Valentín Ceña

Bio: Valentín Ceña is an academic researcher from University of Castilla–La Mancha. The author has contributed to research in topics: Chromaffin cell & Apoptosis. The author has an hindex of 40, co-authored 127 publications receiving 9864 citations. Previous affiliations of Valentín Ceña include National Institutes of Health & French Institute of Health and Medical Research.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Sequential Treatment of SH‐SY5Y Cells with Retinoic Acid and Brain‐Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor‐Dependent, Human Neuron‐Like Cells

Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2′-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

D1 but not D5 Dopamine Receptors Are Critical for LTP, Spatial Learning, and LTP-Induced arc and zif268 Expression in the Hippocampus

Abstract: Recent evidence suggests that glutamatergic and dopaminergic afferents must be activated to induce persistent long-term potentiation (LTP) in the hippocampus. Whereas extensive evidence supports the role of glutamate receptors in long-lasting synaptic plasticity and spatial learning and memory, there is less evidence regarding the role of dopamine receptors in these processes. Here, we used dopamine D 1 receptor knockout (D 1 R −/− ) mice to explore the role of D 1 R in hippocampal LTP and its associated gene expression. We show that the magnitude of early and late phases of LTP (E-LTP and L-LTP) was markedly reduced in hippocampal slices from D 1 R −/− mice compared with wild-type mice. SCH23390, a D 1 /D 5 R antagonist, did not further reduce L-LTP in D 1 R −/− mice, suggesting that D 5 Rs are not involved. D 1 R −/− mice also showed a significant reduction of D 1 R-induced potentiation of N-Methyl-D-aspartic acid-mediated currents, via protein kinase activated by cyclic adenosine 3′,5′-monophosphate activation. Finally, LTP-induced expression of the immediate early genes zif268 and arc in the hippocampal CA1 area was abolished in D 1 R −/− mice, and these mice showed impaired learning. These results indicate that D 1 R but not D 5 R are critical for hippocampal LTP and for the induction of Zif268 and Arc, proteins required for the transition from E-LTP to L-LTP and for memory consolidation in mammals.

Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell

Abstract: Nanoparticles (NPs) and submicron particles are increasingly used as carriers for delivering therapeutic compounds to cells. Their entry into the cell represents the initial step in this delivery process, being most of the nanoparticles taken up by endocytosis, although other mechanisms can contribute to the uptake. To increase the delivery efficiency of therapeutic compounds by NPs and submicron particles is very relevant to understand the mechanisms involved in the uptake process. This review covers the proposed pathways involved in the cellular uptake of different NPs and submicron particles types as well as the role that some of the physicochemical nanoparticle characteristics play in the uptake pathway preferentially used by the nanoparticles to gain access and deliver their cargo inside the cell.

Adenosine released by astrocytes contributes to hypoxia‐induced modulation of synaptic transmission

Abstract: Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia-specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia-induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia-induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.

Receptors for Purines and Pyrimidines

Abstract: ### A. Overview Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

Abstract: Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles.

Abstract: Many types of nanoparticles (NPs) are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging.