Metabolism of extracellular adenine nucleotides in rat hippocampal synaptosomes - sex differences and the roll of female sex steroids

Adenozin-5-trifosfat (ATP) u centralnom nervnom sistemu (CNS) ima ulogu brzog ekscitatornog neurotransmitera i kotransmitera ali i trofičkog faktora, gliotransmitera i signalnog molekula koji učestvuje u komunikaciji između ćelija CNS. Kada se oslobodi u vanćelijski prostor, npr. sinaptičku pukotinu i aktivira odgovarajuće receptore (P1 i P2), ATP se brzo degradira, posredstvom enzima ektonukleotidaza. Primarna uloga ovih enzima je sekvencijalna hidroliza adeninskih nukleotida, kao što su ATP, adenozin-5-difosfat (ADP) i adenozin-5-monofosfat (AMP) čime nastaje nukleozid adenozin, potentni neuromodulator i homeostatski regulator u CNS. S obzirom na to da koncentracije vanćelijskog ATP i adenozina direktno zavise od stepena aktivnosti ektonukleotidaza, svaka promena ekspresije i aktivnosti ovih enzima može biti relevantna u kontekstu fiziologije i patologije. Oskudni literaturni podaci ukazuju da su ektonukleotidaze deo složene molekulske mreže koja je pod uticajem polnih hormona. Kako ženski polni hormoni, pogotovo 17β-estradiol (E2), utiču na gustinu sinapsi, složenost dendritskih grananja i broja trnolikih izraštaja u hipokampusu, ostvarajući tako snažan uticaj na sinaptičku plastičnost, učenje i pamćenje kod pacova oba pola, u ovoj tezi ispitan je uticaj hormonskog statusa i mehanizam regulacije/modulacije sinaptičkih ektonukleotidaza, ektonukleozid 5'-trifosfat difosfohidrolaze 1-3 (NTPD-aza 1-3) i ekto-5'-nukleotidaze (eN) 17β- estradiolom u hipokampusu ženki i mužjaka pacova. Takođe, ispitan je uticaj ponovljenog (sedmodnevnog) tretmana 17-estradiolom (E), E2 i progesteronom (P4) na aktivnost i ekspresiju eN u totalnoj membranskoj frakciji hipokampusa oba pola. Rezultati biohemijskih analiza ukazuju da aktivnost ispitanih ektonukleotidaza diskretno fluktuira tokom estrusnog ciklusa, kao i da uklanjanje jajnika (OVX), primarnog izvora ženskih polnih hormona, smanjuje nivo hidrolize ATP, ADP, AMP u sinaptozomima hipokampusa ženki pacova. Ovi nalazi jasno ukazuju na to da su ektonukleotidaze (NTPDaza 1-3 i eN) pod regulatornom kontrolom endogenih hormona jajnika. Promena stepena hidrolize adeninskih nukleotida uočena je kod OVX ženki nakon jednokratnog tretmana E2, pri čemu je stepen hidrolize ATP i ADP povećan verovatno kao rezultat povećane ekspresije NTPD-aze 1 i 2, dok relativna zastupljenost NTPD-aze 3 ostaje nepromenjena. E2 takođe dovodi do porasta nivoa hidrolize AMP pri čemu reguliše/moduliše eN aktivacijom klasičnih unutarćelijskih estradiolskih receptora (ER i ERβ) koji različitim mehanizmima dovode do povećanja aktivnosti eN u sinaptozomima hipokampusa OVX ženki. E2 moduliše vanćelijski metabolizam adeninskih nukleotida i u sinaptozomima hipokampusa mužjaka pacova. E2 smanjuje proteinsku ekspresiju i/ili aktivnost NTPD-aza 1, 2 i eN, ali ne menja relativnu zastupljenost NTPD-aze 3 u sinaptozomima hipokampusa mužjaka...Adenosine-5´-triphosphate (ATP) is an important extracellular signaling molecule. It acts as neurotransmitter, co-transmitter, gliotransmitter and trophic factor in the central nervous system (CNS). Upon the release, ATP modulates synaptic transmission by acting at either ionotropic (P2X) or metabotropic (P2Y) receptors, and is sequentially catabolized by the action of ectonucleotidases. The first step of ATP inactivation is mediated by the family of ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), which are able to hydrolyze ATP and adenosine-5´-diphosphate (ADP) to adenosine-5´-monophosphate (AMP). The last and the rate-limiting step in of the ATP conversion is catalyzed by ecto-5´- nucleotidase (eN), which hydrolases AMP to adenosine, potent neuromodulator and homeostatic regulator in the CNS. Since ectonucleotidases is crucial for maintaining of physiological levels of extracellular adenine nucleotides in the CNS, any alteration in their activity and expression can be relevant in context of physiology and pathology. Literature data indicate that ectonucleotidases may be part of complex molecular network which may be modulated by ovarian steroids. Based on this findings, and the fact that female sex steroids, particularly 17β-estradiol (E2) play an essential role in the modulation of hippocampal synaptic plasticity therefore influencing hippocampal dependent learning and memory, we hypothesized that steroid hormones, in particular E2 has potential to modulate the activity and expression of ectonucleotidases (NTPDase 1-3 and eN) in hippocampal synaptosomes of male and female rats. Also, the aim of this doctoral dissertation was to determine the impact of steroid hormones on eN after repeated administration of 17α- estradiol, E2 and progesterone (P4) for seven consecutive days in hippocampus of both sexes. It is shown that the activity of the examined ectonucleotidases fluctuates across the estrous cycle in the hippocampal synaptosomes of female rats, while significant reduction level of ATP, ADP and AMP hydrolysis is observed in hippocampal synaptosomes after bilateral ovariectomy. These results clearly indicate that examined ectonucleotidases are regulated/modulated by endogenous female sex steroids. Biochemical analysis indicate that acute E2 treatment in the OVX rats significantly increase the level of ATP and ADP hydrolysis, probably as a result of upregulated NTPDase 1 and 2, while E2 had no effect on NTPDase 3. The level of AMP hydrolysis was also augmented in hippocampal synaptosomes of OVX rats after E2 treatment. Our initial evaluation imply that regulatory E2 action at the activity and protein abundance of eN is mediated by both intracellular estradiol receptors (ERα and ERβ) through different mechanisms in hippocampal synaptosomes of OVX rats..

Time-dependent expression of Bcl-2 and Bax proteins in cortical brain area of adult wistar rats after permanent bilateral occlusions

Model of permanent bilateral occlusion of common carotid arteries (2VO) is generally used to investigate mechanisms of chronic cerebral hypoperfusion that occurs in aging and other neurodegenerative processes. The aim of this study was to determine timedependent modulation of mitochondrial apoptotic signaling in cortical brain area following chronic cerebral hypoperfusion. Using Western blot technique we monitored the changes in the expression of proteins of Bcl-2 family (Bcl-2, Bax) 3, 7 and 90 days following the insult. According to our results the greatest impact of chronic cerebral hipoperfusion occurred on 7th day.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Time-Related Sex Diffrences in Cerebral Hypoperfusion-Induced Brain Injury

Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments), should be tested at this time point

Radiation-mediated modulations of extracelluar nucleotide hydrolysis in adult female rat brain

The present study was performed to investigate whether acute whole-body exposure of female adult rats to low dose (0.5 Gy) of ionizing irradiation (IR) is sufficient to alter ectonucleotidase enzyme activities in the brain. All measurements were done at time points 1, 24 and 72h after irradiation. Neuronal synaptic plasma membranes (SPMs) were isolated from whole brains and enzyme activities were determined by monitoring ATP, ADP and AMP hydrolysis in vitro. Our results indicate that whole-body IR is able to modulate investigated brain enzyme activities in a time-dependent manner.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Erratum to: 17 beta-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes (2016)

17 beta-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.Erratum: [http://vinar.vin.bg.ac.rs/handle/123456789/1460

Ectonucleotidases in the hippocampus: Spatial distribution and expression after ovariectomy and estradiol replacement

Extracellular purine nucleotides, such as adenosine 5′-triphosphate (ATP), are important modulators of hippocampal function and plasticity. In the extracellular space, ATP is inherently short-lived molecule, which undergoes rapid enzymatic degradation to adenosine by ectonucleotidases. Given that ectonucleotidases have distinct and overlapping distribution in the hippocampus, and as ovarian hormones participate in a formation, maturation, and a refinement of synaptic contacts, both during development and in adulthood, the present chapter summarizes known data about spatial distribution of selected ecto-enzymes and estradiol-induced effects on ectonucleotidases in the rat hippocampus

Application of Gray Level Co-Occurrence Matrix Analysis as a New Method for Enzyme Histochemistry Quantification

Enzyme histochemistry is a valuable histological method which provides a connection between morphology, activity, and spatial localization of investigated enzymes. Even though the method relies purely on arbitrary evaluations performed by the human eye, it is still wildly accepted and used in histo(patho)logy. Texture analysis emerged as an excellent tool for image quantification of subtle differences reflected in both spatial discrepancies and gray level values of pixels. The current study of texture analysis utilizes the gray-level co-occurrence matrix as a method for quantification of differences between ecto-5′-nucleotidase activities in healthy hippocampal tissue and tissue with marked neurodegeneration. We used the angular second moment, contrast (CON), correlation, inverse difference moment (INV), and entropy for texture analysis and receiver operating characteristic analysis with immunoblot and qualitative assessment of enzyme histochemistry as a validation. Our results strongly argue that co-occurrence matrix analysis could be used for the determination of fine differences in the enzyme activities with the possibility to ascribe those differences to regions or specific cell types. In addition, it emerged that INV and CON are especially useful parameters for this type of enzyme histochemistry analysis. We concluded that texture analysis is a reliable method for quantification of this descriptive technique, thus removing biases and adding it a quantitative dimension

Two Distinct Hippocampal Astrocyte Morphotypes Reveal Subfield-Different Fate during Neurodegeneration Induced by Trimethyltin Intoxication

Astrocytes comprise a heterogenic group of glial cells, which perform homeostatic functions in the central nervous system. These cells react to all kind of insults by changing the morphology and function that result in a transition from the quiescent to a reactive phenotype. Trimethyltin (TMT) intoxication, which reproduces pathological events in the hippocampus similar to those associated with seizures and cognitive decline, has been proven as a useful model for studying responses of the glial cells to neurodegeneration. In the present study, we have explored morphological varieties of astrocytes in the hippocampal subregions of ovariectomized female rats exposed to TMT. We have demonstrated an early loss of neurons in CA1 and DG subfields. Distinct morphotypes of protoplasmic astrocytes observed in CA1/CA3 and the hilus of control animals developed different responses to TMT intoxication, as assessed by GFAP-immunohistochemistry. In CA1 subregion, GFAP+ astrocytes preserved their domain organization and responded with typical hypertrophy, while the hilar GFAP+ astrocytes developed atrophy-like phenotype and increased expression of vimentin and nestin 7 days after the exposure. Both reactive and atrophied-like astrocytes expressed Kir4.1 in CA1/CA3 and the hilus of DG, respectively, indicating that these cells did not change their potential for normal activity at this time point of pathology. Together, the results demonstrate the persistence of two protoplasmic morphotypes of astrocytes, with distinct appearance, function, and fate after TMT-induced neurodegeneration, suggesting their pleiotropic roles in the hippocampal response to neurodegeneration. © 2019 IBR

Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats

Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance. © 2019 Elsevier B.V

Thermally induced structural transformations of multicomponent Fe72Cu1V4Si15B8 alloy

Thermally induced structural transformations of Fe72Cu1V4Si15B8alloy were examined. Thermal analysis revealed multistep structural stabilization process starting at around 470oC, manifested by two complex exothermic DTA peaks, which were deconvoluted. Microstructure of the as-prepared and thermally treated alloy was studied using XRD. Kinetic triplets of individual steps were determined and further checked by comparing simulated and experimental DTA curves.Physical chemistry 2016 : 13th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 26-30 September 201