Carme Pelegrí

Resveratrol and Neurodegenerative Diseases: Activation of SIRT1 as the Potential Pathway towards Neuroprotection

One of the current problems in medicine research is the development of safe drugs for the treatment of neurological disorders. Furthermore, there is a close relationship between the process of aging and the appearance of neurological disorders, particularly Parkinsons disease and Alzheimers disease. Therefore, an ideal compound would have two characteristics: neuroprotective action and an anti-aging effect. The natural compound resveratrol is a suitable candidate for this purpose due to its low toxicity and antioxidant properties. In addition, recent research has shown that it has an anti-aging effect in rat, yeast, Caenorhabditis elegans, and Drosophila, although the mechanism involved in this process remains to be clarified. One hypothesis is that by activating Sirtuin 1, resveratrol modulates the activity of numerous proteins, including peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1 alpha), the FOXO family, Akt (protein kinase B) and nuclear factor-kappabeta (NFkappabeta). This review summarises recent research on the molecular mechanisms through which resveratrol might exert its therapeutic effects via the interaction with Sirtuin 1, as well as other targets. In addition, we discuss the possibility of using resveratrol in the treatment of neurodegenerative diseases.

Dietary resveratrol prevents Alzheimer’s markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer’s disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Early Amyloid Accumulation in the Hippocampus of SAMP8 Mice

Late-onset Alzheimers disease (AD) is the most common form of AD appearing after 65 years of age. To date, however, there are no non-genetically manipulated rodent models that develop a similar sporadic onset of AD with age-related amyloid-beta (Abeta) deposition. Although the senescence accelerated mouse prone 8 (SAMP8) mice have been proposed as a model of AD, the presence of Abeta deposits remains controversial. In this study, we describe the time course of Abeta deposition in SAMP8 mice as well as in control SAMR1 and ICR-CD1 strains of mice. From as early as 6 months onward, SAMP8 mice show Abeta deposition in the hippocampus that increase in number and extent with age. These deposits are comprised of by clustered granules that contain Abeta{42}, Abeta{40}, and other Abeta protein precursor fragments. By marked contrast, control mice show only low numbers of Abeta clusters that do not develop until 15 months of age. The demonstration that SAMP8 mice present with amyloid deposits in their hippocampus makes this animal model a useful tool to understand the mechanisms involved in Abeta deposition in AD.

Sirtuin activators: Designing molecules to extend life span

Resveratrol (RESV) exerts important pharmacological effects on human health: in addition to its beneficial effects on type 2 diabetes and cardiovascular diseases, it also modulates neuronal energy homeostasis and shows antiaging properties. Although it clearly has free radical scavenger properties, the mechanisms involved in these beneficial effects are not fully understood. In this regard, one area of major interest concerns the effects of RESV on the activity of sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase that has been implicated in aging. Indeed, the role of SIRT1 is currently the subject of intense research due to the antiaging properties of RESV, which increases life span in various organisms ranging from yeast to rodents. In addition, when RESV is administered in experimental animal models of neurological disorders, it has similar beneficial effects to caloric restriction. SIRT1 activation could thus constitute a potential strategic target in neurodegenerative diseases and in disorders involving disturbances in glucose homeostasis, as well as in dyslipidaemias or cardiovascular diseases. Therefore, small SIRT1 activators such as SRT501, SRT2104, and SRT2379, which are currently undergoing clinical trials, could be potential drugs for the treatment of type 2 diabetes, obesity, and metabolic syndrome, among other disorders. This review summarises current knowledge about the biological functions of SIRT1 in aging and aging-associated diseases and discusses its potential as a pharmacological target.

Increased permeability of blood–brain barrier on the hippocampus of a murine model of senescence

SAMP8 mice show several indicative characteristics of accelerated aging and have been used to study the physiological and physiopathological processes that take place during senescence. There is some controversy about the presence of a functional blood-brain barrier (BBB) disturbance on these animals, which could be related to the oxidative stress or the amyloidosis present in their brain. In order to elucidate BBB status in the hippocampus of SAMP8 mice, in this study we have determined the extravasation from brain microvessels of endogenous IgG in SAMP8 mice aged 3, 7 and 12 months and in age-matched control SAMR1 mice. Immunohistochemistry, confocal microscopy and an imaging methodology specially designed to quantify IgG extravasation have been used. The choroid plexus was analyzed as a control for positive extravasation in SAMP8 and SAMR1 mice and, as expected, in all studied ages high IgG immunoreactivity was observed in both strains. We have found significantly higher levels of IgG extravasation in the hippocampus of 12-month-old SAMP8 mice compared to SAMR1 mice, indicating an increased permeability of BBB in aged senescence-accelerated mice.

Cell cycle activation in striatal neurons from Huntington's disease patients and rats treated with 3-nitropropionic acid.

This study was undertaken to investigate the potential role of cell cycle re‐entry in an experimental model of Huntingtons disease and in human brain samples. We found that after treatment of rats with the mitochondrial neurotoxin 3‐nitropropionic acid, the expression of cell cycle markers of G1 phase measured by immunohistochemistry was induced in the striatal brain region. Furthermore, we detected an increase in the nuclear and also cytoplasmatic E2F‐1 expression, suggesting that this protein could activate the apoptotic cascade in rat brain. Western blot analysis of post‐mortem brain samples from patients also showed an increase in the expression of E2F‐1 and cyclin D1 in comparison with control samples. These results indicate that cell cycle re‐entry is activated in Huntingtons disease and may contribute to the neurodegenerative process.

A new method for determining blood–brain barrier integrity based on intracardiac perfusion of an Evans Blue–Hoechst cocktail

A new method for determining brain regions with blood-brain barrier (BBB) alterations is described. In this method, mice were perfused intracardially with Evans Blue (EB) and Hoechst tracers added in a standard formaldehyde fixative solution. This cocktail method was tested after a localized cryolesion induced in the brain had produced an edematous brain region with disrupted BBB in the animals. The results were then compared with the intravenous and intraperitoneal administration of the tracers prior to intracardiac perfusion. When using the cocktail method, red EB fluorescence locates the cryoinjured brain region while the Hoechst tracer stains the nuclei in that same region. EB and Hoechst fluorescence can also be observed in the choroid plexus and circumventricular organs, where there is no functional BBB. The cocktail gives more intense EB staining in zones of disrupted BBB than that given by traditional methods which use this tracer. The Hoechst tracer is also more useful when administered in the cocktail, since when administrated intravenously it stains all the brain nuclei. The cocktail method permits the immunostaining of brain sections, enabling researchers to characterize and analyze structural and cellular changes in regions where BBB disturbances are present. Thus, immunohistochemistry has been used here to determine the nature of intense EB fluorescent cells that appear in the perilesional rim, which were identified here as neuronal cells.

Cerebral Amyloid Angiopathy, Blood-Brain Barrier Disruption and Amyloid Accumulation in SAMP8 Mice

Cerebrovascular dysfunction and β-amyloid peptide deposition on the walls of cerebral blood vessels might be an early event in the development of Alzheimer’s disease. Here we studied the time course of amyloid deposition in blood vessels and blood-brain barrier (BBB) disruption in the CA1 subzone of the hippocampus of SAMP8 mice and the association between these two variables. We also studied the association between the amyloid deposition in blood vessels and the recently described amyloid clusters in the parenchyma, as well as the association of these clusters with vessels in which the BBB is disrupted. SAMP8 mice showed greater amyloid deposition in blood vessels than age-matched ICR-CD1 control mice. Moreover, at 12 months of age the number of vessels with a disrupted BBB had increased in both strains, especially SAMP8 animals. At this age, all the vessels with amyloid deposition showed BBB disruption, but several capillaries with an altered BBB showed no amyloid on their walls. Moreover, amyloid clusters showed no spatial association with vessels with amyloid deposition, nor with vessels in which the BBB had been disrupted. Finally, we can conclude that vascular amyloid deposition seems to induce BBB alterations, but BBB disruption may also be due to other factors.

Characterization of Amyloid-β Granules in the Hippocampus of SAMP8 Mice

The senescence accelerated mouse-prone 8 (SAMP8) strain of mice is an experimental model of accelerated senescence that has also been proposed as a model of Alzheimers disease as it shares several features with this dementia. We have recently reported amyloid-β (Aβ) granules in the hippocampus of SAMP8 mice, which contain Aβ42 and Aβ40 peptides and other amyloid-β protein precursor fragments. These granules appear clustered mainly in the stratum radiatum of the CA1 region and increase in number and size with age. Here we performed several studies to examine whether the Aβ granules in the hippocampus of SAMP8 mice contain other proteins characteristic of neuropathological aggregates, such as tau, MAP2, and α-synuclein. Moreover, we examined whether the Aβ granules in the hippocampus correspond to heparan sulphate proteoglycan (HSPG) positive granules previously described in this animal model. The results showed that Aβ granules correspond to the HSPG granular structures, being syndecan-2, a protein involved in the remodeling of dendritic spines, the type of HSPG found. Tau and MAP2, but not α-synuclein depositions, were also found in Aβ aggregates. Granules do not appear to have an astrocytic origin, since although some Aβ clusters are associated with astrocyte processes, most clusters are not. On the other hand, the presence of tau, MAP2, and NeuN in Aβ granules suggests a neuronal origin. As the components identified in Aβ granules are characteristic of the aggregates present in some neurodegenerative diseases, the SAMP8 model seems to be appropriate for the study of the processes involved in these pathologies.

Comparison of four lymphocyte isolation methods applied to rodent T cell subpopulations and B cells

The aim of this study was to establish the validity of four lymphocyte isolation methods. The effects of three different erythrocyte lysing methods commonly used in the analysis of human cells, namely, lysis by ammonium chloride (AC), Becton Dickinson lysis (BDL) and the Coulter Q-Prep (CQP) preparation system were established by flow cytometry on rat lymphocyte subsets. The results were compared with those obtained with a Ficoll-Isopaque (FI) density gradient procedure adapted for use with rat cells. Lymphocyte isolation by AC or FI gradient was performed before labelling the lymphocyte subpopulations, whereas the BDL and CQP methods were performed after staining the cells in whole blood. The FI gradient yielded the lowest CD5+, CD4+ and CD25+ cell percentages. On the other hand AC lysis produced higher percentages of T cells and lower percentages of B cells than the other methods studied. The percentages obtained after BDL or CQP methods for T lymphocyte subsets and B cells were found to be reproducible. The commercial methods (BDL and CQP) are faster but rather expensive, whereas AC lysis and FI gradient separations are cheap and particularly useful when there is a requirement to culture the cells.