Fingolimod phosphate protection against mitochondrial damage in neuronal cells

Background: Major role of oxidative stress in the pathogenesis of neurodegenerative diseases have been suggested, being mitochondria one of the main sources of ROS. Aim: In the present work, we have studied the antioxidant effect of fingolimod phosphate (FP) on neuronal mitochondrial function and morphology using a model of mitochondrial oxidative damage induced by menadione (Vitk3). Methods: SN4741 neuronal cells were grown (70-80% confluence) and used as control (non-treated cells) or treated cells with Vitk3 15 µM alone or in presence of FP 50 nM during 4 hours. Mitochondrial membrane potential (MMP), cytochrome c oxidase (COX) activity, mitochondrial oxygen consumption rate (OCR), mitochondrial distribution (MTG) and morphology (EM) were analysed. Statistical differences were determined using one-way ANOVA. Results: Vitk3 incubation produces a dramatical decrease in MMP compared to control (43.7 %); this can be almost totally reverted by the co-incubation of Vitk3 in presence of FP (p<0.05). A 20.7 % decrease in COX activity has been found after Vitk3 incubation, again this effect was counteracted when Vitk3 and FP are combined, restoring COX activity to control levels (p<0.05). Vitk3 incubation triggers initially an increase in OCR, decreasing dramatically (61%) after 4 hours. In experiments co-incubating Vitk3 in presence of FP, the OCR decrease found was reduced to only 17% (p<0.05). In experiments with MitoTracker™ Green, we found a change in the network pattern distribution after Vitk3 administration that partially disappears when co-incubated in presence of FP. Almost all the mitochondria treated with Vitk3 show ultrastructural alterations at the electron microscopy level while normal mitochondria can be found when Vitk3 and FP are combined. Conclusion: FP protects against the mitochondrial damage induced by Vitk3, as seen by the results obtained in mitochondrial functional markers, distribution and morphology.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PS13/14: Study of the non-immunological mechanisms of action of Gilenya (Fingolimod) as therapeutic tool in Multiple Sclerosis and/or other neurodegenerative diseases. Novartis Farmacéutica S.A

Role of Insulin-Growth Factor II on mitochondrial recovery in a cellular model of Parkinson's Disease

Insulin-growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. ROS causes damage to cellular macromolecules affecting several cellular processes and resulting in cell death. Mitochondrial ROS damage has a critical role in the pathobiology of PD. The objective was to assess the IGF-II role in the recovery of the oxidative damage produced on mitochondrial in a cellular model of PD. SN4741 cell line was treated as follows: MPP+ alone, in presence of IGF-II and/or co-incubated BMS (Ins/IGF-I receptors antagonist) or AB (anti-IGF-II-receptor). To assess the effect of IGF-II in the recovery of MPP+ damage, this treatment was removed after 2 h and replaced during another 2 h by medium, IGF-II or IGF-II + BMS or IGF-II + AB. Cell death was analysed through annexin-V Mitochondrial structure, localization and morphology was studied by western blot/ immunochemistry of Mitofilin (Mtf) and electron microscopy; function by Mitotracker and oxygen consumption rate. IGF-II prevented MPP+ cell death. In morphological/structural studies, MPP+ treated cells showed swollen mitochondria with loss of cristae, and electron-lucent matrix, inducing a mitochondrial number reduction. IGF-II retrieved normal-shaped mitochondria with intact cristae and outer/inner membranes. Moreover, MPP+ incubation significantly reduced the expression levels of Mtf compared to the CO. This expression was found in areas that had a very weak mark, indicating mitochondrial destruction or dysfunction. IGF-II coincubation, recovered the expression of Mtf, remaining associated with healthy mitochondrial function. Additionally, the decrease in OCR levels after MPP+ administration was recovered in presence of IGF-II. The BMS-receptor blockage did not modify the IGF-II responses, and AB limited its effect. In conclusion, IGF-II recovers mitochondrial structure and function due to MPP+ damage. This improvement is carried out through the specific IGF-II receptor.Supported by M.G-F.&L.J.S. Proyectos I+D+I-Programa Operativo-FEDER Andalucía 2014-2020 (UMA18-FEDERJA-004) Junta de Andalucía. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Papel del receptor S1P sobre el estrés oxidativo mitocondrial en cultivo neuronal

Introducción: Fingolimod, fármaco inmunomodulador, presenta propiedades neuroprotectoras que podrían promover la recuperación de la función cognitiva en enfermedades neurodegenerativas. El estrés oxidativo parece tener un papel fundamental en la patogénesis de dichas enfermedades, siendo la mitocondria una de las fuentes más importantes de especies reactivas de oxigeno (ROS). Objetivo: Determinar la implicación del receptor S1P en los efectos neuroprotectores mostrados por fingolimod fosfato (FP), forma activa de fingolimod, en un modelo celular de estrés oxidativo mitocondrial inducido por menadiona (Vitk3). Material y métodos: La línea celular SN4741 (70-80 % confluencia), se utilizó como control o se trató con Vitk3 15 µM en presencia o ausencia de FP 50 nM o FP 50 nM + W123 10 µM (antagonista S1P) durante 4 horas para estudiar: niveles de ROS mitocondrial según el marcaje de la producción de anión superóxido (O2−.); activación de caspasa-3; niveles de tioles totales (TTLs); marcadores mitocondriales (potencial de membrana mitocondrial-PMM-, actividad citocromo c oxidasa-COX- y consumo de oxígeno-OCR-). Las diferencias estadísticas se determinaron usando ANOVA de un factor. Resultados: W123 revierte parcialmente el efecto protector de FP sobe muerte celular programada, desencadenada por aumento de ROS (p<0,05) y consumo de reserva de antioxidante (p<0,05). El efecto de FP sobre los marcadores mitocondriales PMM, actividad COX y OCR es abolido con W123 (p<0,05). Conclusión: El receptor S1P está implicado en gran parte de los efectos protectores de FP, indicando un papel fundamental de S1P en el mantenimiento de la homeostasis mitocondrial. Proyecto financiado por Novartis Farmacéutica SA (PS13/14).Campus de Excelencia Internacional Andalucía Tech. Proyecto financiado por Novartis Farmacéutica SA (PS13/14). Programa operativo de empleo juvenil; Junta de Andalucía and Fondo Social Europeo (EU). CTS507 and CTS156 from Consejería de Economía Innovación Ciencia y Empresa, Junta de Andalucía and Plan Propio de la Universidad de Málaga 2016

Deep learning-based lung segmentation and automatic regional template in chest X-ray images for pediatric tuberculosis

Tuberculosis (TB) is still considered a leading cause of death and a substantial threat to global child health. Both TB infection and disease are curable using antibiotics. However, most children who die of TB are never diagnosed or treated. In clinical practice, experienced physicians assess TB by examining chest X-rays (CXR). Pediatric CXR has specific challenges compared to adult CXR, which makes TB diagnosis in children more difficult. Computer-aided diagnosis systems supported by Artificial Intelligence have shown performance comparable to experienced radiologist TB readings, which could ease mass TB screening and reduce clinical burden. We propose a multi-view deep learning-based solution which, by following a proposed template, aims to automatically regionalize and extract lung and mediastinal regions of interest from pediatric CXR images where key TB findings may be present. Experimental results have shown accurate region extraction, which can be used for further analysis to confirm TB finding presence and severity assessment. Code publicly available at https://github.com/dani-capellan/pTB_LungRegionExtractor.Comment: This work has been accepted at the SPIE Medical Imaging 2023, Image Processing conferenc

Graphene derivative scaffolds facilitate in vitro cell survival and maturation of dopaminergic SN4741 cells

The emerging carbon nanomaterial Graphene (G), in the form of scaffold structure, has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells, providing a promising approach for neural regeneration. We propose the use of G as a scaffold to re-address the dopaminergic (DA) neurons and the residual axons from dead or apoptotic DA neurons in Parkinson´s disease (PD). G could act as a physical support to promote the axonal sprout as a “deceleration” support for the DA cells derived from neural stem cells or DA direct cell conversion, allowing the propagation of nerve impulses. We cultured a clonal substantia nigra (SN) DA neuronal progenitor cell line (SN4741) in presence of G as scaffold. This cell line derived from mouse embryos was cultured in Dulbecco’s modified Eagle’s medium/10% FCS to about 80% confluence. Cells were incubated in three chemically different G derivatives and two different presentation matrixes as powder and films: 1) G oxide (GO); 2) partially reduced GO (PRGO) which is hydrophobic; and 3) fully reduced GO (FRGO). Cell viability was determined using the MTT assay after adding the following G concentrations: 1mg/ml; 0.1mg/ml; 0.05mg/ml; 0.02mg/ml and 0.01mg/ml, in each type of GO. To study cellular morphology and assessment of cell engraftment into GO films (GO film, PRGO film, FRGO film), we analyzed the immunostaining of the anti-rabbit neuron-specific DNA-binding protein (NeuN) antibody, the anti-rat Beta-3-tubulin antibody in combination with the mitochondrial marker mouse anti-ATP synthase antibody, and the anti-rabbit DCX as immature neuronal marker. Hoechst label was used as nuclei marker. Reactive oxidative species (ROS) were measured by flow cytometry to study the influence of G on the cell redox-state. With this purpose, cells were loaded with dihydroethidium. The mitochondrial membrane potential after JC-1 incubation was studied by flow cytometry. Our results show an increase of survival and metabolism (30-40%) at low concentrations of PRGO and FRGO (0.05-0.01 mg/ml) respect to the higher concentration (1 mg/ml), while no changes were seen in the GO group. LDH concentration was measured in the supernatant using a COBAS analyzer showing a neuroprotective action at low concentrations. Furthermore, either PRGO film or FRGO film show an increase in the effective anchorage capacity to nest into the G matrix and in the maturation of the SN 4741 cells. We conclude that the use of G scaffolds in the research of neurological diseases like PD could offer a powerful platform for neural stem cells, direct cell conversion techniques and neural tissue engineering.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Norwegian Research Council (grant nº 215086

Mesoporous silica particles are phagocytosed by microglia and induce a mild inflammatory response in vitro

Aim: Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses to MSPs of astrocytes and microglia, the two main cellular players in neuroinflammation. Materials & methods: Primary murine cortical mixed glial cultures were treated with rhodamine B-labeled MSPs. Results: MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. MSPs also do not affect mRNA levels of key proinflammatory genes; however, in combination with lipopolysaccharide, they significantly increase extracellular IL-1β levels. Conclusion: These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells. Plain language summary Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses of two types of brain cells, astrocytes and microglia, to MSPs. Mouse astrocytes and microglia were kept alive in cultures and were treated with MSPs that were labeled with a red fluorescent agent to facilitate visualization under the microscope. MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. When given alone, MSPs do not affect mRNA levels of key proinflammatory genes. However, MSPs given in combination with lipopolysaccharide, a strong proinflammatory agent, significantly increase extracellular levels of IL-1β, one of the proinflammatory mediators studied. These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells

Microglial hyperreactivity evolved to immunosuppression in the hippocampus of a mouse model of accelerated aging and Alzheimer’s Disease traits

Neuroinflammation is a risk factor for Alzheimer’s disease (AD). We sought to study the glial derangement in AD using diverse experimental models and human brain tissue. Besides classical pro-inflammatory cytokines, we analyzed chitinase 3 like 1 (CHI3L1 or YKL40) and triggering receptor expressed on myeloid cells 2 (TREM2) that are increasingly being associated with astrogliosis and microgliosis in AD, respectively. The SAMP8 mouse model of accelerated aging and AD traits showed elevated pro-inflammatory cytokines and activated microglia phenotype. Furthermore, 6-month-old SAMP8 showed an exacerbated inflammatory response to peripheral lipopolysaccharide in the hippocampus and null responsiveness at the advanced age (for this strain) of 12 months.This research was funded by Spanish MINECO and European Regional Development Fund, grant number SAF2016- 77703; Spanish MCINN, grant number PID2019-106285RB; Catalan Autonomous Government AGAUR, grant number 2017-SGR-106; Competitiveness Operational Programme 2014-2020, C-Reactive protein therapy for stroke-associated dementia, ID P_37_674, MySMIS code: 103432, contract 51/05.09.2016; and the CERCA Programme/Generalitat de Catalunya. RCo was supported by a post-doctoral research contract of the Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain. AL (PERIS SLT008/18/00061) received funding from Departament de Salut de la Generalitat de Catalunya. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Social networks for reading as a new Literary Training Space

In contrast to the generalized negative vision regarding the use of social networks, this study aims to analyze their present possibilities for the dissemination of literature and for the development of literary education. In that respect, the concepts of social reading and LIJ 2.0 are presented as a case of Internet participation dynamics related to the development of reading habits. Next, the main social reading networks whose main objective is to talk about literature are presented. The main objective of this work is to present several specific uses for the promotion of reading in common social networks such as Facebook, Twitter or Instagram and in specific reading networks such as Goodreads or the Spanish Entrelectores and Leoteca.This study is part of the Research Project: Proyecto de Investigación Emergente de la Universidad de Alicante GRE 16–05: “Literatura Infantil y Juvenil en Internet. LIJ 2.0 Análisis de Aplicaciones y Redes sociales de lectura”

Antioxidant and neuroprotective actions of IGF-II against glucocorticoid-induced toxicity in dopaminergic neurons.

The neurodegenerative Parkinson’s disease (PD) affects 1–3% of the population aged over 65. A wide range of pathways and mechanisms are involved in its pathogenesis, such as oxidative stress, mitochondrial dysfunction, inflammation and neuronal glucocorticoid-induced toxicity, which ultimately produce a progressive loss of nigral dopamine neurons. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT), an endocrine response marker to stress, and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II maintained the tyrosine hydroxylase expression and promotes nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. This work revealed the potential neuroprotective role of IGF-II to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Insulin-like growth factor II neuroprotective effects against mitochondrial-oxidative and neuronal damage induced by CORT and MPP+ in dopaminergic neurons

Aims: Parkinson’s disease (PD) affects 1–3% of the population aged over 65. Stress seems to contribute to PD neuropathology, probably by dysregulation of the hypothalamic–pituitary–adrenal axis. Key factors are oxidative stress, mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. Insulin-like growth factor II (IGF-II) has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Therefore, our aim was to study IGF-II protective effects against oxidative damage on a cellular combined model of PD and mild to moderate stress, based on corticosterone (CORT) and the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Methods: The dopaminergic neuronal cell line SN4741 (RRID:CVCL_S466) derived from mouse substantia nigra were exposed to 200 μM MPP+, 0.5 μM CORT or both, with or without 25 ng/mL IGF-II, for 2.5 or 6 h. Cell viability, oxidative stress parameters, mitochondrial and dopamine markers and intracellular signaling pathways were evaluated. Results: The administration of MPP+ or CORT individually led to cell damage compared to control situations, whereas the combination of both drugs produced very considerable toxic synergistic effect. IGF-II counteracts the mitochondrial-oxidative damage, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Conclusions: IGF-II capacity to protect nigral dopamine neurons against mitochondrial-oxidative damage induced by CORT and MPP+ was demonstrated. Thus, IGF-II is a potential therapeutic tool for prevention and treatment of PD patients suffering mild to moderate emotional stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech