Intrinsic ecto-5'-Nucleotidase/A1R Coupling may Confer Neuroprotection to the Cerebellum in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is widely used animal model of multiple sclerosis (MS). The disease is characterized by demyelination and neurodegeneration triggered by infiltrated autoimmune cells and their interaction with astrocytes and microglia. While neuroinflammation is most common in the spinal cord and brainstem, it is less prevalent in the cerebellum, where it predisposes to rapid disease progression. Because the induction and progression of EAE are tightly regulated by adenosinergic signaling, in the present study we compared the adenosine-producing and -degrading enzymes, ecto-5'-nucleotidase (eN/CD73) and adenosine deaminase (ADA), as well as the expression levels of adenosine receptors A1R and A2AR subtypes in nearby areas around the fourth cerebral ventricle-the pontine tegmentum, the choroid plexus (CP), and the cerebellum. Significant differences in histopathological findings were observed between pontine tegmentum and cerebellum on the same horizontal section level. Reactive astrogliosis and massive infiltration of CD4 + cells and macrophages in CP and pontine tegmentum resulted in local demyelination. In cerebellum, there was no evidence of infiltrates, microgliosis and neuroinflammation at the same sectional level. In addition, Bergman glia showed no signs of reactive gliosis. As for adenosinergic signaling, significant upregulation of eN/CD73 was observed in all areas studied, but in association with different adenosine receptor subtypes. In CP and pons, overexpression of eN/CD73 was coupled with induction of A2AR, whereas in cerebellum, a modest increase in eN/CD73 in resident Bergman glia was accompanied by a strong induction of A1R in the same type of astrocytes. Thus, the presence of specialized astroglia and intrinsic differences in adenosinergic signaling may play a critical role in the differential regional susceptibility to EAE inflammation.

Sustained Systemic Antioxidative Effects of Intermittent Theta Burst Stimulation beyond Neurodegeneration: Implications in Therapy in 6-Hydroxydopamine Model of Parkinson's Disease.

Parkinson's disease (PD) is manifested by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and caudoputamen (Cp), leading to the development of motor and non-motor symptoms. The contribution of oxidative stress to the development and progression of PD is increasingly recognized. Experimental models show that strengthening antioxidant defenses and reducing pro-oxidant status may have beneficial effects on disease progression. In this study, the neuroprotective potential of intermittent theta burst stimulation (iTBS) is investigated in a 6-hydroxydopamine (6-OHDA)-induced PD model in rats seven days after intoxication which corresponds to the occurrence of first motor symptoms. Two-month-old male Wistar rats were unilaterally injected with 6-OHDA to mimic PD pathology and were subsequently divided into two groups to receive either iTBS or sham stimulation for 21 days. The main oxidative parameters were analyzed in the caudoputamen, substantia nigra pars compacta, and serum. iTBS treatment notably mitigated oxidative stress indicators, simultaneously increasing antioxidative parameters in the caudoputamen and substantia nigra pars compacta well after 6-OHDA-induced neurodegeneration process was over. Serum analysis confirmed the systemic effect of iTBS with a decrease in oxidative markers and an increase in antioxidants. Prolonged iTBS exerts a modulatory effect on oxidative/antioxidant parameters in the 6-OHDA-induced PD model, suggesting a potential neuroprotective benefit, even though at this specific time point 6-OHDA-induced oxidative status was unaltered. These results emphasize the need to further explore the mechanisms of iTBS and argue in favor of considering it as a therapeutic intervention in PD and related neurodegenerative diseases.

Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation.

Three novel cytosine-derived α,ß-methylene diphosphonates designated MRS4598, MRS4552, and MRS4602 were tested in the range of 1 × 10-9 to 1 × 10-3 M for their efficacy and potency in inhibiting membrane-bound ecto-5'-nucleotidase/CD73 activity in primary astrocytes in vitro. The compounds were also tested for their ability to attenuate the reactive astrocyte phenotype induced by proinflammatory cytokines. The main findings are as follows: A) The tested compounds induced concentration-dependent inhibition of CD73 activity, with maximal inhibition achieved at ∼1 × 10-3M; B) All compounds showed high inhibitory potency, as reflected by IC50 values in the submicromolar range; C) All compounds showed high binding capacity, as reflected by Ki values in the low nanomolar range; D) Among the tested compounds, MRS4598 showed the highest inhibitory efficacy and potency, as reflected by IC50 and Ki values of 0.11 µM and 18.2 nM; E) Neither compound affected astrocyte proliferation and cell metabolic activity at concentrations near to IC50; E) MRS4598 was able to inhibit CD73 activity in reactive astrocytes stimulated with TNF-α and to induce concentration-dependent inhibition of CD73 in reactive astrocytes stimulated with IL-1ß, with an order of magnitude higher IC50 value; F) MRS4598 was the only compound tested that was able to induce shedding of the CD73 from astrocyte membranes and to enhance astrocyte migration in the scratch wound migration assay, albeit at concentration well above its IC50 value. Given the role of CD73 in neurodegenerative diseases, MRS4598, MRS4552, and MRS4602 are promising pharmacological tools for the treatment of neurodegeneration and neuroinflammation.

Intermittent Theta Burst Stimulation Improves Motor and Behavioral Dysfunction through Modulation of NMDA Receptor Subunit Composition in Experimental Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic system, leading to a variety of motor and nonmotor symptoms. The currently available symptomatic therapy loses efficacy over time, indicating the need for new therapeutic approaches. Repetitive transcranial magnetic stimulation (rTMS) has emerged as one of the potential candidates for PD therapy. Intermittent theta burst stimulation (iTBS), an excitatory protocol of rTMS, has been shown to be beneficial in several animal models of neurodegeneration, including PD. The aim of this study was to investigate the effects of prolonged iTBS on motor performance and behavior and the possible association with changes in the NMDAR subunit composition in the 6-hydroxydopamine (6-OHDA)-induced experimental model of PD. Two-month-old male Wistar rats were divided into four groups: controls, 6-OHDA rats, 6-OHDA + iTBS protocol (two times/day/three weeks) and the sham group. The therapeutic effect of iTBS was evaluated by examining motor coordination, balance, spontaneous forelimb use, exploratory behavior, anxiety-like, depressive/anhedonic-like behavior and short-term memory, histopathological changes and changes at the molecular level. We demonstrated the positive effects of iTBS at both motor and behavioral levels. In addition, the beneficial effects were reflected in reduced degeneration of dopaminergic neurons and a subsequent increase in the level of DA in the caudoputamen. Finally, iTBS altered protein expression and NMDAR subunit composition, suggesting a sustained effect. Applied early in the disease course, the iTBS protocol may be a promising candidate for early-stage PD therapy, affecting motor and nonmotor deficits.

Intermittent Theta Burst Stimulation Ameliorates Cognitive Deficit and Attenuates Neuroinflammation via PI3K/Akt/mTOR Signaling Pathway in Alzheimer's-Like Disease Model.

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 TMT (8 mg/kg), TMT rats subjected to iTBS two times per day for 15 days and TMT sham group. After 3 weeks, we examined exploratory behavior and memory, histopathological and changes on molecular level. TMT-treated rats exhibited severe and cognitive deficit. iTBS-treated animals showed improved cognition. iTBS reduced TMT-induced inflammation and increased anti-inflammatory molecules. We examined PI3K/Akt/mTOR signaling pathway which is involved in regulation of apoptosis, cell growth and learning and memory. We found significant downregulation of phosphorylated forms of Akt and mTOR in TMT-intoxicated animals, which were reverted following iTBS stimulation. Application of iTBS produces beneficial effects on cognition in of rats with TMT-induced hippocampal neurodegeneration and that effect could be mediated via PI3K/Akt/mTOR signaling pathway, which could candidate this protocol as a potential therapeutic approach in neurodegenerative diseases such as AD.

Expression of Ectonucleoside Triphosphate Diphosphohydrolase 2 (NTPDase2) Is Negatively Regulated Under Neuroinflammatory Conditions In Vivo and In Vitro.

Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13 receptors. 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, whereas the recovery of the expression was most profound in the subgranular layer. Concerning the regulation of NTPDase2 gene expression, proinflammatory cytokines IL-6, IL-1ß, TNFα, and IFNγ negatively regulated the expression of NTPDase2 in OLN93 cells, while did not altering the expression in primary astrocytes. Different cell-intrinsic stressors, such as depletion of intracellular energy store, oxidative stress, endoplasmic reticulum stress, and activation of protein kinase C, also massively disturbed the expression of the NTPDase2 gene. Together, our results suggest that the expression and the activity of NTPDase2 transiently cease in neurodegeneration and brain injury, most likely as a part of the acute adaptive response designed to promote cell defense, survival, and recovery.

Altered Topographic Distribution and Enhanced Neuronal Expression of Adenosine-Metabolizing Enzymes in Rat Hippocampus and Cortex from Early to late Adulthood.

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.

Antenatal Dexamethasone Treatment Induces Sex-dependent Upregulation of NTPDase1/CD39 and Ecto-5'-nucleotidase/CD73 in the Rat Fetal Brain.

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5'-nucleotidase (e5'NT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.

Microglial- and Astrocyte-Specific Expression of Purinergic Signaling Components and Inflammatory Mediators in the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration.

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 stage of TMT-induced neurodegeneration might reflect the functional properties, migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. Reactive astrocytes expressed adenosine A1, A2A, and P2Y1 receptors, revealed induction of complement component C3, inducible nitric oxide synthase, nuclear factor-kB, and proinflammatory cytokines at the late stage of TMT-induced neurodegeneration. An increased set of purinergic system components on activated microglia (NTPDase1/CD39, eN/CD73, and P2X7) and astrocytes (A1R, A2AR, and P2Y1), and loss of homeostatic glial and neuronal purinergic pathways (P2Y12 and A1R) may shift purinergic signaling balance toward excitotoxicity and inflammation, thus favoring progression of pathological events. These findings may contribute to a better understanding of the involvement of purinergic signaling components in the progression of neurodegenerative disorders that could be target molecules for the development of novel therapies.

Downregulation of CD73/A2AR-Mediated Adenosine Signaling as a Potential Mechanism of Neuroprotective Effects of Theta-Burst Transcranial Magnetic Stimulation in Acute Experimental Autoimmune Encephalomyelitis.

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-1ß and NF- κB-ir and upregulated IL-10 pointing towards a reduction in the neuroinflammatory process in EAE animals after CTBS treatment. Furthermore, CTBS attenuated EAE-induced glial eN/CD73 expression and activity, while inducing a shift in A2AR expression from glia to neurons, contrary to EAE, where tight coupling of eN/CD73 and A2AR on glial cells is observed. Finally, increased glial A1R expression following CTBS supports anti-inflammatory adenosine actions and potentially contributes to the overall neuroprotective effect observed in EAE animals after CTBS treatment.

Trimethyltin Increases Intracellular Ca2+ Via L-Type Voltage-Gated Calcium Channels and Promotes Inflammatory Phenotype in Rat Astrocytes In Vitro.

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 Ca2+ 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 [Ca2+]i in a dose-dependent manner. Specifically, TMT-induced Ca2+ transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca2+ 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.

Theta burst stimulation ameliorates symptoms of experimental autoimmune encephalomyelitis and attenuates reactive gliosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by inflammatory processes in the central nervous system (CNS). Decades of research led to discovery of several disease-modifying therapeutics strategies with moderate success. Experimental autoimmune encephalomyelitis (EAE) is currently the most commonly used experimental model for MS and for studying various therapeutic approaches. Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neurostimulation technique with multiple beneficial effects on healthy as well as CNS with pathology. However, the molecular and cellular mechanisms of rTMS on acute EAE are scarce. Our study demonstrated beneficial effects of theta-burst stimulation (TBS), an experimental paradigm of rTMS, on disease course of acute EAE. TBS treatment attenuated reactive gliosis, restored myelin sheet and down-regulated expression of vimentin in EAE rats. These effects were reflected through reduced clinical parameters, shorter duration of illness and days spent in paralysis. Based on our research, rTMS deserves further considerations for its neuroprotective effect on EAE, and is an excellent candidate for further research and points that it could be used for more than for simple symptomatic therapy.

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.

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 17ß-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, ERß or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -ß (ERß) 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 ERß, while activation of ERα, -ß 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.

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Complex regulation of ecto-5'-nucleotidase/CD73 and A2AR-mediated adenosine signaling at neurovascular unit: A link between acute and chronic neuroinflammation.

The review summarizes available data regarding the complex regulation of CD73 at the neurovascular unit (NVU) during neuroinflammation. Based on available data we propose the biphasic pattern of CD73 regulation at NVU, with an early attenuation and a postponed up-regulation of CD73 activity. Transient attenuation of CD73 activity on leukocyte/vascular endothelium and leukocyte/astrocyte surface, required for the initiation of a neuroinflammatory response, may be effectuated either by catalytic inhibition of CD73 and/or by shedding of the CD73 molecule from the cell surface, while postponed induction of CD73 is effectuated by transcriptional up-regulation of Nt5e and posttranslational modifications. Neuroinflammatory conditions are also associated with significant enhancement and gain-of-function of A2AR-mediated adenosine signaling. However, in contrast to the temporary prevalence of A2AR over A1R signaling during an acute inflammatory response, prolonged induction of A2AR and resulting perpetual CD73/A2AR coupling may be a contributing factors in the transition between acute and chronic neuroinflammation. Thus, pharmacological targeting of the CD73/A2AR axis may attenuate inflammatory response and ameliorate neurological deficits in chronic neuroinflammatory conditions.

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.

Spatial Distribution and Expression of Ectonucleotidases in Rat Hippocampus After Removal of Ovaries and Estradiol Replacement.

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 ecto-enzymes 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.

Unveiling the Role of Ecto-5'-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools.

CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5'-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,ß-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis.

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.