Ángeles Martín-Requero

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

Cyclin-dependent kinase inhibitor p27(kip1) (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 Alzheimers 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 Ca(2+)/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 pathway.

Enhanced Proliferation of Lymphoblasts from Patients with Alzheimer Dementia Associated with Calmodulin-Dependent Activation of the Na+/H+ Exchanger

We have recently reported that lymphoblasts from late onset Alzheimers disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.

Ca2+/calmodulin-dependent modulation of cell cycle elements pRb and p27kip1 involved in the enhanced proliferation of lymphoblasts from patients with Alzheimer dementia.

Failure of cell cycle regulation in neurons might be critically involved in the process of neurodegeneration in Alzheimers disease (AD). We present here evidence to support the hypothesis that cell cycle alterations occur in cells other than neurons in AD sufferers. Lymphocytes from AD patients immortalized with Epstein-Barr virus showed an enhanced rate of proliferation and increased phosphorylation of the retinoblastoma protein (pRb) and other members of the family of pocket proteins compared with cell lines derived from normal age-matched controls. The calmodulin antagonist calmidazolium, as well as W-7 and W-13, abrogated the enhanced activity of AD cells without altering the normal basal rate of proliferation. The effect of calmidazolium was accompanied by partially dephosphorylation of pRb. No changes were found in the expression levels of the G1 cyclin/Cdks complexes. However, lymphoblasts derived from AD patients showed reduced levels of the Cdk inhibitor p27(kip1), which were restored after anti-calmodulin treatment of the cultures. These observations suggest that in AD cells the enhanced rates of cell proliferation and phosphorylation of pRb and the intracellular content of p27(kip1) may be interrelated events controlled by a mechanism dependent on the Ca(2+)/calmodulin signaling pathway. The distinct functional features of lymphoblastoid cells from AD patients offer an invaluable, noninvasive tool to investigate the etiopathogenesis, and eventually, for the early diagnosis and prognosis of this devastating disease.

HMG-CoA Reductase Inhibitor Simvastatin Inhibits Cell Cycle Progression at the G1/S Checkpoint in Immortalized Lymphocytes from Alzheimer's Disease Patients Independently of Cholesterol-Lowering Effects

Recent work has suggested that statins may exert beneficial effects on patients suffering from Alzheimers 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.

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca2+/calmodulin and ERK1/2 signaling pathways

Abstract.We 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 withdrawal.

Altered transcriptional regulators in response to serum in immortalized lymphocytes from Alzheimer's disease patients

Cell cycle disturbances may precede neuronal death in Alzheimers disease (AD). We described alterations, in lymphocytes from AD patients, on the activity of two transcription factors, E2F and NF-kappaB, involved in cell proliferation and survival regulation, demonstrating that cell cycle dysfunction also occurs in peripheral cells. The analysis of E2F-DNA binding activity revealed lower signal intensity of protein-DNA complexes in AD cells, which correlated with increased phosphorylation of retinoblastoma (pRb) related proteins and enhanced proliferation. The calmodulin (CaM) antagonist calmidazolium (CMZ) abrogated the increased activity of AD cells by partially dephosphorylating pRb and p130. The NF-kappaB-DNA binding activity increased as cell progress through the cell cycle. The reduced NF-kappaB activation observed in AD cells appears not to be related to the increased phosphorylation of the pRb family proteins nor with the enhanced proliferative activity of AD cells, but seems to protect them from death induced by the loss of trophic support. Ca2+/CaM antagonists rescue NF-kappaB-DNA binding activity and sensitize AD cells to serum withdrawal. These observations suggest that disruption of Ca2+/CaM signaling pathway could be linked mechanistically to its pro cell survival actions, promoting enhanced proliferation or decreased cell death depending on the presence of growth-stimulatory signals.

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.

Altered cell cycle‐related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer’s disease [amyloid precursor protein/presenilin 1 (PS1)]

Cumulative evidence indicates that aberrant re‐expression of many cell cycle‐related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer’s disease (AD) pathogenesis. Evidence of cell cycle activation in post‐mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway‐specific RT2Profiler™ PCR Arrays, we detected changes in a number of cell cycle‐related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5′‐bromo‐2′‐deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin‐dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle‐related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

A megalin polymorphism associated with promoter activity and Alzheimer's disease risk†‡§

Elevated cerebral levels of amyloid beta‐protein (Aβ) occur in Alzheimers disease (AD), yet only a few patients show evidence of increased Aβ production. This observation suggests that many, perhaps most, cases of AD are caused by faulty clearance of Aβ. Megalin, which plays an important role in mediating Aβ clearance, is an attractive candidate gene for genetic association with AD. To investigate this hypothesis, we analyzed the megalin gene in a population of 2,183 subjects. Genetic analysis indicated that the rs3755166 (G/A) polymorphism located in the megalin promoter associated with risk for AD, dependently of apolipoprotein E genotype. The rs3755166 AA genotype frequency was significantly greater in AD patients than in control subjects. Furthermore, the luciferase reporter assay indicated that the rs3755166 A variant has 20% less transcriptional activity than the rs3755166 G variant. This study provides strong evidence that this megalin polymorphism confers a greater risk for AD, and supports a biological role for megalin in the neurodegenerative processes involved in AD.

Role of endogenous fatty acids in the control of hepatic gluconeogenesis

Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (less than 0.5 mM). The inhibitory effect of TDGA was overcome by medium and short chain fatty acid or by concentrations of pyruvate about 0.5 mM, but not by 10-fold higher concentrations of lactate. Despite decreased energy demand when gluconeogenesis was inhibited by TDGA, the pyruvate-induced increase in hepatic oxygen consumption was similar to the control, indicating that pyruvate transport across the mitochondrial membrane and/or its decarboxylation was not altered, and therefore can not be responsible for the inhibition of gluconeogenesis. Neither does a deficiency of acetyl-CoA explain the decrease in the gluconeogenic flux since high pyruvate loads (greater than 0.5 mM), beta-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (less than 0.3 mM), presumably physiological, pyruvate concentrations, its rate of mitochondrial utilization is limited by the activity of the monocarboxylate transporter. Agents that reduced the mitochondrial NAD system, and therefore reduced flux through pyruvate dehydrogenase, like beta-hydroxybutyrate or ethanol, stimulated gluconeogenesis when fatty acid oxidation was inhibited. The latter observations indicate that the primary role of endogenous fatty acid, when substrate availability is limiting, is to spare mitochondrial pyruvate by decreasing its oxidation, and therefore shifting the partitioning between the carboxylation and decarboxylation reactions toward the former.