Age-related changes in gonadotropin-releasing hormone (GnRH) splice variants in mouse brain.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of the mammalian reproductive axis. We investigated the spatiotemporal expression of GnRH splice variants (V1, V2, and V3) and splicing factors (Srsf7, Srsf9, and Tra-2) in the male mice brain. Further, using in silico tools, we predicted protein structure and the reason for the low translational efficiency of V2 and V3. Messenger RNA levels of GnRH variants and splicing factors were quantified using real-time reverse transcription-polymerase chain reaction at different age groups. Our data show that expression of almost all the variants alters with aging in all the brain regions studied; even in comparison to the hypothalamus, several brain areas were found to have higher expression of these variants. Hypothalamic expression of splicing factors such as Srsf7, Srsf9, and Tra-2 also change with aging. Computational studies have translation repressors site on the V3, which probably reduces its translation efficiency. Also, V2 is an intrinsically disordered protein that might have a regulatory or signaling function. In conclusion, this study provides novel crucial information and multiple starting points for future analysis of GnRH splice variants in the brain.

A novel in vitro approach to test the effectiveness of fish oil in ameliorating type 1 diabetes.

Diabetes type 1 (T1D) characterized by destruction of pancreatic ß-cells results in inadequate insulin production and hyperglycaemia. Generation of reactive oxygen species and glycosylation end-products stimulates toxic impacts on T1D. Dietary w-3 fatty acids present in Fish oil (FO) might be helpful in the prevention of oxidative stress and lipid peroxidation, thus, beneficial against T1D. But how the cellular secretion from ß-cells under influence of FO affects the glucose homeostasis of peri-pancreatic cells is poorly understood. In the current study, we aimed to introduce an in vitro model for T1D and evaluate its effectiveness in respect of alloxan treatment to pancreatic Min6 cells. We use alloxan in the Min6 pancreatic ß-cell line to induce cellular damage related to T1D. Further treatment with FO was seen to prevent cell death by alloxan and induce mRNA expression of both insulin 1 and insulin 2 isoforms under low-glucose conditions. From the first part of the study, it is clear that FO is effective to recover Min6 cells from the destructive effect of alloxan, and it worked best when given along with alloxan or given after alloxan treatment regime. FO-induced secretion of molecules from Min6 was clearly shown to regulate mRNA expression of key enzymes of carbohydrate metabolism in peri-pancreatic cell types. This is a pilot study showing that an improved in vitro approach of using Min6 along with muscle cells (C2C12) and adipose tissue cells (3T3-L1) together to understand the crosstalk of molecules could be used to check the efficacy of an anti-diabetic drug.

Recent Update on Retinoic Acid-Driven Initiation of Spermatogonial Differentiation.

Germ cells (Gc) propagate the genetic information to subsequent generations. Diploid (2n) Gc get transformed to specialized haploid (n) gametes by mitotic and meiotic divisions in adult gonads. Retinoic acid (RA), an active derivative of vitamin A (retinol), plays a critical role in organ morphogenesis and regulates the meiotic onset in developing Gc. Unlike ovaries, fetal testes express an RA-degrading enzyme CYP26B1, and thereby, male Gc fail to enter into meiosis and instead get arrested at G0/G1 stage, termed as gonocytes/pro-spermatogonia by embryonic (E) 13.5 days. These gonocytes are transformed into spermatogonial stem/progenitor cells after birth (1-3 days of neonatal age). During post-natal testicular maturation, the differentiating spermatogonia enter into the meiotic prophase under the influence RA, independent of gonadotropic (both FSH and LH) support. The first pulse of RA ensures the transition of undifferentiated type A spermatogonia to differentiated A1 spermatogonia and upregulates STRA8 expression in Gc. Whereas, the second pulse of RA induces the meiotic prophase by augmenting MEIOSIN expression in differentiated spermatogonia B. This opinion article briefly reviews our current understanding on the RA-driven spermatogonial differentiation in murine testes.

Expression of polyamines and its association with GnRH-I in the hypothalamus during aging in rodent model.

GnRH-I and GnIH are the key neuropeptides that regulate the hypothalamic-pituitary-gonadal axis in mammals during aging. Polyamines are important aliphatic amines that are expressed in the brain and show variation with aging. The present study demonstrates evidence of variation in the level of expression of polyamines, GnRH-I and GnIH in the hypothalamus of female mice during aging. The study also suggests regulatory effects of polyamines over expression of the hypothalamic GnRH-I. The study shows a significant positive correlation between polyamines, its associated factors and GnRH-I along with significant negative correlation between polyamines, its associated factors and GnIH. This is the first study to report the effect of polyamines along with lactate or TNF-α or both on GnRH-I expression in GT1-7 cell line. TNF-α and lactate significantly decreased hypothalamic GnRH-I mRNA expression in GT1-7 cells when treated for 24 h. Polyamines (putrescine and agmatine) in contrast, significantly increased GnRH-I mRNA expression in GT1-7 cells when treated for 24 h. Also, polyamines increased GnRH-I mRNA expression when treated in presence of TNF-α or lactate thereby suggesting its neuro-protective role. This study also found 3809 differentially expressed genes through RNA-seq done between the hypothalamic GT1-7 cells treated with putrescine only versus TNF-α and putrescine. The present study suggests for the first time that putrescine treatment to TNFα-primed GT1-7 cells upregulates GnRH-I expression via regulation of several pathways such as calcium ion pathway, estrogen signaling, clock genes as well as regulating other metabolic process like neuronal differentiation and neurulation.

Lactate-Dependent Cross-Talk Between Astrocyte and GnRH-I Neurons in Hypothalamus of Aged Brain: Decreased GnRH-I Transcription.

GnRH-I produced by hypothalamic neurosecretory cells is considered a master regulator of mammalian reproduction. Although GnRH-I transcription is well studied, the effect of ageing on transcriptional regulation of GnRH-I has not yet been explored. Here, we elucidate the effects of ageing on the metabolic environment like lactate level and TNF-α and how these affect GnRH-I transcription. Using pathway analysis of transcriptomic data, we found that lactate is upregulated in ageing astrocytes due to the downregulation of cellular respiration pathways possibly resulting in greater pyruvate concentration for lactate production. This lactate could then be shuttled into neurons where it would affect GnRH-I transcription. We showed that supra-physiological level of lactate in young mouse brain can mimic metabolic disturbances in the old brain and cause downregulation in GnRH-I transcription at a young age. In particular, we found upregulation of GnRH-I repressors in the young brain treated with high levels of lactate similar to old brain. Hence, this confirmed that aged metabolic environment can affect GnRH-I transcription even in the young brain. Further downstream analysis using the TRUST database showed NF-Kb signalling which lies downstream of both lactate and TNF-α as being capable of upregulating GnRH-I repressors. Since NF-Kb signalling has been shown in our study as well as others to be induced by TNF-α during ageing, it is likely that GnRH-I transcriptional regulation is mediated through these pathways. Thus, we formed a model for explaining the downregulation of GnRH-I transcription during ageing through differential expression of its TFs in an aged metabolic environment.

Effect of high salinity acclimation on glucose homeostasis in Mozambique tilapia (Oreochromis mossambicus).

During salinity stress, osmoregulatory processes in euryhaline fish need to modify for their survival, and glucose is the preferred mode of extra energy during such conditions. These organisms must have a proper mechanism to maintain glucose homeostasis during such modified osmoregulatory process across different body fluids. Hence, we studied high salinity effect on regulation of glucose homeostasis in Mozambique tilapia. The fish were induced to 15‰ salinity for 21 days. Glucose, glycogen, ion concentrations, Na+-K+-ATPase, pyruvate kinase, γ-amylase activities and GLUT mRNA expressions were investigated in liver, intestine, gill and white muscle tissues. At the end of experiment, Na+ ion concentrations, glucose content and activity of Na+-K+-ATPase especially in the gill and intestine were increased, while decrease in liver and gill glycogen content was seen. Lower concentration of glycogen decrease was observed in the intestine and white muscle of the treated group. High pyruvate kinase activity was noticed in liver and gill tissues that correlates with high Na+-K+-ATPase activity. Elevated γ-amylase activity was observed in the liver and intestine suggesting breakdown of glycogen; however, gill and white muscle did not show any increased activity. Increase in GLUT1 and GLUT4 mRNA expressions was observed especially in the gill and intestine, while increase in GLUT2 mRNA expressions was observed in the liver. Upregulations of GLUTs suggest higher influx of glucose into the cell for catabolism to provide energy and further to drive the enhanced osmoregulatory process. These findings suggest glucose homeostasis being regulated in Mozambique tilapia during salinity acclimation.

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.

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.

Synthesis and photophysical characterization of quasi push-pull dicyanodibenzodioxins and their anti-tumor activity against glioma cell line C6.

Dibenzodioxins bearing multiple electron withdrawing groups were synthesized using a simple one-step methodology including examples of molecules possessing electron acceptor groups in both ends. As a consequence internal charge delocalization occurs and the optical spectra are found to be bathochromically shifted compared to similar examples known thus far. A theoretical analysis of the molecular orbitals reveals the origin of the peaks in the dibenzodioxin optical spectra. Select examples exhibit in vitro neuro-cytotoxicity against glioma cell line C6, a finding which enhances existing knowledge about the pharmacologically relevant structural motifs in dibenzodioxins.