Milka Perovic

Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus- : Evaluation by real time RT-PCR

Accurate normalization is the prerequisite for obtaining reliable results in the quantification of gene expression. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five most commonly used endogenous controls, gapdh, β-actin, 18S rRNA, hprt and cypB, for their presumed stability of expression, in rat cortex and hippocampus, during aging, under dietary restriction and dexamethasone treatment. Valid reference genes (HKGs) were identified using GeNorm and Norm-Finder software packages and by direct comparison of Ct values. Analysis revealed gapdh and β-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and β-actin/hprt are the combination of choice for the single or combined effects of dietary restriction/dexamethasone, respectively. All treatments significantly influenced expression of 18S rRNA and cypB in both structures. In addition, we used gapdh and normalization factor, calculated by GeNorm, to compare the expression of α-syn in the cortex. Our results demonstrate the importance of the right choice of HKG and suggest the appropriate endogenous control to be used for TaqMan RT-PCR analysis of mRNA expression in rat cortex and hippocampus for selected experimental paradigms.

α-Synuclein is expressed in different tissues during human fetal development

Abstractα-Synuclein is a small presynaptic protein associated with both normal synaptic plasticity and neurodegenerative processes. Its normal cellular function, however, remains unknown. Even though it is highly enriched in the brain, its presence was reported in other human adult tissues. In the present study, we examined tissue expression of α-synuclein in human and rat prenatal development. Using Western blot analysis, various peripheral tissues from 15 to 23 gestational weeks, human and E19 rat fetuses, along with human and rat adult tissues, were assayed. α-Synuclein expression was observed in all fetal human organs examined. In adult human tissues the high expression of α-synuclein was maintained in the brain, whereas in other organs the expression was greatly reduced. In contrast, both in fetal and adult rat tissues, α-synuclein was only detected in the brain. In addition to a 19-kDa α-synuclein band, 36- and 52-kDa immunoreactive bands were observed in all fetal and adult human organs, with the exception of the brain, but their identity remains to be determined. These findings suggest that apart from its function in development of the nervous system, α-synuclein has an important function in peripheral tissues as well during normal human prenatal development.

Long-term dietary restriction modulates the level of presynaptic proteins in the cortex and hippocampus of the aging rat.

Brain aging is related to the numerous structural and functional changes including decreased synaptic plasticity. The beneficial effects of dietary restriction (DR) are well known but insufficiently investigated at the level of plasticity-related markers. Therefore, the aim of this study was to examine the expression profiles of proteins structurally and functionally related to synapses-growth-associated protein 43 (GAP-43), synaptophysin (SPH) and alpha-synuclein (alpha-Syn), in the course of aging and in response to long-term DR. The mRNA and protein levels of three presynaptic proteins were assessed by Real Time RT-PCR and Western blotting in the cortex and hippocampus of young (6-month-old), middle-aged (12-month-old), aged (18-month-old) and old (24-month-old) male Wistar rats fed ad libitum and exposed to DR starting from 6 months of age. We observed that long-term DR modulated age-related transcriptional changes by maintaining stable mRNAs levels in the cortex. No major age-related changes of the protein levels were observed in the cortex, while the specific temporal decline was detected in the hippocampus for all three proteins. The SPH levels were decreased across lifespan (0.8-, 0.8- and 0.6-fold change at 12, 18 and 24 months), while the significant decrease of GAP-43 and alpha-Syn protein was detected at 24 months of age (0.6- and 0.7-fold decrease, respectively). Long-term DR eliminated this decline by increasing GAP-43, SPH and alpha-Syn protein levels (1.7-, 1.7- and 1.6-fold, respectively) thus reverting protein levels to the values measured in 6-month-old animals.Specific pattern of changes observed in the hippocampus identifies this structure as more vulnerable to the processes of aging and with a more pronounced response to the DR effects. The observed DR-induced stabilization of the levels of three presynaptic proteins indicates the beneficial effect of DR on age-related decline in the capacity for synaptic plasticity.

Iron protects astrocytes from 6-hydroxydopamine toxicity

The role of iron in 6-hydroxydopamine (6-OHDA) toxicity towards astrocytes was investigated in vitro using rat primary astrocytes, rat astrocytoma cell line C6, and human astrocytoma cell line U251. The assessment of mitochondrial respiration or lactate dehydrogenase release has shown a dose-dependent decrease in the viability of astrocytes treated with 6-OHDA, which coincided with DNA fragmentation and the changes in cellular morphology. This was a consequence of the oxidative stress mediated by 6-OHDA autoxidation products hydrogen peroxide, superoxide anion, and hydroxyl radical. Both FeSO(4) and FeCl(3) markedly alleviated detrimental effects of 6-OHDA treatment, while MgSO(4) was without effect. The protective action of iron was neutralized by a membrane-permeable iron chelator o-phenanthroline, which also augmented astrocyte killing in the absence of exogenous iron. The mechanisms responsible for iron-mediated protection of astrocytes did not involve interference with either 6-OHDA autoxidation, hydrogen peroxide toxicity, or 6-OHDA-induced activation of extracellular signal-regulated kinase. Finally, the addition of iron potentiated and its chelation blocked 6-OHDA toxicity towards neuronal PC12 cells, suggesting the opposite roles for this transition metal in regulating the survival of astrocytes and dopaminergic neurons.

Anthraquinone–chalcone hybrids: Synthesis, preliminary antiproliferative evaluation and DNA-interaction studies

Novel anthraquinone based chalcone compounds were synthesized starting from 1-acetylanthraquinone in a Claisen-Schmidt reaction and evaluated for their anticancer potential against three human cancer cell lines. Compounds 4a, 4b and 4j showed promising activity in inhibition of HeLa cells with IC50 values ranging from 2.36 to 2.73 μM and low cytotoxicity against healthy MRC-5 cell lines. The effects that compounds produces on the cell cycle were investigated by flow cytometry. It was found that 4a, 4b and 4j cause the accumulation of cells in the S and G2/M phases in a dose-dependent manner and induce caspase-dependent apoptosis. All of three compounds exhibit calf thymus DNA-binding activity. The determined binding constants by absorption titrations (2.65 × 10(3) M(-1), 1.36 × 10(3) M(-1)and 2.51 × 10(3) M(-1) of 4a/CT-DNA, 4b/CT-DNA and 4j/CT-DNA, respectively) together with fluorescence displacement analysis designate 4a, 4b and 4j as strong minor groove binders, but no cleavage of plasmid DNA was observed.

The Mechanisms of 6-Hydroxydopamine-Induced Astrocyte Death

Abstract: Treatment with 6‐hydroxydopamine significantly reduced the viability of cultured rat primary astrocytes, rat astrocytoma cell line C6, and human astrocytoma cell line U251. 6‐Hydroxydopamine‐treated astrocytes exhibited altered nuclear morphology, DNA fragmentation, and reduced intracellular esterase activity, which indicated apoptotic cell death. Astrocytes were protected by neutralization of 6‐hydroxydopamine autooxidation products H2O2, O2•−, and •OH, but not by cell‐derived or chemically generated anti‐apoptotic free radical nitric oxide. Finally, 6‐hydroxydopamine activated extracellular signal‐regulated kinase in astrocytes and selective inhibitor of extracellular signal‐regulated kinase activation partially prevented astrocyte death. Taken together, these data indicate that 6‐hydroxydopamine‐triggered oxidative stress induces extracellular signal‐regulated kinase‐dependent apoptotic death of astrocytes.

The impact of aging, dietary restriction, and glucocorticoids on ApoE gene expression in rat brain

The neuroprotective effect of long-term dietary restriction (DR) is widely recognized. Long-term DR is associated with increased production of neurotrophins, enhanced cellular stress response, decreased oxidative stress, increased survival of newly born neuronal cells in the hippocampus, increased glucose utilization, and other neuroprotective and neurorestorative processes. Long-term DR has a positive impact on insulin sensitivity and lipid metabolism. Insulin resistance (type 2 diabetes) and hypercholesterolemia are conditions that are associated with increased risk of Alzheimer’s disease (AD). Glucocorticoids have a profound and complex effect on cognition, particularly on the functioning of the hippocampus, a brain area rich in glucocorticoid receptors and particularly vulnerable in AD. While a transient increase in glucocorticoids can facilitate synaptic efficacy, long-term elevations in circulating glucocorticoids due to exogenous administration are associated with decreased synaptic efficacy, decreased adult neurogenesis, and neuronal atrophy in the hippocampus. Although DR results in increased levels of circulating glucocorticoids, it confers neuroprotection against different acute injuries such as excitotoxic insult and ischemia, a phenomenon known as “the glucocorticoid paradox”. The ability of long-term DR to protect the brain against the negative impact of chronic stress (chronic elevations of glucocorticoids) has not been investigated. The aim of the present study was to examine the effects of aging, DR, and acute glucocorticoid treatment on the expression of ApoE in rat cortex and hippocampus. In the course of the experiment, the animals’ body weight, body temperature, and general well-being were carefully monitored. As expected, DR prevented the ageassociated increase in body weight and was associated with a decrease in basal body temperature [i.e., 34.06±0.43°C for 18-month-old rats on DR compared to 36.0±0.77°C for 18-month-old rats fed ad libitum (AL); P < 0.05]. We measured the levels of free plasma corticosterone in DR rats and AL rats at 6 and 12 months of

Association of Collagen Type I α1 Gene Polymorphism with Bone Mineral Density in Osteoporotic Women in Serbia

BACKGROUND The collagen type I alpha1 (COLIA1) gene encodes for a major bone protein and is a strong candidate for genetic control of bone mineral density (BMD). COLIA1 gene polymorphism is associated with reduced BMD and increased fracture incidence. The aim of this study was to analyze the relationship between COLIA1 gene polymorphism and BMD in Serbian women. METHODS The women were divided into groups according to their DEXA phenotypes. They included 39 osteoporotic, 36 osteopenic, and 33 women with normal BMD. Single nucleotide polymorphism (G to T substitution) within the Sp1-binding site in the first intron of the COLIA1 gene was assessed by polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism (SSCP) analysis. RESULTS The genotype frequencies for the whole group were 67.6% GG homozygotes, 24.1% GT heterozygotes, and 8.3% TT homozygotes and were not in Hardy-Weinberg equilibrium (HWE). Significant deviation from HWE was found only in the osteopenic group (p = 0.0007), where a higher number of homozygotes was found. Comparison of the allele frequencies showed no significant differences between three groups of tested women. CONCLUSIONS The presence of the T allele in the genotype has no influence on BMD in the osteoporotic group of women. The observed deviation in the osteopenic group needs to be investigated further.

Sleep disorder and altered locomotor activity as biomarkers of the Parkinson's disease cholinopathy in rat

HIGHLIGHTSHippocampal sleep disorder is the first and long‐lasting hallmark of PD cholinopathy.High voltage sleep spindle dynamics during REM sleep reflects PD cholinopathy.Hypokinesia reflects impaired cholinergic impact in motor control regulatory network.Amphetamine induces hyperactivity in the hypokinetic rats with PD cholinopathy.Putamen c‐Fos activity reflects re‐organization of motor control in PD cholinopathy. ABSTRACT In order to find out the possible earliest biomarkers of Parkinsons disease (PD) cholinopathy, we followed the impact of bilateral pedunculopontine tegmental nucleus (PPT) lesion in rat on: the cortical and hippocampal sleep/wake states architectures, all sleep states related EEG microstructures, sleep spindles, the basal and stimulated locomotor activity. Sleep and basal locomotor activity in adult Wistar rats were followed during their inactive circadian phase, and throughout the same aging period. The bilateral PPT lesions were done by 0.1 M ibotenic acid (IBO) during the surgical procedure for implantation of the electroencephalographic (EEG) and electromyographic (EMG) electrodes for chronic sleep recording. The cholinergic neuronal loss was identified by NADPH – diaphorase histochemistry. After all sleep and behavioral recording sessions, the locomotor activity was stimulated by d‐amphetamine (d‐AMPH) and the neuronal activity of striatum was followed by c‐Fos immunolabeling. Impaired cholinergic innervation from the PPT was expressed earlier as sleep disorder then as movement disorder, and it was the earliest and long‐lasting at hippocampal and thalamo‐cortical level, and followed by a delayed “hypokinesia”. This severe impact of a tonically impaired PPT cholinergic innervation was evidenced as the cholinergic interneuronal loss of the caudate putamen and as a suppressed c‐Fos expression after stimulation by d‐AMPH. In order how they occurred, the hippocampal non rapid eye movement (NREM) sleep disorder, altered high voltage sleep spindle (HVS) dynamics during rapid eye movement (REM) sleep in the hippocampus and motor cortex, and “hypokinesia” may serve as the biomarkers of PD cholinopathy onset and progression.

Mannich bases of 1,2,4-triazole-3-thione containing adamantane moiety: Synthesis, preliminary anticancer evaluation, and molecular modeling studies

A series of 18 novel N‐Mannich bases derived from 5‐adamantyl‐1,2,4‐triazole‐3‐thione was synthesized and characterized using NMR spectroscopy and X‐ray diffraction technique. All derivatives were evaluated for their anticancer potential against four human cancer cell lines. Several tested compounds exerted good cytotoxic activities on K562 and HL‐60 cell lines, along with pronounced selectivity, showing lower cytotoxicity against normal fibroblasts MRC‐5 compared to cancer cells. The effects of compounds 5b, 5e, and 5j on the cell cycle were investigated by flow cytometric analysis. It was found that these compounds cause the accumulation of cells in the subG1 and G1 phases of the cell cycle and induce caspase‐dependent apoptosis, while the anti‐angiogenic effects of 5b, 5e, and 5j have been confirmed in EA.hy926 cells using a tube formation assay. Further, the interaction of Bax protein with compound 5b was investigated by means of molecular modeling, applying the combined molecular docking/molecular dynamics approach.