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

TTBK1 and CK1 inhibitors restore TDP-43 pathology and avoid disease propagation in lymphoblast from Alzheimer’s disease patients

IntroductionTDP-43 proteinopathy in Alzheimer’s disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism.MethodologyWe have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments.ResultsTDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors (decreasing phosphorylation and increasing nuclear localisation), Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs.ConclusionTTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease

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

Tideglusib, a Non-ATP Competitive Inhibitor of GSK-3β as a Drug Candidate for the Treatment of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is the most common degenerative motor neuron disease in adults. About 97% of ALS patients present TDP-43 aggregates with post-translational modifications, such as hyperphosphorylation, in the cytoplasm of affected cells. GSK-3β is one of the protein kinases involved in TDP-43 phosphorylation. Up-regulation of its expression and activity is reported on spinal cord and cortex tissues of ALS patients. Here, we propose the repurposing of Tideglusib, an in-house non-ATP competitive GSK-3β inhibitor that is currently in clinical trials for autism and myotonic dystrophy, as a promising therapeutic strategy for ALS. With this aim we have evaluated the efficacy of Tideglusib in different experimental ALS models both in vitro and in vivo. Moreover, we observed that GSK-3β activity is increased in lymphoblasts from sporadic ALS patients, with a simultaneous increase in TDP-43 phosphorylation and cytosolic TDP-43 accumulation. Treatment with Tideglusib decreased not only phospho-TDP-43 levels but also recovered its nuclear localization in ALS lymphoblasts and in a human TDP-43 neuroblastoma model. Additionally, we found that chronic oral treatment with Tideglusib is able to reduce the increased TDP-43 phosphorylation in the spinal cord of Prp-hTDP-43A315T mouse model. Therefore, we consider Tideglusib as a promising drug candidate for ALS, being proposed to start a clinical trial phase II by the end of the year

Motor neuron preservation and decrease of in vivo TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment

athogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS

Inactivation of CDK/pRb Pathway Normalizes Survival Pattern of Lymphoblasts Expressing the FTLD-Progranulin Mutation c.709-1G>A

8 figuras, 2 tablasBackground 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-TDPThis work has been supported by grants from Ministry of Education and Science (SAF2007-61701, SAF2010-15700, SAF2011-28603), Fundación Eugenio Rodríguez Pascual, and Basque Government (Saiotek program 2008–2009). NE holds a fellowship of the JAE predoctoral program of the CSICPeer 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

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (<0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: (1) accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; (2) decreased levels of aspartate, glutamate, and alanine; and (3) 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: (1) 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. (2) 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 (<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.This work was supported by grants from the Comisión Asesora de Investigación Científica y Técnica (0652-81 and 1674-82) and the Fondo de Investigaciones Sanitarias, Spain

Rate-limiting steps for hepatic gluconeogenesis. Mechanism of oxamate inhibition of mitochondrial pyruvate metabolism

6 p.-5 fig.-2 tab.Oxamate, structural analog of pyruvate, inhibits gluconeogenesis from pyruvate or substrates yielding pyruvate. The inhibitory effect is the result of a decreased mitochondrial pyruvate utilization. Although the inhibition of gluconeogenesis is competitive for pyruvate, in isolated mitochondria oxamate displays a mixed type kinetics inhibitory pattern of pyruvate utilization. Evidence is presented indicating that this mixed type pattern of inhibition is the result of the action of oxamate on two different sites: noncompetitive inhibition of pyruvate carboxylation, and competitive inhibition of pyruvate entry into the mitochondria. At concentrations of pyruvate above 0.4 mM, although pyruvate carboxylation is decreased by 40% by oxamate, no detectable effects on the gluconeogenic flux were observed. This finding strongly indicates that pyruvate carboxylase is not an important rate-limiting step for hepatic gluconeogenesis. Thus, the inhibition of gluconeogenesis at low pyruvate concentrations (less than 0.4 mM) seems to be the result of an interaction of oxamate with the mitochondrial pyruvate translocator, indicating that pyruvate transport across the mitochondrial membrane is the first nonequilibrium step in the gluconeogenic pathway when low physiological concentrations of this substrate are utilized.This work has been supported in part by Grants 174 and 1674 from the Spanish Comisión Asesora de Investigación Científica y Ténica, and Grants 8 5/839 and 85/1321 from the Fondo de Investigaciones Sanitarias.Peer reviewe