Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, ... more Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG,...
Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to... more Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to pathology progression. It is a feature of many neurological disorders, most common being Alzheimer’s disease (AD). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive stimulation which modulates excitability of stimulated brain areas through magnetic pulses. Numerous studies indicated beneficial effect of rTMS in several neurological diseases, including AD, however, exact mechanism are yet to be elucidated. We aimed to evaluate the effect of intermittent theta burst stimulation (iTBS), an rTMS paradigm, on behavioral, neurochemical and molecular level in trimethyltin (TMT)-induced Alzheimer’s-like disease model. TMT acts as a neurotoxic agent targeting hippocampus causing cognitive impairment and neuroinflammation, replicating behavioral and molecular aspects of AD. Male Wistar rats were divided into four experimental groups–controls, rats subjected to a single dose of...
The present study demonstrates altered topographic distribution and enhanced neuronal expression ... more The present study demonstrates altered topographic distribution and enhanced neuronal expression of major adenosine-metabolizing enzymes, i.e. ecto-5'-nucleotidase (eN) and tissue non-specific alkaline phosphatase (TNAP), as well as adenosine receptor subtype A2A in the hippocampus and cortex of male rats from early to late adulthood (3, 6, 12 and 15 months old males). The significant effect of age was demonstrated for the increase in the activity and the protein expression of eN and TNAP. At 15-m, enzyme histochemistry demonstrated enhanced expression of eN in synapse-rich hippocampal and cortical layers, whereas the upsurge of TNAP was observed in the hippocampal and cortical neuropil, rather than in cells and layers where two enzymes mostly reside in 3-m old brain. Furthermore, a dichotomy in A1R and A2AR expression was demonstrated in the cortex and hippocampus from early to late adulthood. Specifically, a decrease in A1R and enhancement of A2AR expression were demonstrated by immunohistochemistry, the latter being almost exclusively localized in hippocampal pyramidal and cortical superficial cell layers. We did not observe any glial upregulation of A2AR, which was common for both advanced age and chronic neurodegeneration. Taken together, the results imply that the adaptative changes in adenosine signaling occurring in neuronal elements early in life may be responsible for the later prominent glial enhancement in A2AR-mediated adenosine signaling, and neuroinflammation and neurodegeneration, which are the hallmarks of both advanced age and age-associated neurodegenerative diseases.
Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated infl... more Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated inflammation in the central nervous system. Purinergic signaling is critically involved in MS-associated neuroinflammation and its most widely applied animal model—experimental autoimmune encephalomyelitis (EAE). A promising but poorly understood approach in the treatment of MS is repetitive transcranial magnetic stimulation. In the present study, we aimed to investigate the effect of continuous theta-burst stimulation (CTBS), applied over frontal cranial bone, on the adenosine-mediated signaling system in EAE, particularly on CD73/A2AR/A1R in the context of neuroinflammatory activation of glial cells. EAE was induced in two-month-old female DA rats and in the disease peak treated with CTBS protocol for ten consecutive days. Lumbosacral spinal cord was analyzed immunohistochemically for adenosine-mediated signaling components and pro- and anti-inflammatory factors. We found downregulated IL-...
Trimethyltin chloride (TMT) is an organotin neurotoxicant that selectively targets the hippocampu... more Trimethyltin chloride (TMT) is an organotin neurotoxicant that selectively targets the hippocampus, and induces selective and progressive neuronal loss, gliosis, neuroinflammation and cognitive deficits, thus resembling critical features of Alzheimer’s diseases (AD). Flaxseed oil (FSO) is anti-inflammatory agent with potent neuroprotective properties. Therefore, the presented study was designed to evaluate the protective effects of flaxseed oil (FSO) continuous pretreatment to alleviate TMT- (8 mg/kg) induced neurodegeneration. Ovariectomized (OVX) female rats were pretreated with FSO (1 ml/kg, orally) for two weeks. At day 14, part of animals received single dose of TMT (8 mg/kg, i.p.) and application of FSO continued for seven more days. Data have convincingly shown that FSO counteracted TMT effects. Specifically, daily administration of FSO improved TMT- induced behavioral manifestations manifested as hyper-excitability, and hyper-responsiveness, reduced neuronal loss, ameliorate...
The present study examined the involvement of purinergic signaling components in the rat model of... more The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early...
BackgroundThe present study aims to explore the involvement of purinergic signaling in the rodent... more BackgroundThe present study aims to explore the involvement of purinergic signaling in the rodent model of hippocampal degeneration induced by trimethyltin (TMT), which results in behavioral and neurological dysfunction similar to Alzheimer’s disease. Our study has provided novel evidence that TMT induced extracellular depositions of amyloid β, which might be the cause of the well-defined progressive hippocampal neurodegeneration and gliosis. MethodsWe have applied enzyme histochemistry and immunohistochemistry to study spatial and temporal patterns of ectonucleotidase NTPDase1/CD39 and eN/CD73 expression, gene expression analysis and immunochemistry to analyze cellular localization of select purinoreceptors and pro-inflammatory cytokines previously associated with microglia and astrocytes activation. ResultsOur study demonstrated that all Iba1-ir microglial cells, irrespective of the cell shape and localization, upregulated NTPDase1/CD39, while the induction of eN/CD73 has been obs...
Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional tran... more Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional transition referred as reactive gliosis. Every acute or chronic disorder is accompanied by reactive gliosis, which could be categorized as detrimental (A1) of beneficial (A2) for nervous tissue. Another signature of pathological astrocyte activation is disturbed Ca 2+ homeostasis, a common denominator of neurodegenerative diseases. Deregulation of Ca + signaling further contributes to production of pro-inflammatory cytokines and reactive oxygen species. Trimethyltin (TMT) intoxication is a widely used model of hippocampal degeneration, sharing behavioral and molecular hallmarks of Alzheimer’s disease (AD), thus representing a useful model of AD-like pathology. However, the role of astrocyte in the etiopathology of TMT-induced degeneration as well as in AD is not fully understood. In an effort to elucidate the role of astrocytes in such pathological processes, we examined in vitro effects of TMT on primary cortical astrocytes. The application of a range of TMT concentrations (5, 10, 50, and 100 μM) revealed changes in [Ca 2 + ] i in a dose-dependent manner. Specifically, TMT-induced Ca 2 + transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca 2 + and disturbed antioxidative defense of astrocyte in several time points (4, 6, and 24 h) after 10 μM TMT intoxication, inducing oxidative and nitrosative stress. Chronic exposure (24 h) to 10 μM TMT induced strong upregulation of main pro-inflammatory factors, components of signaling pathways in astrocyte activation, A1 markers, and VGCC. Taken together, our results provide an insight into cellular and molecular events of astrocyte activation in chronic neuroinflammation.
Two distinct hippocampal astrocyte morphotypes reveal subfield-different fate during neurodegener... more Two distinct hippocampal astrocyte morphotypes reveal subfield-different fate during neurodegeneration induced by trimethyltin intoxication,
Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and ad... more 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
Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as... more Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory tone within hippocampal neuronal networks. ATP and adenosine are interconnected by ectonucleotidase enzymes, which convert ATP to adenosine. Existing data point to the converging roles of ovarian steroids and purinergic signaling in synapse formation and refinement and synapse activity in the hippocampus. Therefore, in the present study, we have used enzyme histochemistry and expression analysis to obtain data on spatial distribution and expression of ectoenzymes NTPDase1, NTPDase2, and ecto-5′-nucleotidase (eN) after removal of ovaries (OVX) and estradiol replacement (E2) in female rat hippocampus. The results show that target ectonucleotidases are predominantly localized in synapse-rich hippocampal layers. The most represented NTPDase in the hippocampal tissue is NTPDase2, being at the same time the mostly affected ectonucleotidase by OVX and E2. Specifically, OVX decreases the expression of NTPDase2 and eN, whereas E2 restores their expression to control level. Impact of OVX and E2 on ectonucleotidase expression was also examined in purified synaptosome (SYN) and gliosome (GLIO) fractions. Data reveal that SYN expresses NTPDase1 and NTPDase2, both of which are reduced following OVX and restored with E2. GLIO exhibits NTPDase2-mediated ATP hydrolysis, which falls in OVX, and recovers by E2. These changes in the activity occur without parallel changes in NTPDase2-protein abundance. The same holds for eN. The lack of correlation between NTPDase2 and eN activities and their respective protein abundances suggest a non-genomic mode of E2 action, which is studied further in primary astrocyte culture. Since ovarian steroids shape hippocampal synaptic networks and regulate ectonucleotidase activities, it is possible that cognitive deficits seen after ovary removal may arise from the loss of E2 modulatory actions on ectonucleotidase expression in the hippocampus.
17b-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts ... more 17b-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts upon estradiol receptors ERa and ERb, influencing target gene expression and/or modulates intracellular signaling cascades. Another potent modulator of hippocampal function is nucleoside adenosine, the final product of ectonucleotidase cascade, enzymes which hydrolyze extracellular ATP to adenosine. The last and rate-limiting step of the hydrolysis is catalyzed by membrane-bound ecto-5 0-nucleotidase (eN). Previous findings obtained on adenosine metabolism in brain suggest that eN may be modulated by ovarian steroids. Therefore, the present study reports that the activity and protein abundance of membrane-bound eN fluctuates across the estrus cycle in the hippocampal synaptosomes of female rats. Further, we analyzed the role of E2 and its intracellular receptors on the expression of eN in ovariectomized females. We found that E2 upregulated eN activity and protein abundance in the hippocampal synaptosomes. Application of nonspecific ER antagonist, ICI 182,780 and selective ERa and ERb agonists, PPT and DPN, respectively, demonstrated the involvement of both receptor subtypes in observed actions. Selective ERa receptor agonist, PPT, induced upregulation of both the protein level and activity of eN, while application of selective ERb receptor agonist, DPN, increased only the activity of eN. In both cases, E2 entered into the intracellular compartment and activated ER(s), which was demonstrated by membrane impermeable E2-BSA conjugate. Together these results imply that E2induced effects on connectivity and functional properties of the hippocampal synapses may be in part mediated through observed effect on eN.
17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling c... more 17β-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.
related differences in modulating effects of female sex steroids on ecto-5′-nucleotidase expressi... more related differences in modulating effects of female sex steroids on ecto-5′-nucleotidase expression in the rat cerebral cortex and hippocampus, General and Comparative Endocrinology (2016),
Although a substantial number of pre-clinical and experimental studies have investigated effects ... more Although a substantial number of pre-clinical and experimental studies have investigated effects of 17β-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3 μg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochrome C, caspase 3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors-Bcl-2, Bax, and caspase 3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17β-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17β-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17β-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.
Estradiol exerts its physiological function by 2 main pathways: genomic, via the transcription of... more Estradiol exerts its physiological function by 2 main pathways: genomic, via the transcription of certain target genes, and nongenomic, which is independent of direct gene activation . While genomic effects are quite well described , nongenomic estradiol effects are insufficiently known and studied. In different brain regions estradiol affects, in a nongenomic manner, the growth of dendritic branches, activity of GABA receptors, and the concentration of sodium and potassium ions (
To study time-dependent and gender-specific intracellular and biochemical mechanisms that lead to... more To study time-dependent and gender-specific intracellular and biochemical mechanisms that lead to neurodegeneration due to moderate but persistent reduction of cerebral blood flow, 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. Animals were sacrificed 3, 7 or 90 days following the insult. Expression of several apoptotic proteins in synaptic fractions along with Fluoro-Jade B staining and DNA fragmentation assay were used to estimate the apoptotic processes and potential neurodegeneration in cerebral cortex. Data suggest a time-specific increase of Bax as well as time- and gender-associated downregulation in protein expression of Bcl-2, up-regulation of procaspase 3, accompanied with increased cleavage of procaspase 3 and PARP in synaptic terminals. Furthermore, time- but not gender-specific neurodegeneration was observed. Our findings suppo...
Disturbance in blood circulation is associated with numerous pathological conditions characterize... more Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 μg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early postischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.
Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, ... more Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG,...
Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to... more Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to pathology progression. It is a feature of many neurological disorders, most common being Alzheimer’s disease (AD). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive stimulation which modulates excitability of stimulated brain areas through magnetic pulses. Numerous studies indicated beneficial effect of rTMS in several neurological diseases, including AD, however, exact mechanism are yet to be elucidated. We aimed to evaluate the effect of intermittent theta burst stimulation (iTBS), an rTMS paradigm, on behavioral, neurochemical and molecular level in trimethyltin (TMT)-induced Alzheimer’s-like disease model. TMT acts as a neurotoxic agent targeting hippocampus causing cognitive impairment and neuroinflammation, replicating behavioral and molecular aspects of AD. Male Wistar rats were divided into four experimental groups–controls, rats subjected to a single dose of...
The present study demonstrates altered topographic distribution and enhanced neuronal expression ... more The present study demonstrates altered topographic distribution and enhanced neuronal expression of major adenosine-metabolizing enzymes, i.e. ecto-5'-nucleotidase (eN) and tissue non-specific alkaline phosphatase (TNAP), as well as adenosine receptor subtype A2A in the hippocampus and cortex of male rats from early to late adulthood (3, 6, 12 and 15 months old males). The significant effect of age was demonstrated for the increase in the activity and the protein expression of eN and TNAP. At 15-m, enzyme histochemistry demonstrated enhanced expression of eN in synapse-rich hippocampal and cortical layers, whereas the upsurge of TNAP was observed in the hippocampal and cortical neuropil, rather than in cells and layers where two enzymes mostly reside in 3-m old brain. Furthermore, a dichotomy in A1R and A2AR expression was demonstrated in the cortex and hippocampus from early to late adulthood. Specifically, a decrease in A1R and enhancement of A2AR expression were demonstrated by immunohistochemistry, the latter being almost exclusively localized in hippocampal pyramidal and cortical superficial cell layers. We did not observe any glial upregulation of A2AR, which was common for both advanced age and chronic neurodegeneration. Taken together, the results imply that the adaptative changes in adenosine signaling occurring in neuronal elements early in life may be responsible for the later prominent glial enhancement in A2AR-mediated adenosine signaling, and neuroinflammation and neurodegeneration, which are the hallmarks of both advanced age and age-associated neurodegenerative diseases.
Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated infl... more Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated inflammation in the central nervous system. Purinergic signaling is critically involved in MS-associated neuroinflammation and its most widely applied animal model—experimental autoimmune encephalomyelitis (EAE). A promising but poorly understood approach in the treatment of MS is repetitive transcranial magnetic stimulation. In the present study, we aimed to investigate the effect of continuous theta-burst stimulation (CTBS), applied over frontal cranial bone, on the adenosine-mediated signaling system in EAE, particularly on CD73/A2AR/A1R in the context of neuroinflammatory activation of glial cells. EAE was induced in two-month-old female DA rats and in the disease peak treated with CTBS protocol for ten consecutive days. Lumbosacral spinal cord was analyzed immunohistochemically for adenosine-mediated signaling components and pro- and anti-inflammatory factors. We found downregulated IL-...
Trimethyltin chloride (TMT) is an organotin neurotoxicant that selectively targets the hippocampu... more Trimethyltin chloride (TMT) is an organotin neurotoxicant that selectively targets the hippocampus, and induces selective and progressive neuronal loss, gliosis, neuroinflammation and cognitive deficits, thus resembling critical features of Alzheimer’s diseases (AD). Flaxseed oil (FSO) is anti-inflammatory agent with potent neuroprotective properties. Therefore, the presented study was designed to evaluate the protective effects of flaxseed oil (FSO) continuous pretreatment to alleviate TMT- (8 mg/kg) induced neurodegeneration. Ovariectomized (OVX) female rats were pretreated with FSO (1 ml/kg, orally) for two weeks. At day 14, part of animals received single dose of TMT (8 mg/kg, i.p.) and application of FSO continued for seven more days. Data have convincingly shown that FSO counteracted TMT effects. Specifically, daily administration of FSO improved TMT- induced behavioral manifestations manifested as hyper-excitability, and hyper-responsiveness, reduced neuronal loss, ameliorate...
The present study examined the involvement of purinergic signaling components in the rat model of... more The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early...
BackgroundThe present study aims to explore the involvement of purinergic signaling in the rodent... more BackgroundThe present study aims to explore the involvement of purinergic signaling in the rodent model of hippocampal degeneration induced by trimethyltin (TMT), which results in behavioral and neurological dysfunction similar to Alzheimer’s disease. Our study has provided novel evidence that TMT induced extracellular depositions of amyloid β, which might be the cause of the well-defined progressive hippocampal neurodegeneration and gliosis. MethodsWe have applied enzyme histochemistry and immunohistochemistry to study spatial and temporal patterns of ectonucleotidase NTPDase1/CD39 and eN/CD73 expression, gene expression analysis and immunochemistry to analyze cellular localization of select purinoreceptors and pro-inflammatory cytokines previously associated with microglia and astrocytes activation. ResultsOur study demonstrated that all Iba1-ir microglial cells, irrespective of the cell shape and localization, upregulated NTPDase1/CD39, while the induction of eN/CD73 has been obs...
Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional tran... more Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional transition referred as reactive gliosis. Every acute or chronic disorder is accompanied by reactive gliosis, which could be categorized as detrimental (A1) of beneficial (A2) for nervous tissue. Another signature of pathological astrocyte activation is disturbed Ca 2+ homeostasis, a common denominator of neurodegenerative diseases. Deregulation of Ca + signaling further contributes to production of pro-inflammatory cytokines and reactive oxygen species. Trimethyltin (TMT) intoxication is a widely used model of hippocampal degeneration, sharing behavioral and molecular hallmarks of Alzheimer’s disease (AD), thus representing a useful model of AD-like pathology. However, the role of astrocyte in the etiopathology of TMT-induced degeneration as well as in AD is not fully understood. In an effort to elucidate the role of astrocytes in such pathological processes, we examined in vitro effects of TMT on primary cortical astrocytes. The application of a range of TMT concentrations (5, 10, 50, and 100 μM) revealed changes in [Ca 2 + ] i in a dose-dependent manner. Specifically, TMT-induced Ca 2 + transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca 2 + and disturbed antioxidative defense of astrocyte in several time points (4, 6, and 24 h) after 10 μM TMT intoxication, inducing oxidative and nitrosative stress. Chronic exposure (24 h) to 10 μM TMT induced strong upregulation of main pro-inflammatory factors, components of signaling pathways in astrocyte activation, A1 markers, and VGCC. Taken together, our results provide an insight into cellular and molecular events of astrocyte activation in chronic neuroinflammation.
Two distinct hippocampal astrocyte morphotypes reveal subfield-different fate during neurodegener... more Two distinct hippocampal astrocyte morphotypes reveal subfield-different fate during neurodegeneration induced by trimethyltin intoxication,
Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and ad... more 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
Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as... more Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory tone within hippocampal neuronal networks. ATP and adenosine are interconnected by ectonucleotidase enzymes, which convert ATP to adenosine. Existing data point to the converging roles of ovarian steroids and purinergic signaling in synapse formation and refinement and synapse activity in the hippocampus. Therefore, in the present study, we have used enzyme histochemistry and expression analysis to obtain data on spatial distribution and expression of ectoenzymes NTPDase1, NTPDase2, and ecto-5′-nucleotidase (eN) after removal of ovaries (OVX) and estradiol replacement (E2) in female rat hippocampus. The results show that target ectonucleotidases are predominantly localized in synapse-rich hippocampal layers. The most represented NTPDase in the hippocampal tissue is NTPDase2, being at the same time the mostly affected ectonucleotidase by OVX and E2. Specifically, OVX decreases the expression of NTPDase2 and eN, whereas E2 restores their expression to control level. Impact of OVX and E2 on ectonucleotidase expression was also examined in purified synaptosome (SYN) and gliosome (GLIO) fractions. Data reveal that SYN expresses NTPDase1 and NTPDase2, both of which are reduced following OVX and restored with E2. GLIO exhibits NTPDase2-mediated ATP hydrolysis, which falls in OVX, and recovers by E2. These changes in the activity occur without parallel changes in NTPDase2-protein abundance. The same holds for eN. The lack of correlation between NTPDase2 and eN activities and their respective protein abundances suggest a non-genomic mode of E2 action, which is studied further in primary astrocyte culture. Since ovarian steroids shape hippocampal synaptic networks and regulate ectonucleotidase activities, it is possible that cognitive deficits seen after ovary removal may arise from the loss of E2 modulatory actions on ectonucleotidase expression in the hippocampus.
17b-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts ... more 17b-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts upon estradiol receptors ERa and ERb, influencing target gene expression and/or modulates intracellular signaling cascades. Another potent modulator of hippocampal function is nucleoside adenosine, the final product of ectonucleotidase cascade, enzymes which hydrolyze extracellular ATP to adenosine. The last and rate-limiting step of the hydrolysis is catalyzed by membrane-bound ecto-5 0-nucleotidase (eN). Previous findings obtained on adenosine metabolism in brain suggest that eN may be modulated by ovarian steroids. Therefore, the present study reports that the activity and protein abundance of membrane-bound eN fluctuates across the estrus cycle in the hippocampal synaptosomes of female rats. Further, we analyzed the role of E2 and its intracellular receptors on the expression of eN in ovariectomized females. We found that E2 upregulated eN activity and protein abundance in the hippocampal synaptosomes. Application of nonspecific ER antagonist, ICI 182,780 and selective ERa and ERb agonists, PPT and DPN, respectively, demonstrated the involvement of both receptor subtypes in observed actions. Selective ERa receptor agonist, PPT, induced upregulation of both the protein level and activity of eN, while application of selective ERb receptor agonist, DPN, increased only the activity of eN. In both cases, E2 entered into the intracellular compartment and activated ER(s), which was demonstrated by membrane impermeable E2-BSA conjugate. Together these results imply that E2induced effects on connectivity and functional properties of the hippocampal synapses may be in part mediated through observed effect on eN.
17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling c... more 17β-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.
related differences in modulating effects of female sex steroids on ecto-5′-nucleotidase expressi... more related differences in modulating effects of female sex steroids on ecto-5′-nucleotidase expression in the rat cerebral cortex and hippocampus, General and Comparative Endocrinology (2016),
Although a substantial number of pre-clinical and experimental studies have investigated effects ... more Although a substantial number of pre-clinical and experimental studies have investigated effects of 17β-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3 μg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochrome C, caspase 3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors-Bcl-2, Bax, and caspase 3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17β-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17β-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17β-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.
Estradiol exerts its physiological function by 2 main pathways: genomic, via the transcription of... more Estradiol exerts its physiological function by 2 main pathways: genomic, via the transcription of certain target genes, and nongenomic, which is independent of direct gene activation . While genomic effects are quite well described , nongenomic estradiol effects are insufficiently known and studied. In different brain regions estradiol affects, in a nongenomic manner, the growth of dendritic branches, activity of GABA receptors, and the concentration of sodium and potassium ions (
To study time-dependent and gender-specific intracellular and biochemical mechanisms that lead to... more To study time-dependent and gender-specific intracellular and biochemical mechanisms that lead to neurodegeneration due to moderate but persistent reduction of cerebral blood flow, 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. Animals were sacrificed 3, 7 or 90 days following the insult. Expression of several apoptotic proteins in synaptic fractions along with Fluoro-Jade B staining and DNA fragmentation assay were used to estimate the apoptotic processes and potential neurodegeneration in cerebral cortex. Data suggest a time-specific increase of Bax as well as time- and gender-associated downregulation in protein expression of Bcl-2, up-regulation of procaspase 3, accompanied with increased cleavage of procaspase 3 and PARP in synaptic terminals. Furthermore, time- but not gender-specific neurodegeneration was observed. Our findings suppo...
Disturbance in blood circulation is associated with numerous pathological conditions characterize... more Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 μg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early postischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.
Uploads
Papers by Ivana Grković