate strong interaction between TH and the ß-ARs, the underlying mechanism is poorly understood. In the present study, we further explored the influence of TH on ß-AR signaling during the differentiation process. Unlike ß1-AR, binding of (125)I-pindolol to ß2-AR in cell membranes was significantly decreased at 2 h of exposure to TH which came back to control values after 24 h. The initial decrease in ß2-AR in membranes resulted in a concomitant increase in ß2-AR levels in the cytosol, suggesting that TH may induce endocytosis of the receptor. qRT-PCR as well as Western blot analysis demonstrated that unlike ß-adrenergic receptor kinase (ß-ARK)1 and ß-ARK2, the messenger RNA (mRNA) and protein levels of ß-arrestin-1 in the astrocyte cultures increased on exposure to TH. Knockdown of ß-arrestin gene suggested requirement of both ß-arrestin-1 and ß-arrestin-2 isoforms during endocytosis of ß2-AR, thereby facilitating cell differentiation. Endocytic inhibitors blocked the delayed but sustained activation of p-extracellular signal-regulated kinase (ERK) observed during cell differentiation. Observations suggest that TH upregulate ß-arrestin-1 in astrocytes to facilitate endocytosis of ß2-AR, required for endosomal ERK activation to drive the differentiation process.

Essential role of docosahexaenoic acid towards development of a smarter brain.

Evolution of the high order brain function in humans can be attributed to intake of poly unsaturated fatty acids (PUFAs) of which the ω-3 fatty acid, docosahexaenoic acid (DHA) has special significance. DHA is abundantly present in the human brain and is an essential requirement in every step of brain development like neural cell proliferation, migration, differentiation, synaptogenesis etc. The multiple double bonds and unique structure allow DHA to impart special membrane characteristics for effective cell signaling. Evidences indicate that DHA accumulate in areas of the brain associated with learning and memory. Many development disorders like dyslexia, autism spectrum disorder, attention deficit hyperactivity disorder, schizophrenia etc. are causally related to decreased level of DHA. The review discusses the various reports of DHA in these areas for a better understanding of the role of DHA in overall brain development. Studies involving laboratory animals and clinical findings in cases as well as during trials have been taken into consideration. Additionally the currently available dietary source of DHA for supplementation as nutraceutics with general caution for overuse has been examined.

The quinoline compound, S4 effectively antagonizes alcohol intake in mice: Possible association with the histone H3 modifications.

Opioidergic system plays an important role in controlling alcohol seeking behavior. We have previously shown that a quinoline compound, S4 (2-(2-methylquinolin-4-ylamino)-N-phenyl acetamide), having dual affinity for µ- and κ-opioid receptors, could successfully inhibit withdrawal symptoms in mice rendered dependent on morphine. Accordingly, in the present study, we sought to determine the potential of S4 in attenuating voluntary alcohol intake in alcohol-preferring (AP) mice and the mechanism thereof. The study was conducted in different mice strains initially screened for AP and alcohol-avoiding (AA) behavior. S4 was injected subcutaneously (20 mg/kg) to evaluate its efficacy in reducing voluntary alcohol consumption along with prevention of body weight loss during withdrawal from alcohol after discontinuation of the drug. The results showed that S4 significantly reduced the alcohol intake in AP mice and also in a dose dependent manner. Mechanistic studies on the post translational histone H3 modifications in brain of AP mice compared to the AA mice were determined. Compared to AA mice, histone H3 trimethylation at lys9 and its regulators, jumonji domain containing 2A and phosphorylated histones H3 at thr11 as well as the expression of 14-3-3 protein and phosphorylated histones H3 at ser28, were altered in the AP animals, most of which were restored post S4 treatment in the AP mice. Together, the present results suggest that S4 effectively blocked alcohol drinking behavior by restoring the altered epigenetic signature in the AP mice. The study provides a novel compound which could lead to developing effective drugs against alcoholism/alcohol abuse.

Synthesis of iboga-like isoquinuclidines: Dual opioid receptors agonists having antinociceptive properties.

Some novel iboga-analogues consisting of benzofuran moiety and dehydroisoquinuclidine ring connected by -CH2-, (CH2)2 and (CH2)3 linkers have been synthesized with the view to develop potential antinociceptive drugs. The compounds 14 and 21 showed binding at the µ-opioid receptor (MOR), while the compound 11a exhibited dual affinities at both MOR and κ-opioid receptor (KOR). MAP kinase activation indicated all three compounds have opioid agonistic properties. The presence of a double bond and endo-methylcarboxylate group in the dehydroisoquinuclidine ring and the benzofuran and methylene spacer appeared to be essential for opioid receptor binding. Further studies demonstrated 11a caused significant antinociception in mice in the hot-plate test which was comparable to that produced by morphine. The compound 11a was also found to be nontremorigenic unlike various iboga congeners. This study identifies a new pharmacophore which may lead to the development of suitable substitute of morphine in the treatment of pain.

Association of CREB1 gene polymorphism with drug seeking behaviour in eastern Indian addicts.

cAMP response element binding protein (CREB) is a major transcription factor which plays an important role in a wide array of cellular functions. CREB also has a significant function in developing substance abuse. A study was undertaken to identify the single nucleotide polymorphisms (SNP) at selective areas of CREB1 gene in heroin as well as in alcohol addicts in comparison with control population. One hundred and forty control subjects, 112 heroin and 102 alcoholics, all male and residing in Kolkata, a city in eastern India participated in the study. SNPs from several exonic regions of CREB1 gene were assessed to investigate possible associations with addiction. One SNP in exon 3, rs35349697, demonstrated a significant correlation with opioid addiction as well as with alcohol addiction. A novel SNP, also located in exon 3, was identified which showed epistatic interaction with rs35349697 to decrease susceptibility to narcotic addiction in the population. The study is the first report on the identification of a role of CREB1 gene polymorphism with addiction.

BDNF local translation in viable synaptosomes: implication in spine maturation.

The neurotrophic factor, BDNF, is encoded by two transcripts, one short and another long 3' untranslated region containing mRNA. Long BDNF mRNA was found to transport to the dendrites; however report about its translation or regulation of translation in the dendrite remains unknown. Using synaptosomes, to isolate from the nucleus and other subcellular fractions involved in translation, we demonstrate that depolarization by KCl or excitation by glutamate can induce translation of BDNF. Such translation at the synaptosomes was also observed for mRNAs of CaMKllα, Homer and Arc, which are known to travel to dendrite. This synaptosomal translation system is critically dependent on glucose concentration. Other than glucose, BDNF translation in synaptosome is dependent on its own receptor TrkB function as well as on the rise in intra-synaptosomal Ca(2+), both of which are elevated during to depolarization or excitation. As BDNF-TrkB signaling causes maturation of spines by inducing LTP, this study also investigated the possibility of induction of spine maturation signaling in the isolated synaptosomes. Increased phospho-cofilin and phospho-PAK is detected in KCl or glutamate treated synaptosomes compared to control by Western blotting, suggesting a possibility of induction of spine maturation signaling.

Comparison of ex vivo cultivated human limbal epithelial stem cell viability and proliferation on different substrates.

Ocular surface injury causes serious vision-related problems especially when limbal stem cells are affected. Treatment lies in the transplantation of viable donor cells. Various substrates are used for the cultivation of limbal epithelial stem cells. In the present study, viability and proliferation of ex vivo cultured limbal epithelial stem cells were examined on a variety of substrates like collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane. Viability and proliferation of cells were examined by colorimetric assay and [(3)H]-thymidine incorporation study. Furthermore, matrix metalloproteinase is known to be a key regulator in stem cell migration and proliferation. This enzyme activity was studied by gelatinolytic zymography. It was found from this study that although human limbal epithelial stem cells could be cultivated on different substrates such as collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane, maximum growth and proliferation was observed when cultured on intact amniotic membrane. The number of patients suffering from limbal epithelial stem cell deficiency is large compared to donor tissues available for transplantation. Hence, increased cell viability and proliferation is required to serve more patients.

Chronic morphine exposure and its abstinence alters dendritic spine morphology and upregulates Shank1.

Exposure to chronic drugs of abuse has been reported to produce significant changes in postsynaptic protein profile, dendritic spine morphology and synaptic transmission. In the present study we demonstrate alterations in dendritic spine morphology in the frontal cortex and nucleus accumbens of mice following chronic morphine treatment as well as during abstinence for two months. Such alterations were accompanied with significant upregulation of the postsynaptic protein Shank1 in synaptosomal enriched fractions. mRNA levels of Shank1 was also markedly increased during morphine treatment and during withdrawal. Studies of the different postsynaptic proteins at the protein and mRNA levels showed significant alterations in the morphine treated groups compared to that of saline treated controls. Taken together, these observations suggest that Shank1 may have an important role in the regulation of spine morphology induced by chronic morphine leading to addiction.

Effect of cord blood serum on ex vivo human limbal epithelial cell culture.

Limbal cell transplantation is an efficacious procedure for rehabilitation of visual acuity in patients with severe ocular surface disorders. Cultivation of limbal epithelial stem cell with fetal bovine serum for transplantation has been a promising treatment for reconstructing the ocular surface in severe limbal stem cell deficiency caused by Steven Johnson syndrome, chemical or thermal injury. This technique of "cell therapy" has been accepted worldwide but the cost of cultivating the cells for transplantation is high. The objective of this study was to investigate the effect of cord blood serum in place of fetal bovine serum on the growth of human limbal epithelial cell culture. Our group has experimented with human cord blood serum which was obtained free of cost from willing donors. The use of human cord blood serum in place of fetal bovine serum for ex vivo culture of limbal stem cell has helped us in reducing the cost of culture. Fresh human limbal tissues from donor cadavers were cultured on intact and denuded amniotic membrane. Cells were proliferated in vitro with cell culture media containing human cord blood serum. Reverse transcription-polymerase chain reaction and immunofluorescence cytochemistry of cultured human limbal epithelial stem cell was done for characterization of the cells.

Epistatic effects between variants of kappa-opioid receptor gene and A118G of mu-opioid receptor gene increase susceptibility to addiction in Indian population.

OBJECTIVE: Unequivocal evidence suggests contribution of κ-opioid receptor (KOR) in addiction to drugs of abuse. A study was undertaken to identify the single nucleotide polymorphisms (SNP) at selective areas of kappa opioid receptor 1 (OPRK1) gene in heroin as well as in alcohol addicts and to compare them with that in control population. The potential interaction of the identified KOR SNPs with A118G of µ opioid receptor was also investigated. METHODS: Two hundred control subjects, one hundred thirty heroin and one hundred ten alcohol addicts, all male and residing in Kolkata, a city in eastern India, volunteered for the study. Exons 3 and 4 of OPRK1 and the SNP, A118G of mu opioid receptor 1 (OPRM1) in the DNA samples were genotyped by sequencing and restriction fragment length polymorphism respectively. The SNPs identified in the population were analyzed by odds ratio and its corresponding 95% confidence interval was estimated using logistic regression models. SNP-SNP interactions were also investigated. RESULTS: Three SNPs of OPRK1, rs16918875, rs702764 and rs963549, were identified in the population, none of which showed significant association with addiction. On the other hand, significant association was observed for A118G with heroin addiction (χ²=7.268, P=0.0264) as well as with alcoholic addition (χ²=6.626, P=0.0364). A potential SNP-SNP interaction showed that the odds of being addicted was 2.51 fold in heroin subjects [CI (95%)=1.1524 to 5.4947, P=0.0206] and 2.31 fold in alcoholics [CI (95%)=1.025 to 5.24, P=0.0433] with the OPRK1 (rs16918875) and A118G risk alleles than without either. A significant interaction was also identified between GG/AG of A118G and GG of rs702764 [O.R (95%)=2.04 (1.279 to 3.287), P=0.0029] in case of opioid population. CONCLUSION: Our study suggests that set associations of polymorphisms may be important in determining the risk profile for complex diseases such as addiction.

Self-assembling dipeptide-based nontoxic vesicles as carriers for drugs and other biologically important molecules.

Self-assembling short peptides can offer an opportunity to make useful nano-/microstructures that find potential application in drug delivery. We report here the formation of multivesicular structures from self-assembling water-soluble synthetic amphiphilic dipeptides containing a glutamic acid residue at the C-terminus. These vesicular structures are stable over a wide range of pH (pH 2-12). However, they are sensitive towards calcium ions. This causes the rupturing of these vesicles. Interestingly, these vesicles can not only encapsulate an anticancer drug and a fluorescent dye, but also can release them in the presence of calcium ions. Moreover, these multivesicular structures have the potential to carry biologically important molecules like cyclic adenosine monophosphate (cAMP) within the cells keeping their biological functions intact. A MTT cell-survival assay suggests the almost nontoxic nature of these vesicles. Thus, these peptide vesicles can be used as biocompatible delivery vehicles for carrying drugs and other bioactive molecules.

A polymorphism of the CREB binding protein (CREBBP) gene is a risk factor for addiction.

Unequivocal evidences have implicated c-AMP response element binding protein (CREB) in drug addiction. Recent reports indicate that the CREB binding protein (CREBBP), a transcription co-activator, may also be involved in the sensitivity to drugs of abuse. We undertook studies on the single nucleotide polymorphisms (SNP) at selective areas of CREBBP gene in heroin as well as in alcohol addicts and compared them with that in normal population. One hundred fifty healthy controls, one hundred thirty heroin addict and one hundred ten alcohol addicts, all males, Bengali-Hindu, and residing in Kolkata, a city in eastern India, participated in the study. DNA prepared from blood drawn from the subjects was PCR amplified for the regions corresponding to exon 3 and 22 of CREBBP gene followed by sequencing. Three SNPs identified in the population were analyzed to find out the association of these SNPs with addiction. One SNP, rs3025684 in intron 21 having the contig position of 3795363, showed association with addiction. The genotype frequencies of the SNP were significantly different between opioid dependent cases and controls (χ(2)=20.28, p<0.0001) as well as between alcoholics and controls (χ(2)=13.60, p=0.0011). Our studies suggest that rs3025684 polymorphism may be a possible risk factor for developing addiction.

Synthesis and biological evaluation of dibenz[b,f][1,5]oxazocine derivatives for agonist activity at κ-opioid receptor.

A short and high yield synthetic route to dibenz[b,f][1,5]oxazocines has been developed using Pd catalyzed intramolecular cycloamination reaction. Receptor binding assay using [125I]-dynorphin demonstrated that one of the derivative, 5b showed selective κ-opioidergic property.

Thyroid hormones protect astrocytes from morphine-induced apoptosis by regulating nitric oxide and pERK 1/2 pathways.

Unlike neurons and various other non-neuronal cells, astrocytes have been reported to be resistant to morphine induced cytotoxicity. The present work demonstrates that primary cultures of astrocytes are also sensitive to morphine toxicity depending upon the thyroidal status of the culture medium. Chronic morphine treatment of astrocytes, cultured under thyroid hormone (TH)-deficient conditions, induced apoptotic cell death which was characterized by nuclear condensation, DNA fragmentation and activation of caspase-3 like enzymes. Cell death was accompanied with increase in nNOS level, nitration of cellular proteins and down regulation of pAKT level. Phosphorylation of ERK1/2 showed a biphasic response, an initial induction followed by sustained decline during chronic morphine treatment and the initial induction of pERK1/2 level appeared to be critical for apoptosis in the cells. Interestingly, supplementation with normal levels of TH to cells attenuated morphine-induced apoptosis as well as the biphasic response of pERK1/2 in the astrocytes. However, in the presence of glutathione synthetase inhibitor L-buthionine-S,R-sulfoximine, TH failed to protect astrocytes. Overall, the study demonstrates a possible signaling mechanism of morphine induced toxicity to cells and suggests that alteration of glutathione homeostasis by TH protect astrocytes from morphine by regulating NO and pERK1/2 pathways in the cells.

Single-nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu-opioid receptor and may contribute to the genetic risk for addiction.

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single-nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P(trend) < 0.05. The functional significance of G allele in A118G single-nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.

Synthesis and characterizations of novel quinoline derivatives having mixed ligand activities at the kappa and mu receptors: Potential therapeutic efficacy against morphine dependence.

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using (125)I-Dynorphine, (3)H-DAMGO and (125)I-DADLE for kappa, mu, and delta receptors, respectively. Results showed varying degree of activities of the compounds to kappa and mu opioid receptors with negligible interactions at the delta receptor. The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination of IC(50) of S4 revealed a greater affinity towards mu compared to kappa receptor. In conclusion, quinoline derivatives of S4 like structure offer potential tool for treatment of narcotic addictions.