Autophagy, and BiP level decrease are early key events in retrograde degeneration of motoneurons

El pdf del artículo es la versión post-print.-- et al.Disconnection of the axon from the soma of spinal motoneurons (MNs) leads either to a retrograde degenerative process or to a regenerative reaction, depending on the severity and the proximity to the soma of the axonal lesion. The endoplasmic reticulum (ER) is a continuous membranous network that extends from the nucleus to the entire cytoplasm of the neuronal soma, axon and dendrites. We investigated whether axonal injury is sensed by the ER and triggers the activation of protective mechanisms, such as the unfolded protein response (UPR) and autophagy. We found early (at 3 days) accumulation of beclin1, LC3II and Lamp-1, hallmarks of autophagy, in both degenerating MNs after spinal root avulsion and in non-degenerating MNs after distal nerve section, although Lamp-1 disappeared by 5 days only in the former. In contrast, only degenerating MNs presented early activation of IRE1α, revealed by an increase of the spliced isoform of Xbp1 and accumulation of ATF4 in their nucleus, two branches of the UPR, and late BiP downregulation in association with cytoskeletal and organelle disorganization. We conclude that BiP decrease is a signature of the degenerating process, as its overexpression led to an increase in MN survival after root avulsion. Besides, Bcl2 is strongly implicated in the survival pathway activated by BiP overexpression.This work was supported by grants from the Ministerio de Ciencia y Tecnologia (Grants SAF2006-08682, SAF2009-12495), the Ministerio de Sanidad (FIS PI081932) and funds from Red de Terapia Celular (TERCEL) and from the Centro de investigacion biomedica en Red para enfermedades neurodegenerativas (CIBERNED) of Spain.Peer Reviewe

Nanoparticulate architecture of protein-based artificial viruses is supported by protein DNA interactions

Aim & Methods: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine–glycine–aspartic acid cell-binding domains and promote significant transgene expression levels. Results & Conclusion: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of D-helix and the absence of cross-molecular, E-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA–protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.Agencia Nacional de Investigación e Innovació

SIRT1 regulation modulates stroke outcome

El pdf del artículo es la versión post-print.Silent information regulator 1 (SIRT1) is a NAD+−dependent histone deacetylase that represses gene expression and plays a role in longevity. SIRT1 responds to diverse stress conditions and regulates metabolism in nutrient deficiency conditions; therefore, it is involved in adaptive pathways to better fulfill tissue needs in a disturbed environment. SIRT1 overexpression or activation is protective in neurodegenerative diseases. Its role in acute nervous system injury, such as brain ischemia, is emerging, but whether SIRT1 activation improves stroke outcome is still a matter of controversy. In the present review, we will document present knowledge about the contribution of SIRT1 in death/survival in cell and animal models of brain ischemia and discuss whether SIRT1 could be a valuable target for therapeutic intervention in human stroke.The financial support from the Fundació Marato TV3 (110431) and the MICINN (SAF2011-30492).Peer reviewe

In vitro models as tools for the study of neurological diseases

Peer Reviewe

Nimodipine inhibits TMB-8 potentiation of AMPA-induced hippocampal neurodegeneration

Human cerebral calcification has been related to deregulation of intracellular calcium homeostasis. In rat basal ganglia, nimodipine and TMB-8, two commonly used calcium antagonists, worsen the chronic AMPA-induced lesion, whereas only nimodipine potentiates calcification. To investigate whether similar effects are present in the hippocampus, AMPA dose–response and calcium movement blockade were performed. A dose-related increase of both hippocampal lesion and calcification was evident in a saturable mode, mostly different from the continuous globus pallidus response previously observed. The value of 2.7 nmol AMPA, selected as yielding 60% of maximum calcification, was coinjected with nimodipine or/and TMB-8 to determine their influence on tissue damage. TMB-8 increased the AMPA lesion in terms of calcified area, and nimodipine reversed this increase, with no effect alone. These results, divergent from those for the globus pallidus, reveal differences in extra- and intracellular calcium movement between the two neurodegenerative processes. Future work focused on other brain areas is required to understand how control of calcium stores may influence neurodegenerative disease evolution.Funded by: Spanish Ministerio de Sanidad y Consumo (project CIBERNED); Spanish Ministerio de Educación y Ciencia. Grant Number: SAF2005-04314 and Generalitat de Catalunya. Grant Number: 2005SGR00609.Peer reviewe

Development of image analysis tools to characterize and classify cultured microglial cells in healthy and pathological conditions

Trabajo presentado en el 3rd Young Spanish ESMI Group Meeting, celebrado en Barcelona (España), el 13 de mayo de 2019Microglia are the immune cells of the brain and they constantly survey the environment in order to prevent brain infection and phagocytose death cells and cell debris. In physiological conditions, microglia get rid of dysfunctional synapses (pruning) and neuronal molecules bind to microglia receptors to maintain the latter in a harmless state. However, after a brain damage, the balance can be disrupted. Under pathological situations such as neurodegeneration or stroke, damage-associated molecular pattern molecules (DAMPs) are released by injured neurons or astrocytes and activate microglia. The microglia response can cause inflammation and exacerbate neuronal death, but it is also involved in tissue regeneration . In the same brain area, surveillant and activated microglia may coexist. Microglia are highly plastic cells and adopt changes in their morphology in order to quickly reach the damaged area, and to phagocytose apoptotic cells or debris. Recent studies identified a sub-population of ¿neuroprotective¿ microglia among activated microglia in animal models of neurodegenerative diseases based on gene expression analysis suggesting heterogeneity within the microglia population. The goal of the present work is to characterize different subsets of microglia that presumably assume different functions and may coexist after neuronal injury . This objective is quite challenging, particularly in living cells, due to the morphological heterogeneity of microglia, shape transition and the presence of overcrossing cells. Methods We performed live cell imaging in cortical neuron/glia cultures prepared from embryos, resulting from the crossing between ROSA-CAG-tdTomato flox/flox mice and CX3CR1-CRE-ERT2 mice, in control conditions and after an excitotoxic challenge. In these cultures, microglia express tdTomato after exposure to 4-OH-tamoxifen. Time-lapse images were acquired in a Laser Scanning Confocal microscope. We developed plugins for ImageJ to provide an automated measurement of morphological features. Results We developed plugins for ImageJ to segment microglia and track individual cells in a semi-automated fashion. The plugin generates a mask which can be opened in ImageJ and used for morphometric measurements. The data generated were used for the classification of microglia in sub-groups. The novelty of our software is that it allows following-up every single microglia and its phenotype transition along time, and it assigns each cell into a cellular subtype according to the measured parameters. Conclusions Segmentation and dynamic morphometric measurements of cultured microglia in an in vivo time-lapse setting allow clustering the microglial cells into different subclasses

RGD-based cell ligands for cell-targeted drug delivery act as potent trophic factors

El pdf del artículo es el manuscrito revisado de autor.-- et al.Integrin-binding, Arg-Gly-Asp (RGD)-containing peptides are the most widely used agents to deliver drugs, nanoparticles, and imaging agents. Although in nature, several protein-mediated signal transduction events depend on RGD motifs, the potential of RGD-empowered materials in triggering undesired cell-signaling cascades has been neglected. Using an RGD-functionalized protein nanoparticle, we show here that the RGD motif acts as a powerful trophic factor, supporting extracellular signal-regulated kinase 1/2 (ERK1/2)-linked cell proliferation and partial differentiation of PC12 cells, a neuronlike model. From the Clinical Editor: This work focuses on RGD peptides, which are among the most commonly used tags for targeted drug delivery. They also promote protoneurite formation and expression of neuronal markers (MAP2) in model PC12 cells, which is an unexpected but relevant event in the functionalization of drugs and their nanocarriers. © 2012 Elsevier Inc.The authors acknowledge the financial support granted to A. V. from Ministerio de Ciencia e Innovación (MICINN) (ACI2009-0919), Agència de Gestió d'Ajuts Universitaris i de Recerca (2009SGR-108), and Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina, an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund. This work was also supported by MICINN to A. M. P. (SAF2011-30492, Spain) and by ANII (FCE_192, Uruguay) to H. P. U. U. and P. S. received PhD fellowships from ISCIII and J.D.E. from MICINN. A. V. has been distinguished with an Institució Catalana de Recerca i Estudis Avançats Academia award.Peer Reviewe

Nanoparticulate architecture of protein-based artificial viruses is supported by protein-DNA interactions

El pdf del artículo es la versión post-print.-- et al.Aim & Methods: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine-glycine-aspartic acid cell-binding domains and promote significant transgene expression levels. Results & Conclusion: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of α-helix and the absence of cross-molecular, β-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA-protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy. © 2011 Future Medicine Ltd.The authors appreciate the financial support received for the design and production of artificial viruses for gene therapy from MICINN (BIO2007-61194 and ACI2009-0919), AGAUR (2009SGR-108) and CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN, Spain), an initiative funded by the VI National R+D+i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. We also thank ANII, Ministerio de Educacion y Cultura, Uruguay, for financial support.Peer Reviewe

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

El pdf del artículo es la versión post-print.Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.VP is a recipient of a Ramon y Cajal grant and MFN a recipient of a FPI grant from the Spanish Ministry of Education and Science (MEC). This study was supported by grants FIS05/0271 from the Spanish Ministry of Health and SAF2005-05793-C01 from the Ministry of Education and Science.Peer reviewe

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: Involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H 2O2, interferon (INF)-γ and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H2O2, INF-γ or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H2O2, IL-6 and IL-10. Also, H2O2 induced serine (Ser)-727 phosphorylation of Stat1 but not Stat3. The degree of Tyr-701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr-701 but not Ser-727, Stat1 phosphorylation. Also, AG490 inhibited Tyr-705 Stat3 phosphorylation induced by H 2O2 and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-γ, IL-6 and H2O2. These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H2O2, which further shows that H2O2 and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H 2O2-induced cell death.This work was supported by a grant from the Comisión Interministerial de Ciencia y Tecnologia (CICYT; SAF2002-01963). RG has a fellowship from the IDIBAPS and VP is ascribed to the ‘Ramón y Cajal’ programmePeer Reviewe