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

Efficacy of immune checkpoint inhibitors in alveolar soft-part sarcoma: results from a retrospective worldwide registry

Alveolar soft-part sarcoma; Immune checkpoint; ImmunotherapySarcoma alveolar de partes blandas; Puntos de control inmunológico; InmunoterapiaSarcoma alveolar de parts toves; Punts de control immunològic; ImmunoteràpiaBackground Conventional cytotoxic drugs are not effective in alveolar soft-part sarcoma (ASPS). Immune checkpoint (programmed cell death protein 1/programmed death-ligand 1) inhibitors (ICIs) are promising drugs in ASPS. A worldwide registry explored the efficacy of ICI in ASPS. Materials and methods Data from adult patients diagnosed with ASPS and treated with ICI for advanced disease in expert sarcoma centers from Europe, Australia and North America were retrospectively collected, including demographics and data related to treatments and outcome. Results Seventy-six ASPS patients, with a median age at diagnosis of 25 years (range 3-61 years), were registered. All patients received ICI for metastatic disease. Immunotherapy regimens consisted of monotherapy in 38 patients (50%) and combination in 38 (50%) (23 with a tyrosine kinase inhibitor). Among the 68 assessable patients, there were 3 complete responses and 34 partial responses, translating into an overall response rate of 54.4%. After a median follow-up of 36 months [95% confidence interval (CI) 32-40 months] since the start of immunotherapy, 45 (59%) patients have progressed on ICI, with a median progression-free survival (PFS) of 16.3 months (95% CI 8-25 months). Receiving ICI in first line (P = 0.042) and achieving an objective response (P = 0.043) correlated with a better PFS. Median estimated overall survival (OS) from ICI initiation has not been reached. The 12-month and 24-month OS rates were 94% and 81%, respectively. Conclusions This registry constitutes the largest available series of ASPS treated with ICI. Our results suggest that the ICI treatment provides long-lasting disease control and prolonged OS in patients with advanced ASPS, an ultra-rare entity with limited active therapeutic options.DSM is a recipient of a Sara Borrell postdoctoral fellowship funded by the National Institute of Health Carlos III (ISCIII) (CD20/00155). The authors thank SELNET project. SELNET has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 825806

REGISTRI: Regorafenib in first-line of KIT/PDGFRA wild type metastatic GIST: a collaborative Spanish (GEIS), Italian (ISG) and French Sarcoma Group (FSG) phase II trial

Wild type GIST; Biomarker; Clinical trialGIST de tipo salvaje; Biomarcador; Ensayo clínicoGIST de tipus salvatge, Biomarcador; Assaig clínicBackground Approximately 15% of adult GIST patients harbor tumors that are wild-type for KIT and PDGFRα genes (KP-wtGIST). These tumors usually have SDH deficiencies, exhibit a more indolent behavior and are resistant to imatinib. Underlying oncogenic mechanisms in KP-wtGIST include overexpression of HIF1α high IGFR signaling through the MAPK pathway or BRAF activating mutation, among others. As regorafenib inhibits these signaling pathways, it was hypothesized that it could be more active as upfront therapy in advanced KP-wtGIST. Methods Adult patients with advanced KP-wtGIST after central confirmation by NGS, naïve of systemic treatment for advanced disease, were included in this international phase II trial. Eligible patients received regorafenib 160 mg per day for 21 days every 28 days. The primary endpoint was disease control rate (DCR), according to RECIST 1.1 at 12 weeks by central radiological assessment. Results From May 2016 to October 2020, 30 patients were identified as KP-wtGIST by Sanger sequencing and 16 were confirmed by central molecular screening with NGS. Finally, 15 were enrolled and received regorafenib. The study was prematurely closed due to the low accrual worsened by COVID outbreak. The DCR at 12 weeks was 86.7% by central assessment. A subset of 60% experienced some tumor shrinkage, with partial responses and stabilization observed in 13% and 87% respectively, by central assessment. SDH-deficient GIST showed better clinical outcome than other KP-wtGIST. Conclusions Regorafenib activity in KP-wtGIST compares favorably with other tyrosine kinase inhibitors, especially in the SDH-deficient GIST subset and it should be taken into consideration as upfront therapy of advanced KP-wtGIST.GEIS, Bayer and SELNET. SELNET has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement Nº. 825806

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

Neoadjuvant Trastuzumab Emtansine and Pertuzumab in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer: Three-Year Outcomes From the Phase III KRISTINE Study.

PurposeThe KRISTINE study compared neoadjuvant trastuzumab emtansine plus pertuzumab (T-DM1+P) with docetaxel, carboplatin, trastuzumab plus P (TCH+P) for the treatment human epidermal growth factor receptor 2-positive stage II to III breast cancer. T-DM1+P led to a lower pathologic complete response rate (44.4% v 55.7%; P = .016), but fewer grade 3 or greater and serious adverse events (AEs). Here, we present 3-year outcomes from KRISTINE.MethodsPatients were randomly assigned to neoadjuvant T-DM1+P or TCH+P every 3 weeks for six cycles. Patients who received T-DM1+P continued adjuvant T-DM1+P, and patients who received TCH+P received adjuvant trastuzumab plus pertuzumab. Secondary end points included event-free survival (EFS), overall survival, patient-reported outcomes (measured from random assignment), and invasive disease-free survival (IDFS; measured from surgery).ResultsOf patients, 444 were randomly assigned (T-DM1+P, n = 223; TCH+P, n = 221). Median follow-up was 37 months. Risk of an EFS event was higher with TDM-1+P (hazard ratio [HR], 2.61 [95% CI, 1.36 to 4.98]) with more locoregional progression events before surgery (15 [6.7%] v 0). Risk of an IDFS event after surgery was similar between arms (HR, 1.11 [95% CI, 0.52 to 2.40]). Pathologic complete response was associated with a reduced risk of an IDFS event (HR, 0.24 [95% CI, 0.09 to 0.60]) regardless of treatment arm. Overall, grade 3 or greater AEs (31.8% v 67.7%) were less common with T-DM1+P. During adjuvant treatment, grade 3 or greater AEs (24.5% v 9.9%) and AEs leading to treatment discontinuation (18.4% v 3.8%) were more common with T-DM1+P. Patient-reported outcomes favored T-DM1+P during neoadjuvant treatment and were similar to trastuzumab plus pertuzumab during adjuvant treatment.ConclusionCompared with TCH+P, T-DM1+P resulted in a higher risk of an EFS event owing to locoregional progression events before surgery, a similar risk of an IDFS event, fewer grade 3 or greater AEs during neoadjuvant treatment, and more AEs leading to treatment discontinuation during adjuvant treatment

Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4 ) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.National Science Foundation, grant number EAR-2038207EAR-1423939Ministerio de Economía y Competitividad, SPAIN and Fondo Europeo de Desarrollo Regional, FEDER grant numbers CGL2010-18274 and CGL2013-4661

New molecular mechanisms to explain the neuroprotective effects of insulin-like growth factor II in a cellular model of Parkinson's disease

Introduction: One of the hallmarks of Parkinsońs Disease (PD) is oxidative distress, leading to mitochondrial dysfunction and neurodegeneration. Insulin-like growth factor II (IGF-II) has been proven to have antioxidant and neuroprotective effects in some neurodegenerative diseases, including PD. Consequently, there isgrowing interest in understanding the different mechanisms involved in the neuroprotective effect of this hormone. Objectives: To clarify the mechanism of action of IGF-II involved in the protective effect of this hormone. Methods: The present study was carried out on a cellular model PD based on the incubation of dopaminergic cells (SN4741) in a culture with the toxic 1-methyl-4-phenylpyridinium (MPP+), in the presence of IGF-II. This model undertakes proteomic analyses in order to understand which molecular cell pathways might be involved in the neuroprotective effect of IGF-II. The most important proteins found in the proteomic study were tested by Western blot, colorimetric enzymatic activity assay and immunocytochemistry. Along with the proteomic study, mitochondrial morphology and function were also studied by transmission electron microscopy and oxygen consumption rate. The cell cycle was also analysed using 7AAd/BrdU staining, and flow cytometry. Results: The results obtained indicate that MPP+, MPP++IGF-II treatment and IGF-II, when compared to control, modified the expression of 197, 246 proteins and 207 respectively. Some of these proteins were found to be involved in mitochondrial structure and function, and cell cycle regulation. Including IGF-II in the incubation medium prevents the cell damage induced by MPP+, recovering mitochondrial function and cell cycle dysregulation, and thereby decreasing apoptosis. Conclusion: IGF-II improves mitochondrial dynamics by promoting the association of Mitofilin with mitochondria, regaining function and redox homeostasis. It also rebalances the cell cycle, reducing the amount of apoptosis and cell death by the regulation of transcription factors, such as Checkpoint kinase 1

Cocaine detrimentally affects mitochondrial functionality and cell viability in dopaminergic neurons.

An elevated consumption of cocaine (benzoylmethylecgonine), which causes anesthetic and stimulant effects on the central nervous system, may be associated with several neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease (PD). To investigate the impact of cocaine on cell viability and morphology, dopaminergic neurons from the substantia nigra (SN4741) were cultured. Analysis involved assessing cell death (LDH levels) and cell morphology (GIEMSA staining) after a 24-hour treatment period. Additionally, the effects on reactive oxygen species (ROS) generation (DH2), membrane potential (JC-1), oxygen consumption rate (OCR), and mitochondrial stress (Seahorse) were evaluated after a 6-hour treatment. The optimal concentration of cocaine for experimental use (2 mM) was identified, inducing a substantial 39.75% neuronal death. Examination of neuronal death (LDH) revealed a remarkable 280% increase following cocaine treatment. Optical analysis demonstrated heightened mortality and detrimental changes in neuronal morphology post-cocaine treatment, including a globose shape, loss of synapses, extremely thin membrane, and cell aggregation. In the "short time" experiments, mitochondrial oxidative damage was evident in SN cells treated with cocaine, leading to the demise of 75% of the cells. Furthermore, a significant 173.6% increase in reactive oxygen species (ROS) production and a 20% reduction in mitochondrial membrane potential (JC-1 assay) were observed. Cocaine treatment also resulted in a notable 60% decrease in mitochondrial oxygen consumption. In summary, a concentration of 2 mM cocaine induces a considerable rise in mitochondrial oxidative damage, subsequently causing morphological alterations and progressive death of dopaminergic neurons due to the accumulation of reactive oxygen species (ROS).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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