The complex relationship between late-onset caloric restriction and synaptic plasticity in aged Wistar rats.

Age-related reduction in spine density, synaptic marker expression, and synaptic efficiency are frequently reported. These changes provide the cellular and molecular basis for the cognitive decline characteristic for old age. Nevertheless, there are several approaches that have the potential to ameliorate these processes and improve cognition, caloric restriction being one of the most promising and widely studied. While lifelong caloric restriction is known for its numerous beneficial effects, including improved cognitive abilities and increased expression of proteins essential for synaptic structure and function, the effects of late-onset and/or short-term CR on synaptic plasticity have yet to be investigated. We have previously documented that the effects of CR are strongly dependent on whether CR is initiated in young or old subjects. With this in mind, we conducted a long-term study in aging Wistar rats to examine changes in the expression of several key synaptic markers under the regimen of CR started at different time points in life. We found a significant increase in the expression of both presynaptic and postsynaptic markers. However, taking into account previously reported changes in the behavior detected in these animals, we consider that this increase cannot represent beneficial effect of CR.

Dietary restriction alters insulin signaling pathway in the brain.

Insulin is known to be a key hormone in the regulation of peripheral glucose homeostasis, but beyond that, its effects on the brain are now undisputed. Impairments in insulin signaling in the brain, including changes in insulin levels, are thought to contribute significantly to declines in cognitive performance, especially during aging. As one of the most widely studied experimental interventions, dietary restriction (DR) is considered to delay the neurodegenerative processes associated with aging. Recently, however, data began to suggest that the onset and duration of a restrictive diet play a critical role in the putative beneficial outcome. Because the effects of DR on insulin signaling in the brain have been poorly studied, we decided to examine the effects of DR that differed in onset and duration: long-term DR (LTDR), medium-term DR (MTDR), and short-term DR (STDR) on the expression of proteins involved in insulin signaling in the hippocampus of 18- and 24-month-old male Wistar rats. We found that DR-induced changes in insulin levels in the brain may be independent of what happens in the periphery after restricted feeding. Significantly changed insulin content in the hippocampus, together with altered insulin signaling were found under the influence of DR, but the outcome was highly dependent on the onset and duration of DR.

Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L.

The formation of amyloid-ß peptides (Aß), that accumulate in Alzheimer's disease (AD) brains, involves proteolytic processing of the amyloid precursor protein (APP) firstly by ß-secretase (BACE1). Since BACE1 cleaves a plethora of other substrates, in this work we investigated whether the proteolysis and/or distribution of other BACE1 substrates, such as seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), is altered in AD. To test this we used 5xFAD mouse model brains that show an early accumulation of Aß plaques already at 2-months of age. Here we show for the first time that accumulation of BACE1 in peri-plaque regions and its enhanced levels in AD brains does not affect proteolysis of BACE1 substrates other than APP, such as Sez6 and Sez6L. We observed altered distribution of Sez6 and Sez6L in the area of Aß plaques in 5xFAD brains which is distinct to that of APP, BACE1 and/or LAMP1, suggesting different localization and/or function of these BACE1 substrates. While it is necessary to further elucidate the potential role that this may play in the course of AD, it is likely that Aß-targeted therapies may have beneficial effects against accumulation and/or altered distribution of BACE1 and its substrates, in addition to APP.

Late-Onset Calorie Restriction Improves Lipid Metabolism and Aggravates Inflammation in the Liver of Old Wistar Rats.

Aging is a progressive process that could disturb metabolic homeostasis in the liver via ectopic lipid accumulation, oxidative stress, and deterioration of inflammatory response. Although calorie restriction (CR) is recognized as beneficial for life span and health span prolongation, it is still unclear how late-onset CR, characterized by late beginning and short duration, affects age-related processes. The aim of this study was to examine how late-onset CR-induced metabolic adjustments impact lipid status and inflammation in the liver of old rats. The experiments were conducted on aging male Wistar rats fed ad libitum (AL) or exposed to late-onset CR (60% of AL daily intake) from 21st to 24th month. The results showed that late-onset CR reduces body weight, visceral adipose tissue and liver mass, and triglyceride levels when compared to old animals on AL diet. The ameliorating effects of CR on lipid metabolism include increased activity of AMP-activated protein kinase, suppressed de novo fatty acid synthesis, stimulated ß-oxidation, decreased lipotoxicity, and limited triglyceride synthesis and packaging in the liver. Restricted diet regime, however, does not improve expression of antioxidant enzymes, although it leads to progression of age-related inflammation in the liver, partially through lower corticosterone concentration and decreased activation of glucocorticoid receptor. In conclusion, late-onset CR is able to restore age-related imbalance of lipid metabolism in the liver, but has a negative impact on hepatic inflammatory status, implying that the type of diet for older individuals must be balanced and chosen carefully with appropriate duration and start point.

The Less We Eat, the Longer We Live: Can Caloric Restriction Help Us Become Centenarians?

Striving for longevity is neither a recent human desire nor a novel scientific field. The first article on this topic was published in 1838, when the average human life expectancy was approximately 40 years. Although nowadays people on average live almost as twice as long, we still (and perhaps more than ever) look for new ways to extend our lifespan. During this seemingly endless journey of discovering efficient methods to prolong life, humans were enthusiastic regarding several approaches, one of which is caloric restriction (CR). Where does CR, initially considered universally beneficial for extending both lifespan and health span, stand today? Does a lifelong decrease in food consumption represent one of the secrets of centenarians' long and healthy life? Do we still believe that if we eat less, we will live longer? This review aims to summarize the current literature on CR as a potential life-prolonging intervention in humans and discusses metabolic pathways that underlie this effect.

Calorie restriction changes the anxiety-like behaviour of ageing male Wistar rats in an onset- and duration-dependent manner.

Although initially recognized as a universally beneficial approach for the prevention of age-related impairments, the outcome of calorie restriction (CR) is now known to depend on several factors, most notably the age of the subject at the CR commencement, and CR duration. We aimed to examine if and how CR affects anxiety-like behaviour when it is introduced at middle age and late middle age. In addition, as the dopaminergic system is one of the main neurotransmitter systems involved in controlling anxiety, we examined the expression of dopamine receptors (D1R, D2R) in the cortex, striatum, and mesencephalon of male Wistar rats of varying ages. The study was performed on rats fed ad libitum (AL) or exposed to calorie restriction (60% of AL intake). Open field and light-dark tests were used to study anxiety-like behaviour, while PCR and Western blot were used to examine the expression of dopamine receptors. Calorie restriction implemented at middle-age led to variable outcomes on anxiety-like behaviour, while CR implemented at late middle age increased anxiety and decreased the availability of D2R levels in the cortex and mesencephalon. Taken together, these results advise caution when implementing calorie restriction late in life.

Late-Onset Calorie Restriction Worsens Cognitive Performances and Increases Frailty Level in Female Wistar Rats.

The current study aims to determine the potential benefits of calorie restriction (CR), one of the most promising paradigms for life span and healthspan extension, on cognitive performances in female Wistar rats during aging. As a measure of a healthspan, we evaluated the effects of different onset and duration of CR on frailty level. Female Wistar rats were exposed to either ad libitum (AL) or CR (60% of AL daily intake) food intake during aging. Two different CR protocols were used, life-long CR with an early-onset that started at the adult stage (6 months) and 3-month-long CR, started at the middle (15 months) and late-middle (21 months) age, thus defined as a late-onset CR. The effects of CR were evaluated using open-field, Y-maze, and novel object recognition tests. We broadened 2 tools for frailty assessment currently in use for experimental animals, and in alignment with our previous study, we created a physical-cognitive frailty tool that combines both physical and cognitive performances. Our results clearly showed that CR effects are highly dependent on CR duration and onset. While a life-long restriction with an early-onset has been proven as protective and beneficial, short-term restriction introduced at late age significantly worsens an animal's behavior and frailty. These results complement our previous study conducted in males and contribute to the understanding of sex differences in a response to CR during aging.

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, beta-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 NormFinder software packages and by direct comparison of Ct values. Analysis revealed gapdh and beta-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and beta-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 alpha-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.

Dietary restriction modulates alpha-synuclein expression in the aging rat cortex and hippocampus.

Dietary restriction (DR) is one of the promising environmental interventions known to attenuate aging and decrease risk of age-related neurodegenerative disorders. The aim of this study was to assess the effects of DR on expression of alpha-synuclein, a presynaptic protein involved in pathogenesis of Parkinson's and some other neurodegenerative diseases, in the cortex and hippocampus of adult, middle-aged, late middle-aged, and aged rats. Using Real Time RT-PCR, the authors report that aging regulates the expression of alpha-synuclein in a tissue-specific manner and that long-term DR reverts the late age-related changes of alpha-synuclein expression.

The mechanisms of 6-hydroxydopamine-induced astrocyte death.

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 H(2)O(2), O(2)(*-), 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.

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.

Increase of GAP-43 in the rat cerebellum following unilateral striatal 6-OHDA lesion.

In order to further characterize synaptic alterations following a severe lesion of the nigrostriatal system, the expression of synaptic marker proteins, synaptophysin and growth-associated protein-43 (GAP-43), was examined in various brain regions of 6-hydroxydopamine (6-OHDA)-treated rats, an animal model of Parkinson's disease. Unilateral nigrostriatal lesioning induced an increase in synaptophysin protein levels by 68% and 106% in the sensorimotor cortex and striatum, respectively, while changes in the level of GAP-43 were not observed. In contrast, 6-OHDA induced a 73% increase in the level of GAP-43 protein in the cerebellum. This increase was also confirmed with immunohistochemistry. The level of synaptophysin in the cerebellum remained unchanged in response to the lesion. These results suggest that a neurotoxic lesion of the nigrostriatal pathway differentially affects the expression of the two synaptic proteins and that plasticity-related changes in this model are not solely restricted to the nigrostriatal system. In addition, these results provide further evidence of the involvement of the cerebellum in the late response to a 6-OHDA lesion.

[Leiomyomas in pregnancy: ultrasonographic mimicry]. / Lejomiomi u trudnoci: ultrasonografska mimikrija.

Leiomyoma is the most common tumor viewed by echosonography of the uterus. This report presents pregnancy with calcified leiomyoma of the uterus which simulated the head of the fetus, what was verified by echosonography. Woman, nullipara, 41 years old, in week 21 of pregnancy, was referred for echosonographic evaluation. Biochemical screening for Down syndrome, performed in week 17 of pregnancy, was positive. Fetal karyotype, obtained by amniocentesis, was normal (46 XX). In the case presented herein, diameter of leiomyoma was 48 mm, being equal to the diameter of the fetal head in 21st week of pregnancy. Due to calcifications on the surface of leiomyoma which were viewed on echosonography in the form of hyperechogenic border, this tumor looked like a head of the fetus. This echosonographic finding of two fetal heads in a single pregnancy was remarkable even for a gynecologist experienced in echosonographic examination.

Alpha-synuclein is expressed in different tissues during human fetal development.

Alpha-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 alpha-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. alpha-Synuclein expression was observed in all fetal human organs examined. In adult human tissues the high expression of alpha-synuclein was maintained in the brain, whereas in other organs the expression was greatly reduced. In contrast, both in fetal and adult rat tissues, alpha-synuclein was only detected in the brain. In addition to a 19-kDa alpha-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, alpha-synuclein has an important function in peripheral tissues as well during normal human prenatal development.

6-Hydroxydopamine increases the level of TNFalpha and bax mRNA in the striatum and induces apoptosis of dopaminergic neurons in hemiparkinsonian rats.

This study was focused on the apoptosis (programmed cell death) induction involved in the loss of dopaminergic (DA-ergic) neurons in 6-hydroxydopamine (6-OHDA) hemiparkinsonian rats. The apoptosis in the striatum and substantia nigra pars compacta (SNpc), was examined 6, 24 h and 7 days after the 6-OHDA lesions employing the TUNEL method. The changes in mRNA levels of pro-apoptotic protein tumor necrosis factor alpha (TNFalpha) and its "death receptor" TNFalphaRI and then bax mRNA, as an important regulator of apoptotic neurodegeneration were followed by RT-PCR procedure. In situ analysis revealed an increased number of TUNEL-positive neurons in 6-OHDA-treated animals in all examined time points. The highest number of apoptotic neurons was detected 24 h after the lesion, both in the ipsilateral striatum (3.41+/-0.18) and SNpc (5.8+/-0.79). A significant increase in the level of TNFalpha mRNA was observed in 6-OHDA-lesioned striatum, with maximal value after 24 h (46%) comparing to the control. In contrast, 6-OHDA did not significantly change the level of TNFalphaRI mRNA in any time point. Six and 24 h post-operatively, we observed a significant increase of bax mRNA expression (40% and 45%, respectively) in the ipsilateral striatum of treated animals in comparison with the right striatum of the controls. However, the highest level of the bax mRNA expression was reached 7 days after the surgery (94%) in the ipsilateral striatum of 6-OHDA-treated animals. These results suggest that striatal injection of 6-OHDA can induce early changes that would be an important regulator of apoptotic neurodegeneration of dopamine-producing neurons, during the first post-operative week.