Noemí Esteras

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.

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

Frontotemporal 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 G(1)/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 degeneration.

PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers.

Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle-related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca(2+), protein kinase C-dependent, and pertussis toxin-sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca(2+)/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

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

Previous work indicated that changes in Ca(2+)/calmodulin (CaM) signaling pathway are involved in the control of proliferation and survival of immortalized lymphocytes from Alzheimers 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 Ca(2+) 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 Ca(2+)/CaM-dependent signaling pathways, may be important in the etiopathogenesis of AD.

Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer’s disease patients

Previously, we reported a Ca(2+)/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimers disease (AD) lymphoblasts. These cell lines showed downregulation of extracellular signal-regulated kinase (ERK)1/2 activity and elevated content of p21 compared with control cells. The aim of this study was to delineate the molecular mechanism underlying the distinct regulation of p21 content in AD cells. Quantitative reverse transcription polymerase chain reaction analysis demonstrated increased p21 messenger RNA (mRNA) levels in AD cells. The ERK1/2 inhibitor, PD98059, prevented death of control cells and enhanced p21 mRNA and protein levels. The CaM antagonist, calmidazolium, and the CaMKII inhibitor, KN-62, normalized the survival pattern of AD lymphoblasts by augmenting ERK1/2 activation and reducing p21 mRNA and protein levels. Upregulation of p21 transcription in AD cells appears to be the consequence of increased activity of forkhead box O3a (FOXO3a) as the result of diminished ERK1/2-mediated phosphorylation of this transcription factor, which in turn facilitates its nuclear accumulation. Murine double minute 2 (MDM2) protein levels were decreased in AD cells relative to control lymphoblasts, suggesting an impairment of FOXO3a degradation.

Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream strategies for the treatment of progranulin deficient frontotemporal dementia

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disorder marked by mild-life onset and progressive changes in behavior, social cognition, and language. Loss-of-function progranulin gene (GRN) mutations are the major cause of FTLD with TDP-43 protein inclusions (FTLD-TDP). Disease-modifying treatments for FTLD-TDP are not available yet. Mounting evidence indicates that cell cycle dysfunction may play a pathogenic role in neurodegenerative disorders including FTLD. Since cell cycle re-entry of posmitotic neurons seems to precede neuronal death, it was hypothesized that strategies aimed at preventing cell cycle progression would have neuroprotective effects. Recent research in our laboratory revealed cell cycle alterations in lymphoblasts from FTLD-TDP patients carrying a null GRN mutation, and in PGRN deficient SH-SY5Y neuroblastoma cells, involving overactivation of the ERK1/2 signaling pathway. In this work, we have investigated the effects of PGRN enhancers drugs and ERK1/2 inhibitors, in these cellular models of PGRN-deficient FTLD. We report here that both restoring the PGRN content, by suberoylanilide hydroxamic acid (SAHA) or chloroquine (CQ), as blocking ERK1/2 activation by selumetinib (AZD6244) or MEK162 (ARRY-162), normalized the CDK6/pRb pathway and the proliferative activity of PGRN deficient cells. Moreover, we found that SAHA and selumetinib prevented the cytosolic TDP-43 accumulation in PGRN-deficient lymphoblasts. Considering that these drugs are able to cross the blood-brain barrier, and assuming that the alterations in cell cycle and signaling observed in lymphoblasts from FTLD patients could be peripheral signs of the disease, our results suggest that these treatments may serve as novel therapeutic drugs for FTLD associated to GRN mutations.

Systematic Evaluation of Magnetic Resonance Imaging and Spectroscopy Techniques for Imaging a Transgenic Model of Alzheimer's Disease (AβPP/PS1)

Murine models of Alzheimers disease (AD) provide means to detect and follow biomarker changes similar to those observed in humans. Non-invasive biomarkers, such as those provided by magnetic resonance imaging (MRI) and spectroscopy (MRS) methods are highly desirable, however, systematic studies of in vivo MRI/MRS methods to characterize the cerebral morphology and metabolic pattern of these mice remain scarce. We investigated sixteen consecutive slices from the brain of wild-type and AβPP/PS1 mice, obtaining a collection of T2 weighted, diffusion weighted and magnetization transfer weighted images as well as 1H PRESS spectra from the cortical and subcortical areas. Compared to controls, AβPP/PS1 mice show significant regional hyperintensities in T2 weighted images of the cerebral cortex, significant ventricular enlargement, and decreased hippocampal area and fractional magnetization transfer. MRS demonstrated an increase in the ratio of choline (Cho) to creatine (Cr) in the cortical and subcortical areas of the transgenic animals. A logistic regression classifier was implemented considering all parameters investigated, and revealed the most characteristic changes and allowed for the correct classification of control and AβPP/PS1 mice. In summary, the present results provide a useful frame to evaluate optimal MRI/MRS biomarkers for the characterization of AD models, potentially applicable in drug discovery processes, because of their non-invasive and repeatable nature in longitudinal studies.

On the mechanism of inhibition of p27 degradation by 15-deoxy-Δ12,14-prostaglandin J2 in lymphoblasts of Alzheimer's disease patients

Abstract.It has been proposed that neuroinflammation, among other factors, may trigger an aberrant neuronal cell cycle re-entry leading to neuronal death. Cell cycle disturbances are also detectable in peripheral cells from Alzheimer’s disease (AD) patients. We previously reported that the anti-inflammatory 15- deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) increased the cellular content of the cyclin-dependent kinase inhibitor p27, in lymphoblasts from AD patients. This work aimed at elucidating the mechanisms of 15d-PGJ2-induced p27 accumulation. Phosphorylation, half-life, and the nucleo-cytoplasmic traffic of p27 protein were altered by 15d-PGJ2 by mechanisms dependent on PI3K/Akt activity. 15d-PGJ2 prevents the calmodulin-dependent Akt overactivation in AD lymphoblasts by blocking its binding to the 85-kDa regulatory subunit of PI3K. These effects of 15d-PGJ2 were not mimicked by 9,10-dihydro-15-deoxy-Δ12,14- prostaglandin J2, suggesting that 15d-PGJ2 acts independently of peroxisome proliferator-activated receptor γ activation and that the α,β-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ2 is a requisite for the observed effects.

Inactivation of CDK/pRb pathway normalizes survival pattern of lymphoblasts expressing the FTLD-progranulin mutation c.709-1G>A.

Background Mutations in the progranulin (PGRN) gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP), although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK) CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation. Methodology/Principal Findings We performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal. Conclusion/Significance The use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.

Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer’s disease patients

Statins may exert beneficial effects on Alzheimer’s disease (AD) patients. Based on the antineoplastic and apoptotic effects of statins in a number of cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle and/or apoptosis. A growing body of evidence indicates that neurodegeneration involves the cell-cycle activation in postmitotic neurons. Failure of cell-cycle control is not restricted to neurons in AD patients, but occurs in peripheral cells as well. For these reasons, we studied the role of simvastatin (SIM) on cell survival/death in lymphoblasts from AD patients. We report here that SIM induces apoptosis in AD lymphoblasts deprived of serum. SIM interacts with PI3K/Akt and ERK1/2 signaling pathways thereby decreasing the serum withdrawal-enhanced levels of the CDK inhibitor p21Cip1 (p21) and restoring the vulnerability of AD cells to trophic factor deprivation.