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13 p.-4 fig.-1 tab., Deficiencies in Mannosidase β (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3′UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant β-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS., This work was supported by the projects PI16/01259 and PI20/01634, integrated into the Plan Nacional de I+D+I, AES 2013–2016 and 2017–2020; funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) “A way to make Europe”; and by the project PROYECTO/AEI/10.13039/50110001103 from the Agencia Española de Investigación. AGJ holds a Formación de Profesorado Universitario (FPU20/03387) fellowship from Ministerio de Ciencia e Innovación.

43 p.-8 fig.-2 tab., Alzheimer's disease (AD), the leading cause of dementia in the elderly, is a neurodegenerative disorder marked by progressive impairment of cognitive ability. Patients with AD display neuropathological lesions including senile plaques, neurofibrillary tangles, and neuronal loss. There are no disease-modifying drugs currently available. With the number of affected individuals increasing dramatically throughout the world, there is obvious urgent need for effective treatment strategy for AD. The multifactorial nature of AD encouraged the development of multifunctional compounds, able to interact with several putative targets. Here, we have evaluated the effects of two in-house designed cannabinoid receptors (CB) agonists showing inhibitory actions on β-secretase-1 (BACE-1) (NP137) and BACE-1/butyrylcholinesterase (BuChE) (NP148), on cellular models of AD, including immortalized lymphocytes from late-onset AD patients. Furthermore, the performance of TgAPP mice in a spatial navigation task was investigated following chronic administration of NP137 and NP148. We report here that NP137 and NP148 showed neuroprotective effects in amyloid-β-treated primary cortical neurons, and NP137 in particular rescued the cognitive deficit of TgAPP mice. The latter compound was able to blunt the abnormal cell response to serum addition or withdrawal of lymphoblasts derived from AD patients. It is suggested that NP137 could be a good drug candidate for future treatment of AD., This work has been supported by grants from Ministerio de Economía y Competitividad (CTQ2015-66313-R, and RTI2018-096100-B-I00) and CIBERNED to MLC.

16 p.-9 fig.-9 tab.-1 graph. abst., Hybrid compounds containing structural fragments of the Rho kinase inhibitor fasudil and the NRF2 inducers caffeic and ferulic acids were designed with the aid of docking and molecular mechanics studies. Following the synthesis of the compounds using a peptide-coupling methodology, they were characterized for their ROCK2 inhibition, radical scavenging, effects on cell viability (MTT assay), and NRF2 induction (luciferase assay). One of the compounds (1d) was selected in view of its good multitarget profile and good tolerability. It was able to induce the NRF2 signature, promoting the expression of the antioxidant response enzymes HO-1 and NQO1, via a KEAP1-dependent mechanism. Analysis of mRNA and protein levels of the NRF2 pathway showed that 1d induced the NRF2 signature in control and SOD1-ALS lymphoblasts but not in sALS, where it was already increased in the basal state. These results show the therapeutic potential of this compound, especially for ALS patients with a SOD1 mutation., This work was supported by the Comunidad de Madrid (grant B2017/BMD-3813), MINECO (grant RTI2018-097662-B-I00 to J.C.M. and PID2019-105600RB-I00 to A.M. and I.L.-B), ISCiii CIBERNED (CB18/05/00040 to A.M. and Á.M.-R. and CB06/05/0089 to I.L.-B.), and Fundela (2019/00325/001) to I.L.-B.

The goal of this work was to elucidate the pathogenic mechanism of an ALS-associated missense mutation, p.Arg573Gly (R573G), in the TBK1 gene. In particular, we seek to analyze the influence of this variant on the cellular levels and the function of TBK1 in immortalized cells from an ALS patient. The patient (Code# E7) belonged to a Spanish family with autosomal dominant disease manifesting in the sixth decade as either dementia or ALS. Four control individuals without signs of neurological disease were also included in this study. Our results indicate that the R375G TBK1 mutation did not affect the levels of mRNA nor the total TBK1 content; however, we observed a significant decrease in the levels of TBK1 phosphorylation, which is essential for TBK1 activity, as well as a significant reduction in the phosphorylation of p62 and RIPK1, known substrates for TBK1. Lymphoblasts from the R573G TBK1 mutation carrier patient display pathological TDP-43 homeostasis, showing elevated levels of phosphorylated TDP-43 and accumulation of the protein in the cytosolic compartment. In addition, the functional decrease in TBK1 activity observed in the E7 patient did not alter the autophagy flux, but it seems to be enough to increase ROS levels as well as the expression of pro-inflammatory cytokine IL-6.

14 p.-6 fig.-2 tab., 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., This research was funded by Madrid Community (B2017/BMD3813 ELA-Madrid), ISCiii (CIBERNED CB18/05/00040 and CB06/05/0089) and AEI (grant RTI2018-096100-B-I00 to Á.M.-R., PID2019-105600RB-I00 to A.M. and RTI2018-0988885-B-I00 to E.d.L.).

17 p.-6 fig.-1 tab., Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case., This research was funded by Comunidad de Madrid (grant ELA_Madrid B2017/BMD-3813 and S2017/BMD-3688), European Commission MSCA-ITN-ETN (grant DRIVE GA: 765912), ISCiii (CIBERNED CB18/05/000 and CB06/05/0089), Fundela (2019/00325/001) for I.L.-B. and AEI (grant PID2019-105600RB-I00) for, A.M. and I.L.-B.

CdSe quantum dots (QDs) are valuable tools for deciphering molecular mechanisms in cells. Their conjugation with antibodies offers a unique staining source with optimal characteristics, including increased photostability and narrow emission spectra, allowing for improved multiplexing capabilities using a single excitation source. In combination with pathology models derived from patients, they have great potential to contribute to quantitative molecular profiling and promote personalized medicine. However, the commercial availability of diverse CdSe QDs is still limited and characterization techniques must be performed to these materials or the conjugates developed in the lab to assure a proper function and reproducibility. Furthermore, while there is significant data of QDs experiments in cell lines, the literature with primary human cells is scarce, and QD behavior in these systems may be different. Rigorous characterization data of commercially available QDs and their conjugates with biomolecules of interest is needed in order to establish their potential for target labelling and expand their use among research labs. Here we compare the characterization and labelling performance of different QD conjugates in SH-SY5Y cell line, fibroblasts and immortalized lymphocytes derived from amyotrophic lateral sclerosis patients.

12 p.-6 fig.-2 tab., Pathogenesis 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-1delta (protein casein kinase-1delta) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1delta 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-1delta 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-1delta inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1delta inhibitors in a future disease-modifying treatment for ALS., This work was supported by MINECO (grant SAF2015-68580-C2-1-R to E.d.L., J.F.R., and C.R.C., SAF2016-76693-R to A.M. and CTQ2015-66313-R to A.M.R.),CIBERNED (CB06/05/0089 to C.R.C., J.F.R. and E.d.L.and CB18/05/00040 to A.M.R. and A.M.), Madrid Community (grant B2017/BMD3813 ELA-Madrid) and EU structural funds (FSE and FEDER).

Multitarget cannabinoids could be a promising therapeutic strategic to fight against Alzheimer's disease. In this sense, our group has developed a new family of indazolylketones with multitarget profile including cannabinoids, cholinesterase and BACE-1 activity. A medicinal chemistry program that includes computational design, synthesis and in vitro and cellular evaluation has allowed to us to achieve lead compounds. In this work, the synthesis and evaluation of a new class of indazolylketones have been performed. Pharmacological evaluation includes functional activity for cannabinoid receptors on isolated tissue. In addition, in vitro inhibitory assays in AChE/BuChE enzymes and BACE-1 have been carried out. Furthermore, studies of neuroprotective effects in human neuroblastoma SH-SY5Y cells and studies of the mechanisms of survival/death in lymphoblasts of patients with Alzheimer's disease have been achieved. The results of pharmacological tests have revealed that some of these derivatives (5, 6) behave as CB2 cannabinoid agonists and simultaneously show BuChE and/or BACE-1 inhibition., The authors gratefully acknowledge the financial support of Ministerio Español de Economía y Competitividad (CTQ2015- 66313-R) and would like to thank to Guadalupe Pablo and Carmen Merino for their excellent technical assistant. The work of MLC was supported by the Centre for Biomedical Research on Neurodegenerative Diseases (CIBERNED). GV was supported by a fellowship from SENECYT (Ecuador). Work dedicated in memoriam to Mercedes González Hormaizteguy, a great women scientist and wonderful person

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., This research was funded by Spanish MINECO and FEDER, grant number SAF2016-75508; Catalan Autonomous Government AGAUR, grant number 2017-SGR-106; and the CERCA Programme/Generalitat de Catalunya.

48 p.-7 fig.-1 graph. abst., Accumulation of TDP-43 in the cytoplasm of diseased neurons is the pathological hallmark of frontotemporal dementia-TDP (FTLD-TDP) and amyotrophic lateral sclerosis (ALS), two diseases that lack efficacious medicine to prevent or to stop disease progression. The discovery of mutations in the TARDBP gene (encoding the nuclear protein known as TDP-43) in both FTLD and ALS patients provided evidence for a link between TDP-43 alterations and neurodegeneration. Our understanding of TDP-43 function has advanced profoundly in the past several years; however, its complete role and the molecular mechanisms that lead to disease are not fully understood. Here we summarize the recent studies of this protein, its relation to neurodegenerative diseases, and the therapeutic strategies for restoring its homeostasis with small molecules. Finally, we briefly discuss the available cellular and animal models that help to shed light on TDP-43 pathology and could serve as tools for the discovery of pharmacological agents for the treatment of TDP-43-related diseases., Funding from Comunidad de Madrid (B2017/BMD3813 ELA-Madrid), MINECO (SAF2016-76693-R), ISCiii (CIBERNED, CB18/05/00040), and the La Caixa Junior Leader retaining program is acknowledged. V.N. holds a predoctoral FPU grant (FPU16/04466). S.N. was supported by a stipend from the Bio4Med Program (MarieSklodowska-Curie Cofound Grant Agreement 665735).

58 p.-11 fig.-2 tab., Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Aggregates of the nuclear protein TDP-43 have been recognized as a hallmark of proteinopathy in both familial and sporadic cases of ALS. Post-translational modifications of this protein, include hyperphosphorylation, cause disruption of TDP-43 homeostasis and as a consequence, promotion of its neurotoxicity. Among the kinases involved in these changes, cell division cycle kinase 7 (CDC7) plays an important role by directly phosphorylating TDP-43. In the present manuscript the discovery, synthesis, and optimization of a new family of selective and ATP-competitive CDC7 inhibitors based on 6-mercaptopurine scaffold are described. Moreover, we demonstrate the ability of these inhibitors to reduce TDP-43 phosphorylation in both cell cultures and transgenic animal models such as C. elegans and Prp-hTDP43 (A315T) mice. Altogether, the compounds described here may be useful as versatile tools to explore the role of CDC7 in TDP-43 phosphorylation and also as new drug candidates for the future development of ALS therapies., This work was supported by Comunidad de Madrid (grant B2017/BMD-3813), MINECO (grant SAF2016-76693-R and PID2019-105600RB-I00 to A.M., RTI2018-0988885-B-I00 to E.dL. and CTQ2015-66313-R to A.M.R.), ISCii CIBERNED (CB18/05/00040 to A.M., C.G. and A.M.R, and CB06/05/0089 to E.dL.), MECD (FPU14-00204. to E.R.), the National Institutes of Health (R01NS064131 to B.C.K.) and the Department of Veterans Affairs (Merit Review Grants I01BX003755 to B.C.K. and I01BX004044 to N.F.L.), CONICYT-PCI (grant REDES190074 to D.R. and A.M.) and FONDECYT (grant no. 11180604 to D.R.).

31 p.-5 fig.-1 tab., Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder of still unknown etiology that results in loss of motoneurons, paralysis, and death, usually between 2 and 4 years from onset. There are no currently available ALS biomarkers to support early diagnosis and to facilitate the assessment of the efficacy of new treatments. Since ALS is considered a multisystemic disease, here we have investigated the usefulness of immortalized lymphocytes from sporadic ALS patients to study TDP-43 homeostasis as well as to provide a convenient platform to evaluate TDP-43 phosphorylation as a novel therapeutic approach for ALS. We report here that lymphoblasts from ALS patients recapitulate the hallmarks of TDP-43 processing in affected motoneurons, such as increased phosphorylation, truncation, and mislocalization of TDP-43. Moreover, modulation of TDP-43 by an in-house designed protein casein kinase-1δ (CK-1δ) inhibitor, IGS3.27, reduced phosphorylation of TDP-43, and normalized the nucleo-cytosol translocation of TDP-43 in ALS lymphoblasts. Therefore, we conclude that lymphoblasts, easily accessible cells, from ALS patients could be a useful model to study pathological features of ALS disease and a suitable platform to test the effects of potential disease-modifying drugs even in a personalized manner., This work has been supported by grants from MINECO (CTQ2015-66313-R to A.M.-R. and SAF2016-76693-R to A.M.) and Madrid Community (B2017/BMD3813 ELA-Madrid).

43 p.-6 fig.-3 tab. BACKGROUND:Alzheimer's disease is a multifactorial disorder for which there is no disease-modifying treatment yet. CB2 receptors have emerged as a promising therapeutic target for Alzheimer's disease because they are expressed in neuronal and glial cells and their activation has no psychoactive effects. OBJECTIVE:The aim of this study was to investigate whether activation of the CB2 receptor would restore the aberrant enhanced proliferative activity characteristic of immortalized lymphocytes from patients with late-onset Alzheimer's disease. It is assumed that cell-cycle dysfunction occurs in both peripheral cells and neurons in patients with Alzheimer's disease, contributing to the instigation of the disease. METHODS:Lymphoblastoid cell lines from patients with Alzheimer's disease and age-matched control individuals were treated with a new, in-house-designed dual drug PGN33, which behaves as a CB2 agonist and butyrylcholinesterase inhibitor. We analyzed the effects of this compound on the rate of cell proliferation and levels of key regulatory proteins. In addition, we investigated the potential neuroprotective action of PGN33 in β-amyloid-treated neuronal cells. RESULTS:We report here that PGN33 normalized the increased proliferative activity of Alzheimer's disease lymphoblasts. The compound blunted the calmodulin-dependent overactivation of the PI3K/Akt pathway, by restoring the cyclin-dependent kinase inhibitor p27 levels, which in turn reduced the activity of the cyclin-dependent kinase/pRb cascade. Moreover, this CB2 agonist prevented β-amyloid-induced cell death in neuronal cells. CONCLUSION:Our results suggest that the activation of CB2 receptors could be considered a useful therapeutic approach for Alzheimer's disease. This work has been supported by a Grant from Ministerio de Economía y Competitividad (CTQ2015-66313-R). FB is supported by a Sara Borrell fellowship from the Spanish Instituto de Salud Carlos III.

2 p.- Correction to: Scientific Reports https://doi.org/10.1038/s41598-017-01678-4, published online 10 May 2017

Progranulin (PGRN) deficiency is considered the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Recent work unveiled a relationship between Wnt signaling and PGRN in cellular models of FTLD and cells of patients carrying loss-of-function GRN mutations. This study was undertaken to explore the relationship between PGRN deficit and Wnt signaling in the regulation of survival of GRN knockdown neuroblastoma SH-SY5Y cells (GRN KD). We report here that both canonical and noncanonical Wnt signaling cascades are overactivated in GRN KD cells. We detected increased expression levels of Wnt1 and Wnt5a ligands of the Frizzled receptors, as well as evidence for increased signaling of the Wnt/β-catenin and Wnt/Ca2+ cascades in PGRN deficient cells, such as increased nuclear content of β-catenin and higher levels of cyclin D1, or increased levels of the active form of the NFAT1 transcription factor, respectively. Upregulation of either Wnt/β-catenin or Wnt/Ca2+ signaling in GRN KD cells leads to the stimulation of BrdU incorporation into DNA, hyperphosphorylation of the pRb family of proteins and reduced cell viability over time. Blocking the Wnt cascades by specific canonical or noncanonical inhibitors of Wnt-dependent signaling, normalized the rate of DNA synthesis, and what it is more important restored the viability of GRN KD cells. Our results suggest an important role of Wnt activation inducing cell cycle disturbance-mediated neuronal loss in the pathogenesis of PGRN deficiency-linked FTLD-TDP. Therefore, it is plausible that modulation of Wnt signaling could be a promising strategy for developing of new disease-modifying treatments for FTLD-TDP.

In Alzheimer's disease (AD), molecular changes are observed not only in patients' neurons but also in peripheral cells, such as blood lymphocytes. These include changes in the level of oxidative stress markers, mitochondria impairment, and aberrant cell cycle regulation in AD blood lymphocytes. While the concepts of early causes of AD are currently highly controversial, these findings provide support for the cell cycle hypothesis of AD pathomechanism and emphasize the systemic nature of the disease. Moreover, because of difficulties in studying dynamic processes in the human brain, lymphocytes seem to be useful for readout of AD molecular mechanisms. In addition, lymphocytes as easily accessible human cells have potential diagnostic value. We summarize current perspectives for the development of new therapeutic strategies based on oxidative stress and cell cycle dysregulation in AD, and for diagnostic methodologies involving new markers in AD lymphocytes.

30 p.-7 fig.-1 tab., At present, treatment for Parkinson’s disease (PD) is only symptomatic, therefore it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6-associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB), and the m-TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6-hydroxy-dopamine (6-OHDA) treated dopaminergic SH-SY5Y cells and primary rat mesencephalic cultures. Here we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6-OHDA-induced cell death in neuronal cells by preventing the overactivation of the cyclin D3/CDK6/pRb cascade.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 PD., This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces.

16 p.-7 fig.-2 tab., Familial (FAD) and sporadic (SAD) Alzheimer's disease do not share all pathomechanisms, but knowledge on their molecular differences is limited. We previously reported that cell cycle control distinguishes lymphocytes from SAD and FAD patients. Significant differences were found in p21 levels of SAD compared to FAD lymphocytes. Since p21 can also regulate apoptosis, the aim of this study was to compare the response of FAD and SAD lymphocytes to oxidative stress like 2-deoxy-D-ribose (2dRib) treatment and to investigate the role of p21 levels in this response. We report that FAD cells bearing seven different PS1 mutations are more resistant to 2dRib-induced cell death than control or SAD cells: FAD cells showed a lower apoptosis rate and a lower depolarization of the mitochondrial membrane. Despite that basal p21 cellular content was lower in FAD than in SAD cells, in response to 2dRib, p21 mRNA and protein levels significantly increased in FAD cells. Moreover, we found a higher cytosolic accumulation of p21 in FAD cells. The transcriptional activation of p21 was shown to be dependent on p53, as it can be blocked by PFT-α, and correlated with the increased phosphorylation of p53 at Serine 15. Our results suggest that in FAD lymphocytes, the p53-mediated increase in p21 transcription, together with a shift in the nucleocytoplasmic localization of p21, confers a survival advantage against 2dRib-induced apoptosis. This compensatory mechanism is absent in SAD cells. Thus, therapeutic and diagnostic designs should take into account possible differential apoptotic responses in SAD versus FAD cells., This research was supported by the Joint Programming for Neurodegenerative Diseases (JPND) grant 2/BIOMARKAPD/JPND/2012 and the Polish National Science Centre grant NN401 596840 to UW, and by the Spanish Ministry of Economy and Competitiveness (CTQ-2015-66313-R) to AM-R

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.

Previously, we reported a Ca(2+)/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 degradation.

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 RT(2)Profiler™ 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.

Recent 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 G(1)/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 p21(Cip1) and p27(kip1). 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.

Loss-of-function progranulin gene (GRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein 43 (TDP-43) pathology (frontotemporal lobar degeneration [FTLD]-TDP); however, little is known about the association between progranulin (PGRN) deficiency and neuronal loss in individuals with FTLD-TDP. Previously we reported enhanced proliferative activity associated with the activation of WNT5A/CDK6/pRb signalling in PGRN-deficient cells. The objective of this work was to elucidate the association between PGRN deficiency, WNT5A signalling and cell proliferation in immortalized lymphoblasts from carriers of the c.709-1GA GRN mutation (asymptomatic and FTLD-TDP).We assessed cell proliferation in carriers of the c.709-1GA GRN gene mutation and controls without GRN mutation and without sign of neurologic degeneration by cell counting or using an MTT assay. We used a luciferase assay to measure the nuclear factor-κ (NF-κ) activity. We evaluated messenger RNA levels using quantitative real-time polymerase chain reaction and protein levels by immunoblotting. Co-immunoprecipitation was used to analyze the interaction between PGRN and its receptors.We enrolled 19 carriers of the GRN gene mutation and 10 controls in this study. The PGRN-deficient cells showed increased expression of WNT5A due to NF-κB signalling overactivation. We observed a competition between PGRN and tumour necrosis factor-α (TNF-α) for binding both TNF receptors (TNFR) I and II. Blocking NF-κB signalling using wedelolactone or specific antibodies against TNFRs inhibited WNT5A overexpression and proliferation of PGRN-deficient cells. Conversely, the activation of NF-κB signalling by TNF-α increased WNT5A-dependent proliferation of control cells.All cell lines were derived from individuals harboring the same splicing GRN mutation. Nevertheless, most of the known GRN mutations lead to haploinsufficiency of the protein.Our results revealed an important role of NF-κB signalling in PGRN-associated FTLD-TDP and confirm that PGRN can bind to TNF-α receptors regulating the expression of WNT5A, suggesting novel targets for treatment of FTLD-TDP linked to GRN mutations.

[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.

10 páginas, 9 figuras -- PAGS nros. 508-517, Epidemiologic studies indicated that non-steroidal anti-inflammatory drugs (NSAIDs) might prevent or delay the clinical features of Alzheimer disease (AD). The pharmacological activity of NSAIDs is generally attributed to inhibition of cyclooxygenase and peroxisome proliferator-activated receptor γ (PPARγ) activation. Based on the antineoplastic and apoptotic effects of PPARγ activation in a number of cell types, we hypothesized that NSAIDs could protect neurons by controlling the regulation of cell cycle. Recent work suggests that uncoordinated expression of cell cycle molecules and perturbation of cell cycle checkpoints may be one of the mechanisms by which post-mitotic neurons die. Since cell cycle dysfunction is not restricted to neurons in AD, we found it interesting to study the role of PPARγ activation on cell proliferation in immortalized lymphocytes from AD patients. We report here that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), but not NSAIDs or thiazolidinediones inhibited the serum-mediated enhancement of cell proliferation in AD by blocking the events critical for G1/S transition. The cyclopentenone induced a partial inhibition of retinoblastoma protein phosphorylation and increased levels of the CDK inhibitor p27kip1, This work has been supported by grants from the Spanish Fondo de Investigaciones Sanitarias (FIS PI040312), Ministerio de Ciencia y Tecnología (SAF 2003-01458). N.C. and U.M. are fellows from the Fondo de Investigaciones Sanitarias de la Seguridad Social and Ministerio de Educación y Ciencia, respectively

This work has been supported by grants from the Spanish Fondo de Investigaciones Sanitarias (01/1194), Ministerio de Ciencia y Tecnología (SAF 2003-01458) and the “Fundación Rodríguez Pascual”. N.C. and U.M. are fellows from the Fondo de Investigaciones Sanitarias de la Seguridad Social and Ministerio de Educación y Cultura, respectively, Cell cycle disturbances may precede neuronal death in Alzheimer's disease (AD). We described alterations, in lymphocytes from AD patients, on the activity of two transcription factors, E2F and NF-κB, 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-κB–DNA binding activity increased as cell progress through the cell cycle. The reduced NF-κB 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-κB–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, 10 páginas, 7 figuras -- PAGS nros. 615-624

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.

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 Ca2+, 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 Ca2+/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. © 2014 Elsevier Inc., This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces to AM-R. Dr. López de Munain received research support from the Basque Government, Spain (SAIO11.PR11BN002).

This work aimed to investigate the acute effect of the thyroid hormone 3,5,3'-tri-iodo-L-thyronine (T3) in regulating the hepatic metabolism either directly or by controlling the responsiveness to Ca2+-mobilizing agonists. We did not detect any acute metabolic effect of T3 either in perfused liver or in isolated liver cells. However, T3 exerted a powerful inhibitory effect on the alpha1-adrenoreceptor-mediated responses. The promptness of this T3 effect rules out that it was the result of rate changes in gene(s) transcription. T3 inhibited the alpha1-adrenoreceptor-mediated sustained stimulation of respiration and release of Ca2+ and H+, but not the glycogenolytic or gluconeogenic responses, in perfused liver. In isolated liver cells, T3 enhanced the alpha1-agonist-induced increase in cytosolic free Ca2+ and impeded the intracellular alkalinization. Since T3 also prevented the alpha1-adrenoreceptor-mediated activation of protein kinase C, its effects on pH seem to be the result of a lack of activation of the Na+/H+ exchanger. The failure of T3 to prevent the alpha1-adrenergic stimulation of gluconeogenesis despite the inhibition of protein kinase C activation indicates that the elevation of cytosolic free Ca2+ is a sufficient signal to elicit that response. T3 also impaired some of the angiotensin-II-mediated responses, but did not alter the effects of PMA on hepatic metabolism, indicating, therefore, that some postreceptor event is the target for T3 actions. The differential effect of T3 in enhancing the alpha1-adrenoreceptor-mediated increase in cytosolic free Ca2+ and preventing the activation of protein kinase C, provides a unique tool for further investigating the role of each branch of the signalling pathway in controlling the hepatic functions. Moreover, the low effective concentrations of T3 (

The present work aimed to study the influence of corticosteroids on the alpha 1-adrenoreceptor-induced activation of hepatic metabolic functions. The experiments were performed in a nonrecirculating liver perfusion system featuring continuous monitoring of pO2, pCa2+, Ca2+, pH, and portal pressure. The alpha 1-adrenergic-induced stimulation of respiration, H+ and Ca2+ release, glycogen breakdown, and gluconeogenesis, were diminished in livers from adrenalectomized animals. The normal liver responsiveness was restored on administration of exogenous corticosteroids but not mineralocorticoids. The following observations support the conclusion that corticosteroids control a hepatocyte-specific early postreceptor step in the alpha 1-adrenergic signaling pathway: 1) the alpha 1-adrenergic stimulation of vascular smooth muscle contraction was not impaired by corticosteroid deficiency; 2) the alpha 1-adrenoreceptor ligand-binding affinity does not seem to be altered by adrenalectomy; 3) the alpha 1-adrenergic-induced intracellular alkalosis, protein kinase C activation, and Ca2+ mobilization were diminished in hepatocytes from adrenalectomized rats, indicating that both Ca(2+)-dependent and -independent processes were altered; and 4) non-receptor-mediated homeostatic mechanisms of metabolic or intracellular pH control were not impaired by adrenalectomy.

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 RT(2)Profiler™ 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.

17 páginas, 8 figuras, 2 tablas., Murine models of Alzheimer’s disease (AD) provide means to detect and follow biomarker changes similar to thoseobserved in humans. Non-invasive biomarkers, such as those provided by magnetic resonance imaging (MRI) and spectroscopy(MRS) methods are highly desirable, however, systematic studies ofin vivoMRI/MRS methods to characterize the cerebralmorphology and metabolic pattern of these mice remain scarce. We investigated sixteen consecutive slices from the brain ofwild-type and A PP/PS1 mice, obtaining a collection of T2-weighted, diffusion weighted and magnetization transfer weightedimages as well as1H PRESS spectra from the cortical and subcortical areas. Compared to controls, A PP/PS1 mice showsignificant regional hyperintensities in T2-weighted images of the cerebral cortex, significant ventricular enlargement, anddecreased 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 implementedconsidering all parameters investigated, and revealed the most characteristic changes and allowed for the correct classification ofcontrol and A PP/PS1 mice. In summary, the present results provide a useful frame to evaluate optimal MRI/MRS biomarkersfor the characterization of AD models, potentially applicable in drug discovery processes, because of their non-invasive andrepeatable nature in longitudinal studies., This work has been supported by grants from Ministerio de Ciencia e Innovación (SAF2007-624505 andoSAF2010-1700) and Fundación Eugenio Rodríguez Pascual to A.M.R., grants from Instituto de SaludCarlos III (PI060155), Fundación Investigación Médica Mutua Madrileña (2006.125, 2008.93), and CIBERNED to E.C., grants SAF 2008-01327 andBIO2006-170 from Ministerio de Ciencia e Innovación and the Comunidad Autónoma de Madrid to S.C. N.E. holds a fellowship of the JAE predoctoral program of the CSIC

This work aimed to determine the role played by the adrenal gland in the fatty acid control of gluconeogenesis in isolated perfused rat livers. The gluconeogenic substrate concentration responses were not altered in adrenalectomized (ADX) rats. This observation indicates that glucocorticoids are not essential to maintain normal basal gluconeogenic rates. In contrast, fatty acid failed to stimulate gluconeogenesis from lactate and elicited attenuated stimulation with pyruvate as substrate in livers from ADX rats. Fatty acid-induced stimulation of respiration and ketone body production were similar in control and ADX rats. Thus the diminished responsiveness of the gluconeogenic pathway to fatty acid cannot be the result of different rates of energy production and/or generation of reducing power. Fatty acids did not inhibit pyruvate decarboxylation in livers from ADX rats. Even though mitochondria isolated from livers of ADX rats showed normal basal rates of pyruvate metabolism, fatty acids failed to inhibit pyruvate decarboxylation and the activity of the pyruvate dehydrogenase complex. This novel observation of the glucocorticoid effect in controlling the pyruvate dehydrogenase complex responsiveness indicates that the mitochondrial partitioning of pyruvate between carboxylation and decarboxylation reactions may be altered in livers from ADX rats. We propose that the diminished effect of fatty acid in stimulating gluconeogenesis in livers from ADX rats is the result of a limited pyruvate availability for the carboxylase reaction due to a lack of inhibition of flux through the pyruvate dehydrogenase complex., This work was supported in part by grants from Plan Nacional de Investigación Científica y Técnica (SAL 509/91 and SAF 93-0788) and Fondo de Investigaciones Sanitarias (93/0162 and 94/0224). G. Cipres and E. Urcelay were recipients of predoctoral fellowships from the Spanish Secretary of Education and Science. N. Butta was a recipient of a external fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

10 páginas, 1 figura, 1 tabla -- PAGS nros. 121-130, The 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 disease, Work 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 Pascual

429.e7–429.e20 páginas, 8 figuras, 1 tabla, 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 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 degeneration, This 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 Innovation

12 páginas, 9 figuras, 1 tabla -- PAGS nros. 4268-4257, 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, This work was supported by grants from the Ministry of Education and Science (SAF2007-62405) and Fundación Eugenio Rodríguez Pascual. NE holds a fellowship of the JAE predoctoral program of the CSIC. CA was the recipient of a fellowship from the Launching Grants Program of the CIBERER

Elevated cerebral levels of amyloid beta-protein (Aβ) occur in Alzheimer's 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, This work was supported by grants from Fondo de Investigación Sanitaria (FIS) (CP04/00179, PI060155), Fundación Investigación Médica Mutua Madrileña (2006.125), CIBERNED, Obra Social Caja Madrid, Comunidad Autónoma de Madrid (GR/SAL/0783/2004). We thank Drs. P. Gil, P. Coria, and A. Labad-Alquézar for their cooperation in the generation of the case–control samples

12 páginas, 11 figuras -- PAGS nros. 1437-1448, 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, This 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, respectively