Downregulation of ERK1/2 activity by CaMKII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer’s disease patients

11 páginas, 9 figuras, 2 tablas -- PAGS nros. 1090-1100Previously, we reported a Ca2+/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimer’s 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 degradationThis work has been supported by grants from Ministerio de Economía y Competitividad (SAF2007-624505, SAF2011-28603) and Fundación Ramón Areces to A.M.-R. N.E. holds a fellowship of the JAE predoctoral program of the CSICPeer reviewe

Targeting TDP-43 phosphorylation by Casein Kinase-1δ inhibitors: a novel strategy for the treatment of frontotemporal dementia

[Background] Mutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6.[Results] We report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation.[Conclusions] Our results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies.This work has been supported by grants from The Spanish Ministry of Economy and Competitiveness (projects SAF2012-37979-C03-01 to Ana Martinez and SAF2011-28603 to Angeles Martín-Requero). Moreover, Dr. Angeles Martín-Requero was supported by Ramón Areces foundation and Dr. López de Munain received research support from Ilundain Foundation.Peer reviewe

Role of Resveratrol and Selenium on Oxidative Stress and Expression of Antioxidant and Anti-Aging Genes in Immortalized Lymphocytes from Alzheimer's Disease Patients

Oxidative damage is involved in the pathophysiology of age-related ailments, including Alzheimer's disease (AD). Studies have shown that the brain tissue and also lymphocytes from AD patients present increased oxidative stress compared to healthy controls (HCs). Here, we use lymphoblastoid cell lines (LCLs) from AD patients and HCs to investigate the role of resveratrol (RV) and selenium (Se) in the reduction of reactive oxygen species (ROS) generated after an oxidative injury. We also studied whether these compounds elicited expression changes in genes involved in the antioxidant cell response and other aging-related mechanisms. AD LCLs showed higher ROS levels than those from HCs in response to H2O2 and FeSO4 oxidative insults. RV triggered a protective response against ROS under control and oxidizing conditions, whereas Se exerted antioxidant effects only in AD LCLs under oxidizing conditions. RV increased the expression of genes encoding known antioxidants (catalase, copper chaperone for superoxide dismutase 1, glutathione S-transferase zeta 1) and anti-aging factors (sirtuin 1 and sirtuin 3) in both AD and HC LCLs. Our findings support RV as a candidate for inducing resilience and protection against AD, and reinforce the value of LCLs as a feasible peripheral cell model for understanding the protective mechanisms of nutraceuticals against oxidative stress in aging and AD

Papel de la progranulina en los mecanismos de regulación de superviviencia-muerte celular en la demencia frontotemporal

285 p.-17 fig.-8 tab.Frontotemporal lobar degeneration (FTLD) is a genetically complex neurodegenerative disorder, accounting for 20% of patients with an onset of dementia before 65 years (Ikeda, et al., 2004). The clinical symptoms associated with FTLD are diverse, including behavior and personality changes, language disorders of expression and comprehension and cognitive impairment with early preservation of memory (Neary, et al., 1998). To date, the clinicopathologic spectrum of FTLD includes not only the frontal and temporal variants of FTD, frontotemporal degeneration with abnormal behavior (bvFTD), primary progressive aphasia (PPA) or semantic dementia (SD), but also corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), progressive subcortical gliosis (PSG) and FTD with motor neuron disease (FTD-EMN) (Van Langenhove, et al., 2012). Under a microscope, common pathologic changes of FTLD are atrophy brain region presenting neuronal loss and gliosis in cortices of atrophied frontal and temporal lobes. Histochemically, FTLD can be categorized according to the major component of the celular inclusions deposited in the brain. In the majority of cases, the pathological protein is either the microtubule-associated protein tau or the transactive response DNA-binding protein TDP-43 (FTLD-tau, FTLD-TDP respectively), a small number of cases present inclusions of fused in sarcoma (FUS) protein (FTLD-FUS) or inclusions of a protein that can not be identified and that can only be unveiled by immunostaining against the proteins of the ubiquitin proteasome system (UPS) (FTLD-UPS) (Cairns, et al., 2007,Mackenzie, et al., 2011). A positive family history of FTLD is present in 25-50% of cases and the transmission is usually autosomal domi nant. A few genes have been associated with familial FTLD including microtubule-associated protein tau (MAPT), progranulin (GRN) (Baker), transactive response (TAR) DNA-binding protein-43 (TARDBP), chromatin-modifying 2B protein (CHMP2B), Valosin-containing protein (VCP), and chromosome 9 open reading frame 72 (C9ORF72) genes (Sieben, et al., 2012). Mutations in the GRN gene are the major cause of familial FTLD-TDP (Baker, et al., 2006,Cruts, et al., 2006). To date, more than 60 mutations have been reported in GRN gene (Gijselinck, et al., 2008). Most of the pathogenic mutations are predicted to cause functional null alleles with premature termination of the GRN coding sequence, resulting in nonsense-mediated decay (NMD) of the mutant messenger RNAs (mRNAs), suggesting that progranulin (PGRN) haploinsufficiency is a major pathogenic mechanism. Little is known about the normal function of PGRN in the Central Nervous Sistem (CNS) and the mechanism by which PGRN haploinsufficency leads to neurodegeneration in FTLD remained speculative. In the last decade, mounting evidences support the hypothesis that cell cycle reentry of postmitotic neurons and neural stem cells precedes many instances of neuronal dead (Herrup, et al., 2004). Based in the mitogenic and neuotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability.An alternative strategy to study the pathogenesis of FTLD is the use of non-neuronal cells from patients. Numerous observations indicate that, while the predominant clinical expression arises from brain, the neurodegenerative diseases have systemic expression at the cellular and molecular levels. Considerable precedents exist for studying neurodegenerative diseases with peripheral tissues including lymphocytes, fibroblast and platelets (Bialopiotrowicz, et al., 2012). The use of peripheral tissues could complement studies of autopsy samples and provide a useful tool with which to investigate dynamic processes such as signal transduction mechanisms, oxidative metabolism among others. Aims: The main goal of this work was to study the influence of PGRN haploinsufficiency, caused by a prevalent ancestral mutation in GRN gene related to Basque population, c.709- 1G>A, in cell cycle and survival regulation of peripheral blood cells from carriers of the GRN mutation. The possibility to detect molecular alterations in non-neuronal cells, easily accessible, from control, asymptomatic and patients, could provide a useful tool for the early diagnosis of this complex disorder, and particularly to search for treatment or neuroprotective strategies. To this end, we generated lymphoblastoid cell lines from c.709-1G>A carriers diagnosed of FTLD or asymptomatic and control individuals by infecting with the Epstein Barr virus (EBV). Although FTLD symptoms reflect preferential neuronal loss in specific brain regions, PGRN is expressed in almost all tissues, so abnormalities outside the brain might be expected. Moreover, this experimental model has been previously used to study cell cyclerelated events associated with neurodegeneration in other neurodegenerative diseases, e. g. Alzheimer’s disease. Results: Our results show that PGRN haploinsufficiency increased cell cycle activity in immortalized lymphocytes from FTLD patients. 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. In addition, the lymphoblast carriers of the c.709- 1G>A mutation display a small but significant decrease in the nuclear content of TDP-43. Taken together, these observations suggest that a loss of nuclear TDP-43 function in repressing CDK6 may be involved in the increased pRb phosphorylation, and enhanced proliferative activity of PGRN deficient cells. Assuming that the cell cycle disturbances reported here could be peripheral signs of the disease, our results suggest that neurons of c.709- Degeneración Lobar Frontotemporal asociada a mutaciones en progranulina 1G>A mutation carriers are at high risk of entering an unscheduled cell cycle that would then drive them to death. It is suggested that therapeutic interventions aimed at ameliorating cell cycle progression activity may have a positive impact in FTLD. Using the same cells of FTLD patients, we performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carries of c.709-1G>A GRN mutation. Our results suggest that the CDK6/pRb pathway also is enhanced after the serum deprivation. 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 normal response of lymphoblasts from FLTD-TDP patients to trophic factor withdrawal or cell proliferation. Our results suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.The next step in this work was analyzing potential alterations in signaling pathways induced by PGRN haploinsufficency to explain the altered cellular response to serum stimulation. To this end we use specific inhibitors of main pathways as well as conditioned medium from control and PGRN-deficient lymphoblast or exogenous recombinant human PGRN (rhPGRN). Our results show that the specific signaling cascade implicated in the upregulation of CDK6 activity and cell proliferation of GRN mutation bearing lymphoblasts is Ca2+, protein kinase C (PKC), and pertussis toxin (PTX)-dependent and involves activationof the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathway. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that non-canonical 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 (pCaMKII). Moreover, treatment of control cells with exogenous Wnt5a activated Ca2+/calmodulin kinase II (CaMKII), increased activity of the ERK1/2 pathway 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 FTLDTDP pathology and suggest a novel target for therapeutic intervention. One of the aims in this thesis was to search for new clinical approximations and therapies for the treatment of FTLD-TDP using lymphoblastoid cell lines form carriers of c.709-1G>A GRN mutation. In this regard, we investigated the effects of drugs able to increase PGRN levels, either activating the GRN gene expression such as SAHA, or at posttranslational level as chloroquine (CQ), as well as specific inhibitors of the ERK1/2 signaling cascade such as Selumentinib or MEK162. Considering the haploinsufficiency mechanism, GRN is a particularly appealing gene for drug targeting. On the other hand, given that PGRN deficit appears to induce alterations in the ERK1/2 cascade, it is possible to envision new avenues for therapeutic intervention in PGRN-deficient FTLD. In this regard, the use of highly selective inhibitors of BRAF (v-raf-1 murine leukemia viral oncogene homolog B1) and MEK1/2 (Mitogen-activated protein kinase kinase or ERK kinase 1 and 2), the upstream activators of ERK1/2, have shown promising results in clinical trials, including in previously intratable diseases such as melanoma. Our results indicate that SAHA and CQ were effective in restoring the PGRN levels of c.709-1G>A carriers, and prevented the enhanced activation of ERK1/2 activity. Selumetinib, and MEK160, inhibitors of the ERK1/2 cascade, normalized the response of PGRN deficient lymphoblasts to serum activation. Both sets of drugs ultimately modulate the CDK6/pRb pathway and therefore regulate cell survival/death mechanisms. Considering that these drugs are already used in clinic for treatment of other diseases, with good tolerance, it is plausible that they may serve as novel therapeutic drugs for FTLD associated to GRN mutations. Conclusions: 1.- Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A mutation bearing lymphoblasts and this feature is controlling the mechanism of regulation of proliferation/death in progranulin deficient cells. 2.- Our results established a molecular link between misslocalization of TDP-43 and increased CDK6 levels, pRb phosphorylation, and the control of proliferation/death in lymphoblasts from c.709-1G>A carriers 3.- We provided evidence that PGRN haplinsufficiency aberrantly increased noncanonical Gαi dependent-Wnt signaling leading to activation of ERK1/2-mediated stimulation of proliferation of lymphoblast derived from individuals bearing the FTLD-associated GRN mutation. 4.-Our results indicate that the use of drugs able to increase PGRN levels or blocking the ERK1/2 pathway, restore the normal cell response to serum stimulation, by preventing the overactivation of the CDK6/pRb cascade in c.709-1G>A GRN mutation carriers. 5.- The distinct functional features of lymphoblastoid cell from c.709-1G>A carriers offer an inavaluable, noninvasive tool to investigate the etiopathogenesis of FTLDEl trabajo aquí presentado ha sido realizado con cargo a los siguientes proyectos de investigación: MICINN SAF2010-15700 (2010-2011). “Papel de Progranulina en los mecanismos de regulación de supervivencia/muerte en la demencia frontotemporal”. MICINN SAF2011-28630 (2012-2014). “Mecanismos moleculares neurodegenerativos en la demencia frontotemporal (FTLD-TDP): Déficit de progranulina, desregulación del ciclo celular y acumulación de TDP-43”. FUNDACIÓN RAMÓN ARECES (2012-2015). “Mecanismos moleculares, modelos experimentales y aproximaciones terapéuticas en la demencia lobar frontotemporal (DLFT-TDP)”.Peer reviewe

Progranulin deficiency reduces CD K4/6/pRb activation and survival of human neuroblastoma SH-SY5Y cells

36 p.-10 fig.-1 tab.Null mutations in GRN are associated with Frontotemporal Lobar with TDP-43 inclusions (FTLDTDP).However the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/pRb pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis. (i) Inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN deficient cells. (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induced death of control SH-SY5Y cells. (iii) PGRN deficient cells show reduced stimulation of PI3K/Akt, ERK1/2 and CDK4/6 activities compared with control cells in the absence of serum. (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces.Peer 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

Control of cell adhesion and migration by Podocalyxin. Implication of Rac1 and Cdc42

23 p.-4 fig.-1 fig.supl.Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell–cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesionThe work was funded with Grants from the Spanish Plan of R&D, SAF2007-61701 and BFU2010-15237Peer reviewe

Calmodulin levels in blood cells as a potential biomarker of Alzheimer's disease

Abstract Introduction The clinical features of Alzheimer’s disease (AD) overlap with a number of other dementias and conclusive diagnosis is only achieved at autopsy. Accurate in-life diagnosis requires finding biomarkers suitable for early diagnosis, as well as for discrimination from other types of dementia. Mounting evidence suggests that AD-dependent processes may also affect peripheral cells. We previously reported that calmodulin (CaM) signaling is impaired in AD lymphoblasts. Here, we address the issue as to whether the assessment of CaM levels in peripheral cells could serve as a diagnostic biomarker. Methods A total of 165 subjects were enrolled in the study, including 56 AD patients, 15 patients with mild cognitive impairment, 7 with frontotemporal dementia associated with progranulin mutations, 4 with dementia with Lewy bodies, 20 patients with Parkinson’s disease, 10 with amyotrophic lateral sclerosis, 5 with progressive supranuclear palsy, and 48 cognitively normal individuals. CaM levels were then analyzed in lymphoblasts, peripheral blood mononuclear cells and plasma. Receiver operating characteristic (ROC) curve analyses were employed to evaluate the diagnostic performance of CaM content in identifying AD patients. Results Compared with control individuals, CaM levels were significantly increased in AD cells, but not in the other neurodegenerative disorders. CaM levels differentiated AD from control with a sensitivity of 0.89 and a specificity of 0.82 and were not dependent on disease severity or age. MCI patients also showed higher levels of the protein. Conclusions CaM levels could be considered a peripheral biomarker for AD in its early stage and help to discriminate from other types of dementia.This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces.Peer 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