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Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary–gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis. This is the peer reviewed version of the following article: Milosevic A, Janjic MM, Lavrnja I, Savic D, Bozic ID, Tesovic K, Jakovljevic M, Pekovic S, Stojilkovic SS, Bjelobaba I. The sex-specific patterns of changes in hypothalamic-pituitarygonadal axis during experimental autoimmune encephalomyelitis. Brain Behav Immun. 2020 [http://dx.doi.org/10.1016/j.bbi.2020.06.025]

The growing body of data implies that SARS-CoV-2 infection may affect the nervous system. We here present a short, taciturn overview of described neurological impairments related to SARS-CoV-2 infection. While it is obvious that neurological impairments can be diagnosed in a portion of COVID-19 patients, evidence of SARS-CoV-2 neurovirulence in humans is still lacking. The existing data on the incidence of neurological impairments among COVID-19 patients is highly variable, probably because they (most often) come from small, single-center retrospective studies. These data are practically published in real-time, and the question remains when larger studies will be available, given that the pandemic is continuing. We here also shortly address the other issues related to neurological disorders and COVID-19 pandemic, including the concern for people with existing chronic neurological disorders and possible long-term neurological consequences of SARS-CoV-2 infection.

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

Pituitary gonadotrophs play a key role in reproductive functions by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The LH secretory activity of gonadotroph is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) via GnRH receptors and is accompanied by only minor effects on high basal Lhb gene expression. The secretory profiles of GnRH and LH are highly synchronized, with the latter reflecting a depletion of prestored LH in secretory vesicles by regulated exocytosis. In contrast, FSH is predominantly released by constitutive exocytosis, and secretory activity reflects the kinetics of Fshb gene expression controlled by GnRH, activin, and inhibin. Here is a review of recent data to improve the understanding of multiple patterns of gonadotroph gene expression and hormone secretion.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic–pituitary–adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Multiple sclerosis (MS) is a chronic, inflammatory, neurodegenerative disease with an autoimmune component. It was suggested that potassium channels, which are involved in crucial biological functions may have a role in different diseases, including MS and its animal model, experimental autoimmune encephalomyelitis (EAE). It was shown that voltage-gated potassium channels Kv1.5 are responsible for fine-tuning in the immune physiology and influence proliferation and differentiation in microglia and astrocytes. Here, we explored the cellular distribution of the Kv1.5 channel, together with its transcript and protein expression in the male rat spinal cord during different stages of EAE. Our results reveal a decrease of Kv1.5 transcript and protein level at the peak of disease, where massive infiltration of myeloid cells occurs, together with reactive astrogliosis and demyelination. Also, we revealed that the presence of this channel is not found in infiltrating macrophages/microglia during EAE. It is interesting to note that Kv1.5 channel is expressed only in resting microglia in the naive animals. Predominant expression of Kv1.5 channel was found in the astrocytes in all experimental groups, while some vimentin+ cells, resembling macrophages, are devoid of Kv1.5 expression. Our results point to the possible link between Kv1.5 channel and the pathophysiological processes in EAE.

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

Continuous but not pulsatile delivery of GnRH leads to marked down regulation of luteinizing hormone (LH) secretion and blockade of reproductive functions. However, the relationship between LH beta subunit gene (Lhb) expression and LH secretion has not been studied in detail. Here we studied the relationship between Lhb expression, LH release and intracellular LH content under different experimental paradigms. Experiments were performed in vivo and in vitro using female and male rats and in cultured rat pituitary cells. In vitro experiments included evaluation of Lhb expression during the prolonged absence of GnRH, influence of culture medium on gene expression, continuous vs repetitive GnRH application, and ELISA vs Western blot analysis of LH content. Experiments showed that Lhb expression declined progressively after pituitary removal and cell dispersion, indicating the loss of regulated gene expression. Addition of GnRH receptor agonists did not rescue Lhb expression, suggesting that other factor(s) account(s) for regulated gene expression. On the other hand, GnRH stimulated LH secretion in vitro, leading to gradual depletion of the secretory pool during continuous receptor activation. Inhibition of transcription by actinomycin D had only a minor effect on in vitro LH secretion, in contrast to cycloheximide, a protein synthesis inhibitor. In vivo administration of a GnRH receptor agonist was accompanied with a rapid increase in serum LH levels and a progressive depletion of the intrapituitary LH levels, without affecting Lhb expression. These results indicate that GnRH controls LH synthesis and exocytosis post-transcriptionally and that pulsatile GnRH release protects gonadotrophs from depletion of the LH secretory pool.

It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1β release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc.

Summary Active membrane remodeling during myelination relies on phospholipid synthesis and membrane polarization, both of which are known to depend on inositol phospholipids. Here, we show that sciatic nerves of mice lacking phosphatidylinositol 4-kinase alpha (PI4KA) in Schwann cells (SCs) show substantially reduced myelin thickness with grave consequences on nerve conductivity and motor functions. Surprisingly, prolonged inhibition of PI4KA in immortalized mouse SCs failed to decrease plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels or PI 3-kinase (PI3K) activation, in spite of large reductions in plasma membrane PI4P levels. Instead, it caused rearrangements of the actin cytoskeleton, which was also observed in sciatic nerves of knockout animals. PI4KA inactivation disproportionally reduced phosphatidylserine, phosphatidylethanolamine, and sphingomyelin content in mutant nerves, with similar changes observed in SCs treated with a PI4KA inhibitor. These studies define a role for PI4KA in myelin formation primarily affecting metabolism of key phospholipids and the actin cytoskeleton.

[No abstract available]

Discovered 45 years ago, ribavirin still proves useful as a broad-spectrum anti-viral drug against different RNA and DNA viruses. Although many cellular and molecular mechanisms of ribavirin action have been proposed during several decades of the extensive research, the complete spectrum of its actions is still not fully known. The direct mechanisms of ribavirin anti-viral action involve RNA polymerase inhibition and lethal mutagenesis. The main intracellular target of ribavirin action is inosine 5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in a de novo synthesis of guanine nucleotides. The inhibition of IMPDH activity leads to depletion of the guanine nucleotide pool and the consequent attenuation of GTPdependent cellular processes, inducing cell cycle arrest, and the interruption of DNA and RNA synthesis. Since these processes are essential for the normal as well as mitogenic functions of all cells, the inhibition of IMPDH probably represents the central mechanism of ribavirin action, including its cytotoxic effect. Recent data suggest that ribavirin may also be effective as an anti-cancer drug. By inhibiting the eukaryotic translation initiation factor e4E (eIF4E), ribavirin impedes eIF4E-mediated oncogenic transformation. Additionally, immunomodulatory and immunosuppressive actions of ribavirin have been shown in different in vivo and in vitro models of neuroinflammation. Accumulating evidence points to important cell type-specific differences in response to ribavirin that may arise from cell specific variations in ribavirin metabolism, as well as the functional status of targeted cells. This chapter reviews the antiviral, anticancer and anti-inflammatory activities of ribavirin and its metabolites, and discusses the possible mechanisms of action. Frontiers in Drug Design & Discovery Volume 9. Sharjah: Bentham Science Publishers; 2018. p. 113–75.

Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling.

Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis. Serbian Ministry for Education and Science Project {[}III41014]; DAAD; Serbian Ministry of Education and Science; DAAD - Scientific Internship in Germany for Undergraduates and Graduates from Serbia

BACKGROUND: Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS: We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS: We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients' pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells. CONCLUSIONS: We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions. This is a post-peer-review, pre-copyedit version of an article published in Neurochemical Research. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s11064-018-2509-8]

Multiple sclerosis (MS) is a chronic, progressive disorder of the central nervous system (CNS) that affects more than two million people worldwide. Several animal models resemble MS pathology; the most employed are experimental autoimmune encephalomyelitis (EAE) and toxin- and/or virus-induced demyelination. In this review we will summarize our knowledge on the utility of different animal models in MS research. Although animal models cannot replicate the complexity and heterogeneity of the MS pathology, they have proved to be useful for the development of several drugs approved for treatment of MS patients. This review focuses on EAE because it represents both clinical and pathological features of MS. During the past decades, EAE has been effective in illuminating various pathological processes that occur during MS, including inflammation, CNS penetration, demyelination, axonopathy, and neuron loss mediated by immune cells. "This is the peer reviewed version of the following article: Bjelobaba I, Begovic-Kupresanin V, Pekovic S, Lavrnja I. Animal models of multiple sclerosis: Focus on experimental autoimmune encephalomyelitis. J Neurosci Res. 2018, which has been published in final form at [https://doi.org/10.1002/jnr.24224]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), acting via its receptors (GnRHRs) expressed in pituitary gonadotrophs, represents a critical molecule in control of reproductive functions in all vertebrate species. GnRH-activated receptors regulate synthesis of gonadotropins in a frequency-dependent manner. The number of GnRHRs on the plasma membrane determines the responsiveness of gonadotrophs to GnRH and varies in relation to age, sex, and physiological status. This is achieved by a complex control that operates at transcriptional, translational, and post-translational levels. This review aims to overview the mechanisms of GnRHR gene (Gnrhr) transcription in mammalian gonadotrophs. In general, Gnrhr exhibits basal and regulated transcription activities. Basal Gnrhr transcription appears to be an intrinsic property of native and immortalized gonadotrophs that secures the presence of a sufficient number GnRHRs to preserve their functionality independently of the status of regulated transcription. On the other hand, regulated transcription modulates GnRHR expression during development, reproductive cycle, and aging. GnRH is crucial for regulated Gnrhr transcription in native gonadotrophs but is ineffective in immortalized gonadotrophs. In rat and mouse, both basal and GnRH-induced Gnrhr transcription rely primarily on the protein kinase C signaling pathway, with subsequent activation of mitogen activated protein kinases. However, continuous GnRH application, after a transient stimulation, shuts off regulated but not basal transcription, suggesting that different branches of this signaling pathway control transcription. Pituitary adenylate cyclase activating polypeptide, but not activins, contributes to the regulated transcription utilizing the protein kinase A signaling pathway, whereas a mechanism by which steroid hormones modulate Gnrhr transcription has not been characterized.

Ectonucleotidases are cell surface-located enzymes responsible for the extracellular degradation of nucleotides. They are comprised of several protein families: ectonucleoside triphosphate diphosphohydrolases (E-NTPDase), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPases) and ecto-5’-nucleotidase. Previously we showed that cortical stab injury alters ectonucleotidase activities in the rat brain, but that the specific enzymes responsible for these changes were not identified. In this study we investigated the gene expression of the specific ectonucleotidase enzymes, NTPDase1- 3, NPP1-3 and ecto-5’-nucleotidase, two and seven days after cortical stab injury in rats, using real-time PCR. Two days after the injury we observed only one significant change: the downregulation in NTPDase2 mRNA expression. Our results indicate that traumatic brain injury induces significant upregulation of NTPDase1, NTPDase2 and ecto-5’-nucleotidase transcripts, and the downregulation of NPP1, seven days after the injury. Thus, traumatic brain injury has diverse impacts on ectonucleotidases gene expression, which may be reflected in the enzyme activities and extracellular nucleotide concentrations in the perilesional tissue. [Projekat Ministarstva nauke Republike Srbije, br. III41014]

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Biochimica et Biophysica Acta (BBA) - Biomembranes (2017)

Persistent neuroinflammation is now recognized as a chief pathological component of practically all neurodegenerative diseases. Neuroinflammation in the central nervous system (CNS), is accompanied with immune responses of glial cells. Glial cells respond to pathological stimuli through antigen presentation, and cytokine and chemokine signaling. Therefore, limiting CNS inflammation represents prospective therapeutic approach in diseases like Alzheimer's, amyotrophic lateral sclerosis, Parkinson's, ischemia, various psychiatric disorders and Multiple sclerosis (MS). As a complex disease, MS is characterized by neuroinflamation, demyelination and sequential axonal loss. Due to unknown etiology and the heterogeneous presentation of the disease, MS is hard to treat and the search for potential therapeutics is wide and meticulous. However, finding a proper anti-neuroinflammatory drug may bring an advance in selecting noveltreatment regimens of ample of neurodegenerative diseases and neurological disorders. The present review gives the overview of the existing and potential therapies in MS, aimed to modulate neuroinflammation and ensure neuroprotection. Current Pharmaceutical Design (2017), 23(5): 693-730

Summary Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury.

Acetylcholine (ACh) has been established as a paracrine factor in the anterior pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order beta 2 > beta 1 = alpha 9 > alpha 4 in cultured rat pituitary cells. The expression of the subunits in immortalized L beta T2 mouse gonadotrophs followed the order beta 2 > alpha 4 = alpha 1. M4 > M3 muscarinic receptor mRNA were also identified in pituitary and L beta T2 cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of alpha 9 and alpha 4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of beta 2-subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity, and Ca2+ influx were identified in single gonadotrophs and L beta T2 cells. In both cell types, the M3 receptor-mediated, phospholipase C-dependent Ca2+ mobilization activated an outward apamin-sensitive K+ current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release in a pertussis toxin-sensitive manner. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4 receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs. (Endocrinology 154: 421-433, 2013) Intramural Research Program of the National Institute of Child Health and Human Development; Internal Grant Agency of Academy of Sciences [IAA500110910, P304/12/G069]; Serbian Ministry of Education, Science, and Technological Development [41014]

Injury and other pathological conditions induce a massive release of ATP and ADP that initiate an immune response. Extracellular nucleotides are degraded by ectonucleotidases: enzymes from E-NTPDase and E-NPP families sequentially hydrolyze ATP and ADP to AMP, which is further hydrolyzed by ecto-5’-nucleotidase to adenosine that exerts suppressive effects on immune cells. We investigated the ectonucleotidase activities of peripheral lymphocytes at different post-injury times after an unilateral brain injury in the rat. Significant and dynamic changes in the lymphocytic ectonucleotidase activities were obtained. ATP- and ADP-hydrolysis changes, together with their calculated ratios, indicate the major contribution of E-NTPDase 1 and its comparable upregulation between sham operation and injury. AMP hydrolysis changes were more brain-injury specific, with a longer-lasting lymphocytic response induced by cortical stab injury (CSI). In summary, CSI and sham operation induce the upregulation of the whole enzyme chain for adenine nucleotide hydrolysis in lymphocytes, suggesting an important roles of ectonucleotidases in the course of recovery after brain injury. [Projekat Ministarstva nauke Republike Srbije, br. III41014]

Cushing’s disease (CD) in children is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Germline or somatic mutations in genes such as MEN1, CDKIs, AIP, and USP8 have been identified in pediatric CD, but the genetic defects in a significant percentage of cases are still unknown. In this study, we investigated the orphan G-protein-coupled receptor GPR101, a gene known to be involved in somatotropinomas, for its possible involvement in corticotropinomas. We performed GPR101 sequencing, expression analyses by RT-qPCR and immunostaining, and functional studies (cell proliferation, pituitary hormone secretion, and cAMP measurement) in a series of patients with sporadic CD secondary to ACTH-secreting adenomas in whom we extracted DNA from peripheral blood and pituitary tumor samples (n=36). No increased GPR101 expression was observed in tumors compared with normal pituitary (NP) tissues, nor did we find a correlation between GPR101 and ACTH expression levels. Sequence analysis revealed a very rare germline heterozygous GPR101 variant (p.G31S) in one patient with CD. Overexpression of the p.G31S variant did not lead to increased growth and proliferation, although modest effects on cAMP signaling were observed. GPR101 is not overexpressed in ACTH-secreting tumors compared with NPs. In conclusion, rare germline GPR101 variant was found in one patient with CD, but in vitro studies did not support a consistent pathogenic effect. GPR101 is unlikely to be involved in the pathogenesis of CD.

We recently showed that Xq26.3 microduplications cause X-linked acrogigantism (X-LAG). X-LAG patients mainly present with growth hormone and prolactin-secreting adenomas and share a minimal duplicated region containing at least four genes. GPR101 was the only gene highly expressed in their pituitary lesions, but little is known about its expression patterns. In this work, GPR101 transcripts were characterized in human tissues by 5′-Rapid Amplification of cDNA Ends (RACE) and RNAseq, while the putative promoter was bioinformatically predicted. We investigated GPR101 mRNA and protein expression by RT-quantitative PCR (qPCR), whole-mount in situ hybridization, and immunostaining, in human, rhesus monkey, rat and zebrafish. We identified four GPR101 isoforms characterized by different 5′-untranslated regions (UTRs) and a common 6.1kb long 3′UTR. GPR101 expression was very low or absent in almost all adult human tissues examined, except for specific brain regions. Strong GPR101 staining was observed in human fetal pituitary and during adolescence, whereas very weak/absent expression was detected during childhood and adult life. In contrast to humans, adult monkey and rat pituitaries expressed GPR101, but in different cell types. Gpr101 is expressed in the brain and pituitary during rat and zebrafish development; in rat pituitary, Gpr101 is expressed only after birth and shows sexual dimorphism. This study shows that different GPR101 transcripts exist and that the brain is the major site of GPR101 expression across different species, although divergent species- and temporal-specific expression patterns are evident. These findings suggest an important role for GPR101 in brain and pituitary development and likely reflect the very different growth, development and maturation patterns among species.

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LβT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LβT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development.

Nucleoside triphosphate diphosphohydrolase3 (NTPDase3) is membrane-bound ecto-enzyme which hydrolyzes extracellular ATP, thus modulating the function of purinergic receptors and the pattern of purinergic signaling. Here we analyzed the developmental expression of NTPDase3 in female hypothalamus, cerebral cortex and hippocampal formation at different postnatal ages (PD7-PD90) by qRT-PCR and immunohistochemistry. In hypothalamus and hippocampus, a similar developmental profile was seen: NTPDase3 gene expression was stable during postnatal development and increased in adults. In the cortex, upregulation of NTPDase3 mRNA expression was seen at PD15 and further increase was evidenced in adults. Immunohistochemical analysis at PD7 revealed faint neuronal NTPDase3 localization in a dorsal hypothalamus. The immunoreactivity (ir) gradually increased in PD15 and PD20, in clusters of cells in the lateral, ventral and dorsomedial hypothalamus. Furthermore, in PD20 animals, NTPDase3-ir was detected on short fibers in the posterior hypothalamic area, while in PD30 the fibers appeared progressively longer and markedly varicose. In adults, the strongest NTPDase3-ir was observed in collections of cells in dorsomedial hypothalamic nucleus, dorsal and lateral hypothalamus and in several thalamic areas, whereas the varicose fibers traversed entire diencephalon, particularly paraventricular thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus and the prefornical part of the lateral hypothalamus. The presumably ascending NTPDase3-ir fibers were first observed in PD20; their density and the varicose appearance increased until the adulthood. Prominent enhancement of NTPDase3-ir in the hypothalamus coincides with age when animals acquire diurnal rhythms of sleeping and feeding, supporting the hypothesis that this enzyme may be involved in regulation of homeostatic functions. (C) 2016 Elsevier B.V. All rights reserved.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), a human inflammatory and demyelinating disease. Microglia and astrocytes are glial cells of the central nervous system (CNS) that play a dual role in MS and EAE pathology. The aim of this study was to examine the effect of combined treatment with two nucleoside analogues, ribavirin and tiazofurin, on microglia and astrocytes in actively induced EAE. Therapeutic treatment with a combination of these two nucleoside analogues reduced disease severity, mononuclear cell infiltration and demyelination. The obtained histological results indicate that ribavirin and tiazofurin changed activated microglia into an inactive type and attenuated astrocyte reactivity at the end of the treatment period. Since reduction of reactive microgliosis and astrogliosis correlated with EAE suppression, the present study also suggests that the obtained beneficial effect of ribavirin and tiazofurin could be a consequence of their action inside as well as outside the CNS. [Acknowledgments. This work was supported by the Serbian Ministry of Education and Science, Project No: III41014.]

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate-limiting enzyme in the ectonucleotidase pathway is ecto-5 '-nucleotidase (e-5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e-5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e-5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double-immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e-5NT-positive astrocytes, accounting for up-regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e-5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI. (C) 2011 Wiley-Liss, Inc. Serbian Ministry of Science and Technology [41014]

The ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), an enzyme involved in degradation of extracellular adenosine triphosphate (ATP), is expressed on nerve fibers in different brain regions, including cortex. Here we studied the expression and role of this enzyme after unilateral cortical stab injury in rats. In cortical sections of control rats, NTPDase3 immunoreactivity was associated with two types of fibers: thin processes, occasionally with small mushroom-like protrusions and slightly thicker fibers with more pronounced and more frequent varicosities, whereas immunopositive neuronal perycaria were never observed. Although NTPDase3-positive thin processes and thicker fibers, by general appearance, size and shape, could be dendrites and axons, respectively, they were never immunopositive for microtubule associated protein-2 or neurofilament H subunit. Cortical stab injury induced rapid (within 4 hours) focal varicose swelling that evolved over time to prominent beading of NTPDase3-positive fibers. The NTPDase3-positive fibers in all experimental groups also abundantly express NTPDase1, ecto-5'-nucleotidase and P2X2 receptor channels. Because the brain injury causes a massive ATP release, it is reasonable to conclude that purinoreceptors and ectonucleotidases play an important role in the process of neuritic beading. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Serbian Ministry of Science and Technology [143005]