Dunja Drakulić

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca(2+) transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca(2+) influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca(2+) influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na(+)/Ca(2+) exchanger for Na(+) inhibited mitochondrial Ca(2+) efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, we obtained that E2s action on mitochondrial Ca(2+) efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na(+)/Ca(2+) exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca(2+) concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca(2+) in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca(2+) transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβs involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development.

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

Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus.

The present study attempted to investigate how chronic cerebral hypoperfusion (CCH) and repeated low-dose progesterone (P) treatment affect gene and protein expression, subcellular distribution of key apoptotic elements within protein kinase B (Akt) and extracellular signal-regulated kinases (Erk) signal transduction pathways, as well as neurodegenerative processes and behavior. The results revealed the absence of Erk activation in CCH in cytosolic and synaptosomal fractions, indicating a lower threshold of Akt activation in brain ischemia, while P increased their levels above control values. CCH induced an increase in caspase 3 (Casp 3) and poly (ADP-ribose) polymerase (PARP) gene and protein expression. However, P restored expression of examined molecules in all observed fractions, except for the levels of Casp 3 in synapses which highlighted its possible non-apoptotic or even protective function. Our study showed the absence of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) response to this type of ischemic condition and its strong activation under the influence of P. Further, the initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by CCH was significantly reduced by P. Finally, P reversed the CCH-induced reduction in locomotor activity, while promoting a substantial decrease in anxiety-related behavior. Our findings support the concept that repeated low-dose post-ischemic P treatment reduces CCH-induced neurodegeneration in the hippocampus. Neuroprotection is initiated through the activation of investigated kinases and regulation of their downstream molecules in subcellular specific manner, indicating that this treatment may be a promising therapy for alleviation of CCH-induced pathologies.

Time-related sex differences 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. [Projekat Ministarstva nauke Republike Srbije, br. 173044 i br. 41014]

Effect of EDTA on copper-induced inhibition of rat myometrial ecto-ATPase activity

AbstactThe aim of this study was to examine in vitro chelator’s ability to prevent copper-induced inhibition of rat myometrial ecto-ATPase activity. The effects of increasing CuSO4 concentrations, in the absence and presence of 1 mmol/l EDTA, showed sigmoidal and complete inhibition relative to the control enzyme activity. IC50 values, 1.15 × 10−4 and 1.71 × 10−3 mol/l in the absence and presence of EDTA, respectively, were determined by Hill analysis from experimental curves. According to the results presented in this work, 1 mmol/l EDTA increased by one order of magnitude CuSO4 concentration for half-maximal inhibition (IC50), by decreasing Cu2+ concentrations, available to form inactive CuATP2− complex.

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.

Prooxidant–antioxidant balance, advanced oxidation protein products and lipid peroxidation in Serbian patients with Parkinson's disease

ABSTRACT Background: Biomarkers of oxidative stress are relevant in the evaluation of the disease status and prooxidant–antioxidant balance, advanced oxidation protein products and lipid peroxidation products (malondialdehyde and 4-hydroxynonenal) are being extensively evaluated regarding their relationship with clinical presentation and disease severity. Aim of the study: The aim of this study was to evaluate the levels of the above-mentioned parameters in plasma of 39 men and 17 women with Parkinsons disease, originated from the Republic of Serbia and their relation to clinicopathological characteristics (gender, age at examination, duration of the disease, and Hoehn and Yahr score) and oxidative status. Results: The incidence of disease was 2:1 towards males. The investigated oxidative parameters were gender and Hoehn and Yahr related. Significant association of higher Hoehn and Yahr scores was observed for malondialdehyde (p = 0.01) and prooxidant–antioxidant balance (p = 0.02). Relation between oxidant–antioxidant status was further supported by observed positive correlation between 4-hydroxynonenal (p = 0.04) and prooxidant–antioxidant balance (p = 0.03). Finally, the multivariate analysis indicated that prooxidant–antioxidant balance and malondialdehyde were partially determined by gender (10.6% and 7.6%) and Hoehn and Yahr scores (13.6% and 18.8%), while Hoehn and Yahr scores contributed to the variance of advanced oxidation protein products with 13.2%. Conclusion: Our results indicate the higher level of oxidative stress (oxidant–antioxidant imbalance) and possible relation of several markers with gender and disease stage in patients with Parkinsons disease. The analyzed markers could be used to specify the severity of oxidative stress; however, their potential value should be analyzed in further studies.

Regional-specific effects of cerebral ischemia/reperfusion and dehydroepiandrosterone on synaptic NMDAR/PSD-95 complex in male Wistar rats

Excessive glutamate efflux and N-methyl-D-aspartate receptor (NMDAR) over-activation represent well-known hallmarks of cerebral ischemia/reperfusion (I/R) injury, still, expression of proteins involved in this aspect of I/R pathophysiology show inconsistent data. Neurosteroid dehydroepiandrosterone (DHEA) has been proposed as potent NMDAR modulator, but its influence on I/R-induced changes up to date remains questionable. Therefore, I/R-governed alteration of vesicular glutamate transporter 1 (vGluT1), synaptic NMDAR subunit composition, postsynaptic density protein 95 (PSD-95) and neuronal morphology alone or following DHEA treatment were examined. For that purpose, adult male Wistar rats were treated with a single dose of vehicle or DHEA (20 mg/kg i.p.) 4 h following sham operation or 15 min bilateral common carotid artery occlusion. Western blot was used for analyses of synaptic protein expressions in hippocampus and prefrontal cortex, while neuronal morphology was assessed using Nissl staining. Regional-specific postischemic changes were detected on protein level i.e. signs of neuronal damage in CA1 area was accompanied with hippocampal vGluT1, NR1, NR2B enhancement and PSD-95 decrement, while histological changes observed in layer III were associated with decreased NR1 subunit in prefrontal cortex. Under physiological conditions DHEA had no effect on protein and histological appearance, while in ischemic milieu it restored hippocampal PSD-95 and NR1 in prefrontal cortex to the control level. Along with intact neurons, ones characterized by morphology observed in I/R group were also present. Future studies involving NMDAR-related intracellular signaling and immunohistochemical analysis will reveal precise effects of I/R and DHEA treatment in selected brain regions.

Cell proliferation assay – method optimisation for in vivo labeling of DNA in the rat forestomach

Abstract The study of cell proliferation is a useful tool in the fields of toxicology, pathophysiology and pharmacology. Cell proliferation and its degree can be evaluated using 5-bromo-2′-deoxyuridine which is incorporated into the newly synthesized DNA. The aim of this study was the optimization of subcutaneous application of 5-bromo-2′-deoxyuridine implantation for continuous and persistent marking of proliferating cells in the rat forestomach. 3-tert-Butyl-4-hydroxyanisole was used as the agent that ensures cell proliferation. In order to determine the optimal dose for proliferating cells labeling, 5-bromo-2′-deoxyuridine doses of 50 mg, 100 mg, 200 mg or 350 mg were implemented 2 days prior to sacrifice by flat-faced cylindrical matrices. Immunohistochemical analysis using 5-bromo-2′-deoxyuridine in situ detection kit was performed for the detection of 5-bromo-2′-deoxyuridine labeled cells. The results showed that for adult rats, the optimum 5-bromo-2′-deoxyuridine dose is 200 mg per animal for subcutaneous application. The here described manner of 5-bromo-2′-deoxyuridine in vivo labeling provides a simple, efficient, and reliable method for cell labeling, and at the same minimizes stress to animals.