Control de supervivencia/muerte celular en la patología de Alzheimer. Estudios en células extraneurales

Leída en la Universidad Complutense de Madrid. Facultad de Ciencias Biológicas el 06-24-2009; 240 págs.La enfermedad de Alzheimer (EA) es la forma más común de demencia en los ancianos. Una de las características más notables de la EA es la pérdida neuronal en áreas selectivas del cerebro, por causas aún no bien conocidas. Recientemente, se ha considerado la posibilidad de que un intento de las neuronas por entrar en un ciclo celular abortivo puede, eventualmente, conducirlas a la muerte celular por apoptosis. La EA afecta fundamentalmente al Sistema Nervioso Central, pero presenta también manifestaciones sistémicas que, aunque no tienen gran repercusión clínica, pueden ser de utilidad para el estudio de ciertos aspectos relacionados con la patología. Trabajos previos de nuestro laboratorio y de otros han demostrado que también se producen a lteraciones en la maquinaria del ciclo celular en células extraneurales. Utilizando linfocitos inmortalizados de pacientes de EA esporádica y controles de la misma edad hemos podido demostrar una respuesta alterada al suero, una mayor actividad proli ferativa y una menor vulnerabilidad a la muerte celular inducida por la ausencia de factores tróficos. En este trabajo, hemos estudiado alguno de los mecanismos moleculares implicados en el control de supervivencia/muerte celular. La alteración de la vía de señalización Ca2 /Calmodulina (CaM) parece ser responsable de la respuesta anómala de las células de EA. La adición de antagonistas de CaM restaura la respuesta celular normal ante la presencia o ausencia de suero. La interacción entre Ca2 /C aM y la ruta PI3K/Akt controla la tasa de proliferación celular, regulando a la baja los niveles del inhibidor de CDK, p27kip1. Por el contrario, la mayor resistencia a la ausencia de factores tróficos en los linfoblastos de EA se debe a la estimulac ión exacerbada y sostenida en el tiempo de Erk1/2. La estimulación de Erk1/2 depende también de Ca2 /CaM y se ejerce a través de cambios en el balance de proteínas pro y anti-apoptóticas. Por otra parte, hemos observado que el tratamiento de linfobl astos de EA con simvastatina, previene el aumento de proliferación y la activación de PI3K/Akt, así como la disminución de p21 y p27, inducidos por el suero en linfoblastos de pacientes con EA, por un mecanismo independiente del metabolismo del coles terol.Aunque nuestros resultados no se pueden extrapolar directamente al SNC, es posible que parte de los efectos beneficiosos de las estatinas para los pacientes de EA pudieran explicarse por la actividad antiproliferativa de la droga, que podría imFondo de Investigaciones Sanitarias (01/1194) y PI04/0312 Del Ministerio de Sanidad y Consumo por el Ministerio de Educación y Cienca (S AF2007-62405), por Caja Segovia Obra Social y por la Fundación E. Rodriguez PascualPeer reviewe

Distinct regulation of cell cycle and survival in lymphocytes from patients with Alzheimer´s disease and amyotrophic lateral sclerosis

9 páginas, 6 figuras, 1 tabla -- PAGS nros. 390-398Alterations in cell cycle progression seem to be associated with neuronal death in Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). We previously reported disturbances in the control of cell survival/death fate in immortalized lymphocytes from AD patients. These cell cycle dysfunction and impaired apoptosis were considered systemic manifestations of AD disease. The purpose of this study was to evaluate whether these abnormalities are characteristic of AD, or they may be seen in other neurodegenerative disorders such ALS. Our results indicate that alterations in signaling molecules, Akt and ERK1/2, and in the cyclin-dependent kinase complex inhibitors (CDKis) p21Cip1 and p27Kip1 are detectable in lymphoblasts from AD patients, but not in ALS patients, suggesting that these variables may be considered for the development of biomarkers of AD. However, lymphocytes from ALS patients do not represent a useful model to study cell cycle-related events associated with neurodegeneration of motoneuronsThis work has been supported by grants from the Spanish Ministry of Science and Innovation (SAF2007-62405) and Fundación Rodríguez Pascual. NE holds a fellowship of the JAE predoctoral program of the CSICPeer reviewe

Altered calmodulin degradation and signaling in non-neuronal cells from Alzheimer’s disease patients

11 páginas -- PAGS nros. 267-277Previous work indicated that changes in Ca2+/calmodulin (CaM) signaling pathway are involved in the control of proliferation and survival of immortalized lymphocytes from Alzheimer’s disease (AD) patients. We examined the regulation of cellular CaM levels in AD lymphoblasts. An elevated CaM content in AD cells was found when compared with control cells from age-matched individuals. We did not find significant differences in the expression of the three genes that encode CaM: CALM1, 2, 3, by real time RT-PCR. However, we observed that the half-life of CaM was higher in lymphoblasts from AD than in control cells, suggesting that degradation of CaM is impaired in AD lymphoblasts. The rate of CaM degradation was found to be dependent on cellular Ca2+ and ROS levels. CaM degradation occurs mainly via the ubiquitin-proteasome system. Increased levels of CaM were associated with overactivation of PI3K/Akt and CaMKII. Our results suggest that increased levels of CaM synergize with serum to overactivate PI3K/Akt in AD cells by direct binding of CaM to the regulatory α-subunit (p85) of PI3K. The systemic failure of CaM degradation, and thus of Ca2+/CaM-dependent signaling pathways, may be important in the etiopathogenesis of ADPeer reviewe

Enhanced proteasome-dependent degradation of the CDK inhibitor p27kip1 in immortalized lymphocytes from Alzheimer´s dementia patients

11 páginas, 11 figuras -- PAGS nros. 1474-1484Cyclin-dependent kinase inhibitor p27kip1 (p27), a critical determinant for cell cycle progression, is an important regulation target of mitogenic signals. We have recently reported the existence of a molecular link between decreased p27 levels and enhanced phosphorylation of pRb protein and proliferation of immortalized lymphocytes from Alzheimer's disease (AD) patients. These cell cycle disturbances might be considered systemic manifestations, which mirror changes thought to occur in the brain, where post-mitotic neurons have been shown to display various cell cycle markers prior to degeneration. This work was undertaken to delineate the molecular mechanisms underlying the p27 down-regulation associated with AD. To this end, we evaluated the p27 protein stability in control and AD lymphoblasts. Half-life of p27 protein was markedly reduced in lymphoblasts from AD patients compared with that in control cells. The increased phosphorylation of p27 at Thr187, rather than changes in the 26S proteasome activity, is likely responsible for the enhanced degradation of p27 in AD cells. The serum-induced enhanced proliferation of AD lymphoblasts and decreased levels of p27 were abrogated by calmodulin (CaM) antagonists. The findings presented here suggest that Ca2+/CaM-dependent overactivation of PI3K/Akt signaling cascade in AD cells, plays an important role in regulating p27 abundance by increasing its degradation in the ubiquitin-proteasome pathwayThis work has been supported by grants from the Spanish Fondo de Investigaciones Sanitarias (FIS PI040312). UM is a fellow from the Spanish Ministry of Education and Science, and FB holds a contract from the I3P program of the CSICPeer reviewe

G1/S cell cycle checkpoint dysfunction in lymphoblasts from sporadic Parkinson’s disease patients.

38 p.-8 fig.Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease among aging individuals, affecting greatly the quality of their life. However, the pathogenesis of Parkinson's disease is still incompletely understood to date. Increasing experimental evidence suggests that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Since cell cycle dysfunction is not restricted to neurons, we investigated this issue in peripheral cells from patients suffering from sporadic PD and age-matched control individuals. Here, we describe increased cell cycle activity in immortalized lymphocytes from PD patients, that is associated to enhanced activity of the cyclin D3/CDK6 complex, resulting in higher phosphorylation of the pRb family protein and thus, in a G1/S regulatory failure. Decreased degradation of cyclin D3, together with increased p21 degradation, as well as elevated levels of CDK6 mRNA and protein were found in PD lymphoblasts. Inhibitors of cyclin D3/CDK6 activity like sodium butyrate, PD-332991, and rapamycin were able to restore the response of PD cells to serum stimulation. We conclude that lymphoblasts from PD patients are a suitable model to investigate cell biochemical aspects of this disease. It is suggested that cyclin D3/CDK6-associated kinase activity could be potentially a novel therapeutic target for the treatment of PD.This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces to AMR, and Fundación Neurociencias y Envejecimiento to JAM.Peer reviewe

Alterations in cell cycle-related proteins in lymphoblasts from carries of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia

429.e7–429.e20 páginas, 8 figuras, 1 tablaFrontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G1/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degenerationThis work has been supported by grants from Ministry of Education and science (SAF2007-62405, SAF2010-15700), Fundación Eugenio Rodríguez Pascual, Diputación Foral de Gipuzkoa (76/08) and Basque Government (Saiotek program). N.E. holds a fellowship of the JAE predoctoral program of the CSIC. J.J. Merino holds a contract of the Ramon y Cajal Program of the Spanish Ministry of Science and InnovationPeer reviewe

On the mechanism of inhibition of p27 degradation by 15-deoxy-Δ12,14-prostaglandin J 2 in lymphoblasts of Alzheimer’s disease patients

13 páginas, 10 figuras -- PAGS nros. 3507-3519We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca2+/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca2+/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawalThis work has been supported by grants from Ministry of Education and Science (SAF2007–62405) and Fundación Eugenio Rodríguez Pascual. N.E. holds a fellowship of the JAE predoctoral program of the CSICPeer reviewe

3HMG-CoA reductasa inhibitor, simvastatin, inhibits cell cycle progression at the G1-S checkpoint in immortalized lymphocytes from Alzheimer´s disease patients independently of cholesterol lowering effects

8 páginas, 8 figuras -- PAGS nros. 352-359Recent work has suggested that statins may exert beneficial effects on patients suffering from Alzheimer's disease (AD). The pharmacological effects of statins extend beyond their cholesterol-lowering properties. Based on the antineoplastic and apoptotic effects of statins in several cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle. A growing body of evidence indicates that neurodegeneration involves the activation of cell cycle machinery in postmitotic neurons. We and others have presented direct evidence to support the hypothesis that the failure of cell cycle control is not restricted to neurons in AD patients, but that it occurs in peripheral cells as well. For these reasons, we found it worthy to study the role of simvastatin on cell proliferation in immortalized lymphocytes from AD patients. We report here that simvastatin (SIM) inhibits the serum-mediated enhancement of cell proliferation in AD by blocking the events critical for G1/S transition. SIM induces a partial blockade of retinoblastoma protein phosphorylation and inhibition of cyclin E/cyclin-dependent kinase (CDK)2 activity associated with increased levels of the CDK inhibitors p21Cip1 and p27kip1. These effects of SIM on AD lymphoblasts are dependent on inhibition of the proteasome-mediated degradation of p21 and p27 proteins. The antiproliferative effect of this natural statin may provide a therapeutic approach for AD disease.Statin therapy is a widely used treatment for hypercholesterolemia, reduces the risk of stroke, and improves cardiovascular functions (Farnier and Davignon, 1998). The statin family of drugs is competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in the synthesis of cholesterol (Corsini et al., 1995), which converts HMG-CoA to mevalonate (MEV). In the last decade, epidemiological, clinical, and experimental evidence has accumulated that links cholesterol to the development of AD, and recent studies showed that statin therapy might be of benefit in treating AD (Wolozin, 2004), although the efficacy of statins at slowing the cognitive decline and the progression of AD remains controversial. The link between cholesterol and AD is not surprising because the brain is the most cholesterol-rich organ, and disturbances in cholesterol homeostasis have been found associated with all major neuropathological features of AD (Shobab et al., 2005). Data from the Canadian Study of Health and Aging revealed a 74% reduced risk of developing AD in statin users compared with the total population (Rockwood et al., 2002). In other studies, a reduction in the risk of AD was observed in patients treated with statins compared with those receiving other medications typically used in cardiovascular disease (Wolozin et al., 2000), suggesting that statins in particular, rather than low cholesterol levels or lipid-lowering agents in general, are responsible for the reduction in the risk of AD.A number of nonlipid-dependent or pleiotropic effects of statins have been reported (Takemoto and Liao, 2001). By preventing the synthesis of isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in the mevalonate pathway, statins may alter the subcellular localization and function of multiple proteins, including protein kinases, the subunit of trimeric G proteins, and Ras and Ras-related GTPases (Danesh et al., 2002). Pleiotropic effects of statins include anti-inflammatory properties as well as antiproliferative and proapoptotic effects (Koyuturk et al., 2004), all of these potentially relevant in treating AD. Growing evidence suggests that neuronal cell cycle regulatory failure, leading to apoptosis, may be a significant component of the AD pathogenesis (Herrup et al., 2004; Nagy, 2005). Neuronal changes supporting alteration on cell cycle control in the etiology of AD include the ectopic expression of cell cycle markers, or cytoskeletal alterations (Busser et al., 1998; Copani et al., 2001; Nagy, 2005). Moreover, it was reported that a significant number of hippocampal pyramidal and basal forebrain neurons in AD brain have undergone full or partial DNA replication (Yang et al., 2001). These events occur early in the progression of AD (Yang et al., 2003; Yang and Herrup, 2007), suggesting that cell cycle-induced death is a central mechanistic feature of the disease. There is an expanding body of evidence supporting the ability of some statins to exert direct antiproliferative and proapoptotic effects on various types of human cells (Katz, 2005). On these grounds, we have considered the possibility that the beneficial effects of statin therapy in AD could be related to their ability to interfere with cell cycle machinery. To this aim, we have investigated the effects of simvastatin, a lipophilic statin, on the distinct features of control of cell proliferation in lymphoblasts derived from late-onset AD patients. Previous work from this and other laboratories has demonstrated that cell cycle regulatory deficit is not restricted to neurons in AD; it is also observed in peripheral cells such as lymphocytes or fibroblasts (Tatebayashi et al., 1995; Nagy et al., 2002; de las Cuevas et al., 2003), thus providing a useful tool to study the involvement of cell cycle-related events in the pathogenesis of AD. A number of studies have found AD-specific changes in molecules and signaling pathways in peripheral lymphocytes that mirror changes in the brain (Eckert et al., 1998; Nagy et al., 2002; Muñoz et al., 2007). Moreover, these cells have also been used to study molecular changes in response to therapy in AD (Casademont et al., 2003; Reale et al., 2005). Conversely, Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B-lymphoblastoid cell lines that resemble activated B cells (Neitzel, 1986). In fact, we have previously demonstrated identical cellular response to serum addition or withdrawal in peripheral lymphocytes or EBV-transformed lymphocytes from control and AD patients (Bartolomé et al., 2007; Muñoz et al., 2007). Taken together, these reports support a rationale for the use of peripheral cells, and in particular EBV lymphoblasts from AD patients as a model to further understand disease biology, progression, and therapeutic actions.We report here that simvastatin selectively blocked the serum-enhanced proliferation of lymphoblasts from AD patients by regulating critical events of the G1/S transition, suggesting potential additional molecular targets for the clinical efficacy of this drug in treating ADThis work was supported by Spanish Fondo de Investigaciones Sanitarias Grant FIS PI040312 and Ministry of Education and Science Grant SAF2007-62405). F.B. holds a contract of the Consejo Superior de Investigaciones Cientificas (I3P Program). U.M. is a fellow from the Spanish Ministry of Education and SciencePeer reviewe

Impaired apoptosis in lymphoblasts from Alzheimer´s disease patients: Cross-talk of Ca(2+)/calmodulin and ERK1/2 signaling pathways

12 páginas, 11 figuras -- PAGS nros. 1437-1448We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca2+/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca2+/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawalThis work has been supported by grants from the Spanish Fondo de Investigaciones Sanitarias (FIS PI040312). F.B. holds a contract of the CSIC (I3P program). N.C. and U.M. are fellows from the Fondo de Investigaciones Sanitarias de la Seguridad Social and Ministerio de Educación y Ciencia, respectivelyPeer reviewe

Cell cycle and Alzheimer´s disease. Studies in non-neuronal cells

10 páginas, 1 figura, 1 tabla -- PAGS nros. 121-130The most common cause of dementia in the elderly is Alzheimer disease (AD). In Europe, AD is a leading cause of death. The prevalence of this disease in developed countries is increasing because of very significant shifts in life expectancy and demographic parameters. AD is characterized by progressive cognitive impairment, resulting from dysfunction and degeneration of neurons in the limbic and cortical regions of the brain. Two prominent abnormalities in the affected brain regions are extracellular deposits of beta-amyloid, and intracellular aggregates of tau protein in neurofibrillary tangles. The role of these features in AD pathogenesis and progression is not yet completely elucidated. Research over the last decade has revealed that the activation of cell cycle machinery in postmitotic neurons is one of the earliest events in neuronal degeneration in AD. Here we summarize evidence to support the hypothesis that cell cycle alterations occur in cells other than neurons in AD sufferers. Immortalized lymphocytes from AD patients have show an enhanced rate of proliferation associated with G1/S regulatory failure induced by alterations in the cyclin/CDK/pRb/E2F pathway. In addition, these cells have a higher resistance to serum deprivation-induced apoptosis. These neoplastic-like features, cell cycle dysfunction and impaired apoptosis can be considered systemic manifestations of AD diseaseWork in the authors's laboratory has been supported from grants from the Spanish Fondo de Investigaciones Sanitarias (FIS 01/1194 and PI040312), the Spanish Ministry of Science and Innovation (SAF 2003-01458 and SAF 2007-62405), and the Fundacion E. Rodriguez PascualPeer reviewe