Lysosome-dependent cell death and deregulated autophagy induced by amine-modified polystyrene nanoparticles

Nanoparticles (NPs) typically accumulate in lysosomes. However, their impact on lysosomal function, as well as autophagy, a lysosomal degradative pathway, is still not well known. We have previously reported in the 1321N1 cell line that amine-modified polystyrene (NH2-PS) NPs induce apoptosis through damage initiated in the lysosomes leading ultimately to release of lysosomal content in the cytosol, followed by apoptosis. Here, by using a combination of biochemical and cell biological approaches, we have characterized in a mouse embryonic fibroblast cell line that the lysosomal alterations induced by NH2-PS NPs is progressive, initiating from mild lysosomal membrane permeabilization (LMP), to expansion of lysosomal volume and intensive LMP before the summit of cell death. Though the cells initially seem to induce autophagy as a surviving mechanism, the damage of NH2-PS NPs to lysosomes probably results in lysosomal dysfunctions, leading to blockage of autophagic flux at the level of lysosomes and the eventual cell death

Altered blood gene expression of tumor-related genes (PRKCB, BECN1 and CDKN2A) in Alzheimer’s disease

22 p.-3 fig.-3 tab. Antonell, Anna et al.Alzheimer’s disease (AD) is the most common of the neurodegenerative diseases. Recent diagnostic criteria have defined a preclinical disease phase during which neuropathological substrates are thought to be present in the brain. There is an urgent need to find measurable alterations in this phase as well as a good peripheral biomarker in the blood. We selected a cohort of 100 subjects (controls = 47; preclinical AD = 11; patients with AD = 42) and analyzed whole blood expression of 20 genes by quantitative polymerase chain reaction. The selected genes belonged to calcium-signaling, senescence and autophagy, and mitochondria/oxidative stress pathways. Additionally, two genes associated with an increased risk of developing AD (CLU and BIN1) were also analyzed. We detected significantly different gene expressions of BECN1 and PRKCB between the control and the AD groups; and, of CDKN2A between the control and the preclinical AD groups. Notably, these three genes are also considered tumor suppressor (CDKN2A and BECN1) or tumor promoter (PRKCB) genes. Gene-gene expression Pearson correlations were computed separately for controls and patients with AD. The significant correlations (p<0.001) were represented in a network analysis with Cytoscape tool, which suggested an uncoupling of mitochondriarelated genes in AD group. Whole blood is emerging as a valuable tissue in the study of the physiopathology of AD.This study was supported by grants to JLM (Consolider CSD2010-00045) and to ALL (PI14/00282, ISCIII, Cofinancia FEDER, Unión Europea, Otra manera de hacer Europa) from the Spanish Ministry of Economy and Competitiveness. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115568, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution.Peer reviewe

How autophagy is related to programmed cell death during the development of the nervous system

5 páginas, 3 figuras -- PAGS nros. 813-817Programmed cell death, together with proliferation and differentiation, is an essential process during the development of the nervous system. During neurogenesis, neurons and glia are generated in large numbers and, subsequently, they die in a process that depends on trophic signalling that refines the cytoarchitecture and connectivity of the nervous system. In addition, programmed cell death also affects proliferating neuroepithelial cells and recently differentiated neuroblasts. Autophagy is a lysosomal degradative pathway that allows the recycling of cell constituents, and seems to be able to play a dual role. It may serve to protect the cell by preventing the accumulation of deleterious products and organelles and supplying energy and amino acids. On the other hand, it has been considered a type of cell death. The role of autophagy during development is little characterized. The retina provides an excellent model system to study autophagy in the context of neural development, and to establish its relationship with proliferation, differentiation and cell death. In the present review, we summarize recent findings showing that autophagy contributes to the development of the nervous system by providing energy for cell corpse removal after physiological cell death, a process associated with retinal neurogenesisResearch in our laboratory is supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2006-00508 and BFU2006-26073-E to P.B. and SAF2007-66175 to E.J. de la R.). M.A.M. is an FPU (Formación de Profesorado Universitario) Fellow and P.B. is a Ramón y Cajal Fellow (both programmes are financed by the Ministerio de Educación y Ciencia)Peer reviewe

Interferon alfa subtypes and levels of type I interferons in the liver and peripheral mononuclear cells in patients with chronic hepatitis C and controls

Viral infections stimulate the transcription of interferon type I, which includes IFN-alfa (IFN-alpha) (13 subtypes) and IFN-beta (a single substance). Hepatitis C virus (HCV) infection is remarkable by its ability to evade host antiviral defenses; however, there is little information as to whether endogenous IFN is activated or not in this disease. Additionally, despite the fact that the various IFN-alpha subtypes may differ in biological activity, there are no data concerning the IFN-alpha subtypes specifically expressed in normal and diseased liver tissue. Thus, we have analyzed the IFN-alpha subtypes and the mRNA levels of type I IFNs in samples of normal liver tissue and in liver from patients with chronic hepatitis C. Similar studies were performed in peripheral blood mononuclear cells (PBMC) from patients and controls. After amplification and cloning of IFN-alpha cDNA, we observed that 98 of the 100 clones from normal liver tissue corresponded to the IFN-alpha5 subtype. However, in livers with chronic hepatitis C and in PBMC from controls and patients, a variety of subtypes, in addition to IFN-alpha5, were detected, suggesting a participation of infiltrating leukocytes in the production of IFN-alpha in livers with chronic hepatitis C. As compared with controls, patients with chronic hepatitis C showed a significant increase in IFN-beta mRNA in both the liver and PBMC, while IFN-alpha mRNA was significantly increased in PBMC but markedly reduced in liver tissue. In conclusion, IFN-alpha5 is the sole IFN-alpha subtype expressed in normal liver tissue. The hepatic levels of IFN-alpha are reduced in chronic hepatitis C, an event that may favor viral persistence

Viral proteins targeting mitochondria: controlling cell death

AbstractMitochondrial membrane permeabilization (MMP) is a critical step regulating apoptosis. Viruses have evolved multiple strategies to modulate apoptosis for their own benefit. Thus, many viruses code for proteins that act on mitochondria and control apoptosis of infected cells. Viral proapoptotic proteins translocate to mitochondrial membranes and induce MMP, which is often accompanied by mitochondrial swelling and fragmentation. From a structural point of view, all the viral proapoptotic proteins discovered so far contain amphipathic α-helices that are necessary for the proapoptotic effects and seem to have pore-forming properties, as it has been shown for Vpr from human immunodeficiency virus-1 (HIV-1) and HBx from hepatitis B virus (HBV). In contrast, antiapoptotic viral proteins (e.g., M11L from myxoma virus, F1L from vaccinia virus and BHRF1 from Epstein–Barr virus) contain mitochondrial targeting sequences (MTS) in their C-terminus that are homologous to tail-anchoring domains. These domains are similar to those present in many proteins of the Bcl-2 family and are responsible for inserting the protein in the outer mitochondrial membrane leaving the N-terminus of the protein facing the cytosol. The antiapoptotic proteins K7 and K15 from avian encephalomyelitis virus (AEV) and viral mitochondria inhibitor of apoptosis (vMIA) from cytomegalovirus are capable of binding host-specific apoptosis-modulatory proteins such as Bax, Bcl-2, activated caspase 3, CAML, CIDE-B and HAX. In conclusion, viruses modulate apoptosis at the mitochondrial level by multiple different strategies

Muerte neural temprana: un proceso inadvertido en el desarrollo del sistema nervioso.

15 p.-7 fig.[EN]During the development of the vertebrate nervous system, multiple physiological processes are involved in the generation of its complex cytoarchitecture and functionality. Among them, programmed cell death has been recognized as a key process that affects connecting neurons. By contrast, there is limited information available regarding the cell death that affects neuroepithelial cells, and recently born neurons and glia, hindering the comprehensive understanding of neural development. We have demonstrated that exquisitely regulated PCD occurs during early stages of neural development such as neurulation and neurogenesis. We have characterized how survival signals from proteins like proinsulin/insulin, c-Raf, and HSC70 counteract caspase-dependent apoptosis, which affects neuroepithelial cells proliferation and the generation of retinal ganglion cells. Furthermore, the characterization of these physiological signals during retinal neurogenesis has the potential to provide new therapeutic tools to attenuate retinal neurodegeneration.[ES]Durante el desarrollo del sistema nervioso de vertebrados, múltiples procesos fisiológicos participan en la generación de su compleja arquitectura celular y funcionalidad. Entre ellos, la muerte celular programada que afecta a neuronas de conexión está reconocido como un proceso fundamental. Por otro lado, hay escasa información disponible acerca de la muerte celular que afecta a células neuroepiteliales y a neuronas y glía recién nacidas, lo que impide que tengamos una noción completa sobre el desarrollo neural. Los estudios de nuestro laboratorio han demostrado que la muerte celular programada se encuentra finamente regulada y ocurre en etapas tan tempranas del desarrollo como la neurulación o la neurogénesis. Hemos caracterizado el papel que moléculas de supervivencia, como la proinsulina/insulina, c-Raf o HSC70, desempeñan bloqueando la apoptosis dependiente de caspasas, proceso que afecta a células neuroepiteliales proliferativas, así como a la generación de las células ganglionares de la retina. Es más, la caracterización de estas señales fisiológicas originadas durante la neurogénesis de la retina nos ha proporcionado una nueva herramienta terapéutica potencial para el tratamiento y atenuación de las neurodegeneraciones retinianas.Research in the laboratory is funded by grants-in-aid from the Spanish Ministerio de Educación y Ciencia (SAF2007-66175-C02-01 to EJdlR, BFU2007-61055/BMC to FdP, and BFU2006-00508 to PB).Peer reviewe

Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles

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Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-alpha is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-alpha subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-alpha2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-alpha subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-alpha mRNAs and in the production of IFN-alpha protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-alpha5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-alpha8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-alpha subtypes, IFN-alpha5 being the specific subtype induced in PBMC from patients with chronic HCV infection

Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal

17 p.-6 figRelatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand,replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly,autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.F.R. is supported by Marie Skłodowska-Curie Cofund [713660], Marie Skłodowska-Curie ITN [765912], ALW Open Program [ALWOP.310]and ZonMW VICI [016.130.606] grants. R.C. and C.R. are supported by the Dutch Cancer Society [Grant number 5792 and 12458]. Patricia Boya is supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI),the Fondo Europeo de Desarrollo Regional (FEDER) [PGC2018-098557-B-I00] and a Marie Skłodowska-Curie ITN grant [765912]. Idil Orhon is a recipient of a FEBS postdoctoral fellowship and Beatriz Villarejo-Zori of a Fundacion Tatiana Perez de Guzman el Bueno predoctoral fellowhip.Peer reviewe