Javier Gutierrez-Cuesta

Hyperphosphorylation of microtubule-associated protein tau in senescence-accelerated mouse (SAM).

Tau is a neuronal microtubule-associated protein found predominantly on axons. Tau phosphorylation regulates both normal and pathological functions of this protein. Hyperphosphorylation impairs the microtubule binding function of tau, resulting in the destabilization of microtubules in brain, ultimately leading to the degeneration of the affected neurons. Numerous serine/threonine kinases, including GSK-3beta and Cdk5 can phosphorylate tau. SAMR1 and SAMP8 are murine strains of senescence. We show an increase in hyperphosphorylated forms of tau in SAMP8 (senescent mice) in comparison with resistant strain SAMR1. Moreover, an increase in Cdk5 expression and activation is described but analysis of GSK3beta isoforms failed to show differences in SAMP8 in comparison to age-matched SAMR1. In conclusion, tau hyperphosphorylation occurs in SAMP-8 (early senescent) mice, indicating a link between aging and tau modifications in this murine model.

Evaluation of potential pro‐survival pathways regulated by melatonin in a murine senescence model

Abstract:  We examined the effect of melatonin on pro‐survival processes in three groups of mice. Untreated senescence‐accelerated mice (SAMP8), melatonin‐treated SAMP8 and untreated senescence‐accelerated resistant mice (SAMR1) of 10 months old were studied. Melatonin (10 mg/kg) or vehicle (ethanol at 0.066%) was supplied in the drinking water from the end of the first month until the end of the ninth month of life. Differences in the Akt/Erk1‐2 pathway and downstream targets were examined and no significant changes were observed, except for β‐catenin. However, sirtuin 1 expression was significantly lower in SAMP8 than in SAMR1. In addition, acetylated p53 and NFκB expression were lower in SAMP8 than in SAMR1. These changes were prevented by melatonin. Moreover, the concentration/expression of α‐secretase was lower and that of amyloid β aggregates (Aβ) was higher in untreated SAMP8 than in SAMR1. Likewise, the levels of Bid were higher, whereas Bcl‐2XL levels were lower in SAMP8 than in SAMR1. Melatonin reduced all these changes. We conclude that melatonin improves pro‐survival signals and reduces pro‐death signals in age‐related impairments of neural processes.

Modulation of Sirtuins: New Targets for Antiageing

Aging is characterized by a progressive deterioration of physiological functions and metabolic processes. Healthy aging remains one of the ideals of modern society. In aging and in diseases associated with the elderly, such as Alzheimers or Parkinsons, the loss of cells in vital structures or organs may be related to several factors, among which the production of reactive oxygen species (ROS) by mitochondria is a common denominator, one that leads to DNA damage, apoptosis and death. Although a diet rich in antioxidants seems to offer hope in delaying the onset of unhealthy disorders that accompany aging, no clinical treatment as such has yet been developed and anti-aging drugs are still unavailable. It is well established that reducing food intake (caloric restriction) extends the life-span in a wide range of species. The protein implicated in this protective process is the silent information regulator 2 (SIR2, SIRT1 in mammals), an enzyme that belongs to a nicotinamide adenine dinucleotide (NAD)+-dependent protein deacetylases. SIRs regulate gene silencing, DNA repair, rDNA recombination, and ageing, apart from regulating programmed cell death. In this context, increasing SIRT1 has been found to protect cells against amyloid-beta-induced ROS production and DNA damage, thereby reducing apoptotic death in vitro. Moreover, it has been demonstrated that Alzheimers and Huntingtons disease neurons are rescued by the over-expression of SIRT1, induced by either caloric restriction or administration of resveratrol, a potential activator of this enzyme. The therapeutic use of resveratrol (a polyphenol present in red wines) and other related compounds, which utilize SIRT1 pathway modulators, in treating aging-related brain disorders will be discussed in this review. Provided herein are novel new compound related with resveratrol or sirtinol that are able to modulate sirtuin activity that will be tested to treat and/or prevent a wide variety of diseases including, disorders related to aging or neurodegenerative diseases.

Anti-aging properties of melatonin in an in vitro murine senescence model: involvement of the sirtuin 1 pathway

Abstract:  Sirtuin 1 is a member of the sirtuin family of protein deacetylases, which have attracted considerable attention as mediators of lifespan extension in several model organisms. Induction of sirtuin 1 expression also attenuates neuronal degeneration and death in animal models of Alzheimer’s disease and Huntington’s disease. In this study, an in vitro model of neuronal aging was used to test in several ways whether melatonin acts as a sirtuin 1 inducer and if this effect could be neuroprotective. It is shown that melatonin is able to increase the level of this deacetylase in young primary neurons, as well as in aged neurons. We also observed an increase in the deacetylation of several substrates of sirtuin 1, such as p53, PGC‐1α, FoxO1, ADAM10 and NFκB. In addition, there was a reduction in its nuclear translocation and, subsequently, an improvement in transcriptional activity. Sirtinol, a sirtuin 1 inhibitor, was used to correlate these effects with sirtuin. It is shown that sirtinol reduces sirtuin 1 expression and impairs the beneficial action of melatonin on cell viability and apoptosis prevention. Moreover, some of the sirtuin 1 substrates studied also reversed the melatonin effect when sirtinol is added to the cells, mainly p53. Globally, these results add weight to the findings of previous reports, indicating a new role for melatonin in improving cell function gated to an increased neuroprotective role for the sirtuin 1 pathway.

Modulation of SIRT1 expression in different neurodegenerative models and human pathologies

We examined the expression of SIRT1 in several experimental paradigms of human pathologies. We used a neuroblastoma cell line (B65), neuronal primary cultures (hippocampus and cerebellar granule cells) and in vivo approaches in rat and senescence murine models (SAM). Cell cultures and rats were treated with several well-know neurotoxins, i.e. rotenone, MPP(+), kainate and 3-nitropropionic acid. Subsequently, SIRT1 expression was compared in these different paradigms of neurotoxicity. The pattern of expression of SIRT1 in proliferating cell cultures (B65) was different to that in quiescent cell cultures. In the murine model of senescence (senescence-accelerated mice prone, SAMP8), SIRT1 expression progressively decreased, while in the control strain (senescence-accelerated mice resistant, SAMR1) it increased. Finally, we studied human samples of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and Huntingtons diseases (HD). SIRT1 expression decreased dramatically in HD, but there were no significant changes in Parkinson-related illnesses. In conclusion, SIRT1 expression may be a good sensor of toxic neuronal processes.

Favorable effects of a prolonged treatment with melatonin on the level of oxidative damage and neurodegeneration in senescence-accelerated mice

Abstract:  Senescence‐accelerated mice (SAMP8) and senescence‐accelerated resistant mice (SAMR1) were studied at 5 and 10 months of age, respectively. In the animals, neurodegenerative processes and how they were influenced by melatonin were examined. Melatonin (10 mg/kg) or vehicle (ethanol at 0.066%) treatments were administrated from the age of 1 to 9 months in the drinking water. Differences in the neurodegenerative markers examined were found between the two strains with a more damaged protein, phosphorylated Tau at Ser392, increased neurofibrillary tangles (NT) and higher α‐synuclein expression in SAMP8 versus SAMR1 mice overall, when the mice were 10 months of age. Changes in density of receptors and oxidative stress‐related signaling with age were found in the brains of SAM strains at 10 months as shown by a marked decrease in the level of MT‐1 melatonin receptor and retinoic acid receptor‐related orphan receptor (ROR)‐α1. This diminution was earlier and more pronounced in SAMP8 mice. Likewise, the levels of nuclear factor‐kappa B (NF‐kB) transcriptional factor were higher in SAMP8 mice compared with SAMR1 mice regardless of age confirming the direct role of oxidative stress in the aging process. Treatment with melatonin in SAMP8 and SAMR1 mice reduced the neurodegenerative changes with an increase of ROR‐α1 levels without an apparent influence in the levels of MT‐1 receptor. However, different melatonin effects on NF‐kB signaling were observed suggesting that NF‐kB could trigger inflammatory processes in a different way, being SAM strain‐dependent and associated with age‐related oxidative stress levels. The effectiveness of melatonin in improving age‐related neural impairments is corroborated.

Changes in oxidative stress parameters and neurodegeneration markers in the brain of the senescence-accelerated mice SAMP-8

The senescence-accelerated strains of mice (SAMP) are well-characterized animal models of senescence. Senescence may be related to enhanced production or defective control of reactive oxygen species, which lead to neuronal damage. Therefore, the activity of various oxidative-stress related enzymes was determined in the cortex of 5 months-old senescence-accelerated mice prone-8 (SAMP-8) of both sexes and compared with senescence-accelerated mice-resistant-1 (SAMR-1). Glutathione reductase and peroxidase activities in SAMP-8 male mice were lower than in male SAMR-1, and a decreased catalase activity was found in both male and female SAMP-8 mice, which correlates with the lower catalase expression found by Western blotting. Nissl staining showed marked loss of neuronal cells in the cerebral cortex of five month-old SAMP-8 mice. SAMP-8 mice also had marked astrogliosis and microgliosis. We also found an increase in caspase-3 and calpain activity in the cortex. In addition, we observed morphological changes in the immunostaining of tau protein in SAMP-8, indicative of a loss of their structural function. Altogether, these results show that, at as early as 5 months of age, SAMP-8 mice have cytological and molecular alterations indicative of neurodegeneration in the cerebral cortex and suggestive of altered control of the production of oxidative species and hyper-activation of calcium-dependent enzymes.

Chronic administration of melatonin reduces cerebral injury biomarkers in SAMP8.

Abstract:  Certain effects of melatonin on senescence were investigated. The experimental model used was 10‐month‐old senescence‐accelerated mouse prone 8 (SAMP8). The mice in the experiment were administered melatonin (10 mg/kg) from the age of 1 month. Results showed that chronic administration of melatonin decreased cell loss in the cerebral cortex and reduced oxidative damage in protein and lipids. There are several studies suggesting that the activation of the cdk5/p35 pathway at its cleavage to cdk5/p25 may play a role in hyperphosphorylation of tau during aging and neurodegenerative diseases. Melatonin not only reduced the cerebral aging disturbances, but also prevented tau hyperphosphorylation present in the experimental model used in this study. Melatonin reduced cdk5 expression, as well as the cleavage of p35 to p25. The other tau kinase studied, GSK3β, showed a reduction in this activity in comparison with SAMP8 nontreated SAMP8. These data indicate that melatonin possesses neuroprotective properties against cerebral damage gated to senescence. Moreover, these data suggest that the cdk5/GSKβ signaling cascade has a potential role as a target for neurodegenerative diseases related to aging.

Lithium treatment decreases activities of tau kinases in a murine model of senescence.

Lithium modulates glycogen synthase kinase 3β (GSK-3β), a kinase involved in Alzheimer disease-related tau pathology. To investigate mechanisms of aging and the potential therapy of lithium in neurodegenerative disease, we treated senescence-accelerated mouse (SAM)P8 mice, a murine model of senescence, and mice of the control SAMR1 strain with lithium. The treatment reduced hippocampal caspase 3 and calpain activation, indicating that it provides neuroprotection. Lithium also reduced both the levels and activity of GSK-3β and the activity of cyclin-dependent kinase 5 and reduced hyperphosphorylation of 3 different phosphoepitopes of tau: Ser199, Ser212, and Ser396. In lithium-treated primary cultures of SAMP8 and SAMR1 cerebellar neurons, there was a marked reduction in protease activity mediated by calpain and caspase 3. Both lithium and SB415286, a specific inhibitor of GSK-3β, reduced apoptosis in vitro. Taken together, these in vivo and in vitro findings of lithium-mediated reductions in GSK-3β and cyclin-dependent kinase 5 activities, tau phosphorylation, apoptotic activity, and cell death provide a strong rationale for the use of lithium as a potential treatment in neurodegenerative diseases.

Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence‐accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brains susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence‐accelerated‐resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser199 and Ser396 in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3β and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3β by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser396. Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1–5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.