Bioinformatic analysis of the human brain extracellular matrix proteome in neurodegenerative disorders.

Alzheimer's, Parkinson's, and Huntington's diseases are characterized by selective degeneration of specific brain areas. Although increasing number of studies report alteration of the extracellular matrix on these diseases, an exhaustive characterization at the brain's matrix level might contribute to the development of more efficient cell restoration therapies. In that regard, proteomics-based studies are a powerful approach to uncover matrix changes. However, to date, the majority of proteomics studies report no or only a few brain matrix proteins with altered expression. This study aims to reveal the changes in the brain extracellular matrix by integrating several proteomics-based studies performed with postmortem tissue. In total, 67 matrix proteins with altered expression were collected. By applying a bioinformatic approach, we were able to reveal the dysregulated biological processes. Among them are processes related to the organization of the extracellular matrix, glycosaminoglycans and proteoglycans' metabolism, blood coagulation, and response to injury and oxidative stress. In addition, a protein was found altered in all three diseases-collagen type I alpha 2-and its binding partners further identified. A ClueGO network was created, depicting the GO groups associated with these binding partners, uncovering the processes that may consequently be affected. These include cellular adhesion, cell signaling through membrane receptors, inflammatory processes, and apoptotic cell death in response to oxidative stress. Overall, we were able to associate the contribution of the modification of extracellular matrix components to essential biological processes, highlighting the investment needed on proteomics studies with specific focus on the extracellular matrix in neurodegeneration.

The lack of neuropeptide Y-Y1 receptor signaling modulates the chemical and mechanical properties of bone matrix.

Genetic and pharmacological functional studies have provided evidence that the lack of Neuropeptide Y-Y1  receptor (Y1 R) signaling pathway induces a high bone mass phenotype in mice. However, clinical observations have shown that drug or genetic mediated improvement of bone mass might be associated to alterations to bone extracellular matrix (ECM) properties, leading to bone fragility. Hence, in this study we propose to characterize the physical, chemical and biomechanical properties of mature bone ECM of germline NPY-Y1 R knockout (Y1 R-/- ) mice, and compare to their wild-type (WT) littermates. Our results demonstrated that the high bone mass phenotype observed in Y1 R-/- mice involves alterations in Y1 R-/-  bone ECM ultrastructure, as a result of accelerated deposition of organic and mineral fractions. In addition, Y1 R-/- bone ECM displays enhanced matrix maturation characterized by greater number of mature/highly packed collagen fibers without pathological accumulation of immature/mature collagen crosslinks nor compromise of mineral crystallinity. These unique features of Y1 R-/-  bone ECM improved the biochemical properties of Y1 R-/-  bones, reflected by mechanically robust bones with diminished propensity to fracture, contributing to greater bone strength. These findings support the future usage of drugs targeting Y1 R signaling as a promising therapeutic strategy to treat bone loss-related pathologies.

OXPHOS dysfunction regulates integrin-ß1 modifications and enhances cell motility and migration.

Mitochondria are central organelles for cellular metabolism. In cancer cells, mitochondrial oxidative phosphorylation (OXPHOS) dysfunction has been shown to promote migration, invasion, metastization and apoptosis resistance. With the purpose of analysing the effects of OXPHOS dysfunction in cancer cells and the molecular players involved, we generated cybrid cell lines harbouring either wild-type (WT) or mutant mitochondrial DNA (mtDNA) [tRNAmut cybrids, which harbour the pathogenic A3243T mutation in the leucine transfer RNA gene (tRNAleu)]. tRNAmut cybrids exhibited lower oxygen consumption and higher glucose consumption and lactate production than WT cybrids. tRNAmut cybrids displayed increased motility and migration capacities, which were associated with altered integrin-ß1 N-glycosylation, in particular with higher levels of ß-1,6-N-acetylglucosamine (GlcNAc) branched N-glycans. This integrin-ß1 N-glycosylation pattern was correlated with higher levels of membrane-bound integrin-ß1 and also with increased binding to fibronectin. When cultured in vitro, tRNAmut cybrids presented lower growth rate than WT cybrids, however, when injected in nude mice, tRNAmut cybrids produced larger tumours and showed higher metastatic potential than WT cybrids. We conclude that mtDNA-driven OXPHOS dysfunction correlates with increased motility and migration capacities, through a mechanism that may involve the cross talk between cancer cell mitochondria and the extracellular matrix.

The respiratory chain inhibitor rotenone affects peroxisomal dynamics via its microtubule-destabilising activity.

Peroxisomes and mitochondria in mammalian cells are closely linked subcellular organelles, which maintain a redox-sensitive relationship. Their interplay and role in ROS signalling are supposed to impact on age-related and degenerative disorders. Whereas the generation of peroxisome-derived oxidative stress can affect mitochondrial morphology and function, little is known about the impact of mitochondria-derived oxidative stress on peroxisomes. Here, we investigated the effect of the mitochondrial complex I inhibitor rotenone on peroxisomal and mitochondrial membrane dynamics. We show that rotenone treatment of COS-7 cells alters peroxisome morphology and distribution. However, this effect is related to its microtubule-destabilising activity rather than to the generation of oxidative stress. Rotenone also induced alterations in mitochondrial morphology, which-in contrast to its effect on peroxisomes-were dependent on the generation of ROS but independent of its microtubule-active properties. The importance of our findings for the peroxisome-mitochondria redox relationship and the interpretation of in cellulo and in vivo studies with rotenone, which is widely used to study Parkinson's disease, are discussed.

Caffeine-mediated BDNF release regulates long-term synaptic plasticity through activation of IRS2 signaling.

Caffeine has cognitive-enhancing properties with effects on learning and memory, concentration, arousal and mood. These effects imply changes at circuital and synaptic level, but the mechanism by which caffeine modifies synaptic plasticity remains elusive. Here we report that caffeine, at concentrations representing moderate to high levels of consumption in humans, induces an NMDA receptor-independent form of LTP (CAF LTP) in the CA1 region of the hippocampus by promoting calcium-dependent secretion of BDNF, which subsequently activates TrkB-mediated signaling required for the expression of CAF LTP. Our data include the novel observation that insulin receptor substrate 2 (IRS2) is phosphorylated during induction of CAF LTP, a process that requires cytosolic free Ca2+ . Consistent with the involvement of IRS2 signals in caffeine-mediated synaptic plasticity, phosphorylation of Akt (Ser473) in response to LTP induction is defective in Irs2-/- mice, demonstrating that these plasticity changes are associated with downstream targets of the phosphoinositide 3-kinase (PI3K) pathway. These findings indicate that TrkB-IRS2 signals are essential for activation of PI3K during the induction of LTP by caffeine.

Local iron homeostasis in the breast ductal carcinoma microenvironment.

BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.

FABP4 Enhances Lipidic and Fibrotic Cardiac Structural and Ca2+ Dynamic Changes.

BACKGROUND: Adipocyte FABP4 (fatty acid-binding protein 4) is augmented in the epicardial stroma of patients with long-standing persistent atrial fibrillation. Because this molecule is released mainly by adipocytes, our objective was to study its role in atrial cardiomyopathy, focusing our attention on fibrosis, metabolism, and electrophysiological changes. These results might clarify the role of adiposity as a mediator of atrial cardiomyopathy. METHODS: We used several preclinical cellular models, epicardial and subcutaneous stroma primary cell cultures from patients undergoing open heart surgery, human atrial fibroblasts, atrial cardiomyocytes derived from human induced pluripotent stem cells and isolated from adult mice, and Nav1.5 transfected Chinese hamster ovary cells. Fibrosis, glucose, mitochondrial and adipogenesis activity, gene expression, and proteomics were determined by wound healing, enzymatic, colorimetric, fluorescence assays, real-time quantitative polymerase chain reaction, and TripleTOF proteomics. Molecular changes were analyzed by Raman confocal microspectroscopy, calcium dynamics by confocal microscopy, and ion currents by patch clamp. Epicardial, subcutaneous, and atrial fibroblasts and cardiomyocytes were incubated with FABP4 at 100 ng/mL. RESULTS: Our results showed that FABP4 induced fibrosis, glucose metabolism, and lipid accumulation on epicardial and subcutaneous stroma cells and atrial fibroblasts. Besides, it modified lipid content and calcium dynamics in atrial cardiomyocytes without effects on INa. CONCLUSIONS: FABP4 exerts fibrotic and metabolic changes on epicardial stroma and modifies lipid content and calcium dynamic on atrial cardiomyocytes. These results suggest its possible role as an atrial cardiomyopathy mediator.

Impact of Long-Term Swimming Exercise on Rat Femur Bone Quality.

Considering the conflicting evidence regarding the potential long-term detrimental effect of swimming during growth on femur quality and fracture risk, our aim was to investigate the effect of eight months of swimming on femur quality. Twenty male eight-week-old Wistar rats were assigned into a swimming (SW; n = 10; 2 h/day, 5 days/week) or active control group (CG; n = 10, housed with running wheel) for eight months. Plasma osteocalcin and C-terminal telopeptide of type I collagen concentrations (ELISA) were assessed at baseline, four, and eight months of protocol. Femur structure (micro-computed tomography), biomechanical properties (three-point bending), and cellular density (histology) were determined after the protocol. SW displayed a lower uncoupling index, suggesting higher bone resorption, lower empty lacunae density, cortical and trabecular femur mass, femur length and cortical thickness, and higher cortical porosity than CG (p < 0.05). Although both biomarkers' concentrations decreased in both groups throughout the experiment (p < 0.001), there were no significant differences between groups (p > 0.05). No differences were also found regarding biomechanical properties, bone marrow adiposity, and osteocyte and osteoclast densities (p > 0.05). Long-term swimming was associated with unbalanced bone turnover and compromised femur growth, lower femur mass, and deteriorated cortical bone microarchitecture. However, femur trabecular microarchitecture and biomechanical properties were not affected by swimming.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

High-resolution micro-CT for 3D infarct characterization and segmentation in mice stroke models.

Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.

Data-Driven Modeling of the Cellular Pharmacokinetics of Degradable Chitosan-Based Nanoparticles.

Nanoparticle drug delivery vehicles introduce multiple pharmacokinetic processes, with the delivery, accumulation, and stability of the therapeutic molecule influenced by nanoscale processes. Therefore, considering the complexity of the multiple interactions, the use of data-driven models has critical importance in understanding the interplay between controlling processes. We demonstrate data simulation techniques to reproduce the time-dependent dose of trimethyl chitosan nanoparticles in an ND7/23 neuronal cell line, used as an in vitro model of native peripheral sensory neurons. Derived analytical expressions of the mean dose per cell accurately capture the pharmacokinetics by including a declining delivery rate and an intracellular particle degradation process. Comparison with experiment indicates a supply time constant, τ = 2 h. and a degradation rate constant, b = 0.71 h-1. Modeling the dose heterogeneity uses simulated data distributions, with time dependence incorporated by transforming data-bin values. The simulations mimic the dynamic nature of cell-to-cell dose variation and explain the observed trend of increasing numbers of high-dose cells at early time points, followed by a shift in distribution peak to lower dose between 4 to 8 h and a static dose profile beyond 8 h.

Characterization of the Striatal Extracellular Matrix in a Mouse Model of Parkinson's Disease.

Parkinson's disease's etiology is unknown, although evidence suggests the involvement of oxidative modifications of intracellular components in disease pathobiology. Despite the known involvement of the extracellular matrix in physiology and disease, the influence of oxidative stress on the matrix has been neglected. The chemical modifications that might accumulate in matrix components due to their long half-live and the low amount of extracellular antioxidants could also contribute to the disease and explain ineffective cellular therapies. The enriched striatal extracellular matrix from a mouse model of Parkinson's disease was characterized by Raman spectroscopy. We found a matrix fingerprint of increased oxalate content and oxidative modifications. To uncover the effects of these changes on brain cells, we morphologically characterized the primary microglia used to repopulate this matrix and further quantified the effects on cellular mechanical stress by an intracellular fluorescence resonance energy transfer (FRET)-mechanosensor using the U-2 OS cell line. Our data suggest changes in microglia survival and morphology, and a decrease in cytoskeletal tension in response to the modified matrix from both hemispheres of 6-hydroxydopamine (6-OHDA)-lesioned animals. Collectively, these data suggest that the extracellular matrix is modified, and underscore the need for its thorough investigation, which may reveal new ways to improve therapies or may even reveal new therapies.

Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells.

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.

Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.

Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a "basic" group (pH range 6.2-11) with about 46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to 16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis-trans isomerase B (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogen granules and not contaminants of the preparation, we conducted a series of validation experiments. Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like bona fide peripheral membrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructural and immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase-YFP fusion protein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins, proteoglycan-binding properties have been reported. The importance of our findings for sorting and packaging during ZG formation is discussed.

Proteomic analysis of zymogen granules.

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. ZG constituents play important roles in pancreatic injury and disease. The molecular mechanisms underlying these processes are still poorly defined. Thus, there is currently great interest in the identification and characterization of ZG components. Recent proteomic studies have greatly enhanced our knowledge regarding potential new 'players' in ZG biogenesis and regulated secretion. In this article, we present the latest advancements in and insights into the analysis of the ZG proteome by the combination of organelle isolation, protein separation, mass spectrometry and validation of protein identification. Recent developments in the analysis of ZG proteins from pancreatic juice and related proteins from saliva are also discussed.

Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions.

Helicobacter pylori, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the gastric mucosa, H. pylori continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that H. pylori targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection, H. pylori induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the H.pylori virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell-cell and cell-matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of H. pylori-host interactions and associated diseases. Additionally, it raises awareness for potential interference between H. pylori infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK i: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK i: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK d values comparable to the pK i values. The fluorescent probes 21, 23, and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA.

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells.

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.

Immunomodulation of Human Mesenchymal Stem/Stromal Cells in Intervertebral Disc Degeneration: Insights From a Proinflammatory/Degenerative Ex Vivo Model.

STUDY DESIGN: Ex vivo experimental study. OBJECTIVE: To investigate the effect of proinflammatory/degenerative intervertebral disc (IVD) microenvironment on the regenerative and immunomodulatory behavior of mesenchymal stem/stromal cells (MSCs), using an ex vivo model from bovine origin. SUMMARY OF BACKGROUND DATA: Low back pain is a cause of disability worldwide, most frequently associated with IVD degeneration and inflammation, and characterized by increased levels of inflammatory mediators, often disregarded. MSC-based therapies to low back pain have been advocated, but the involvement of inflammation in IVD remodeling mechanism, promoted by MSCs has not yet been explored. METHODS: Bovine IVD organ cultures of nucleus pulposus punches were stimulated with needle puncture and culture medium supplementation with 10 ng/mL of interleukin (IL)-1ß, to induce a proinflammatory/degenerative environment, as previously established. Human bone marrow-derived MSCs were cultured on top of transwells, placed above nucleus pulposus punches, for up to 16 days. MSCs were analyzed by screening cell viability/apoptosis, metabolic activity, migration, and inflammatory cytokines production in response to the proinflammatory environment. IVD extracellular matrix (ECM) remodeling, gene expression profile of IVD cells, and inflammatory cytokine profile in the presence of MSCs in basal versus proinflammatory conditions were also evaluated. RESULTS: Proinflammatory/degenerative IVD conditions did not affect MSCs viability, but promoted cell migration, while increasing IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 and reducing transforming growth factor-ß1 production by MSCs. MSCs did not stimulate ECM production (namely type II collagen or aggrecan) in neither basal nor inflammatory conditions, instead MSCs downregulated bovine proinflammatory IL-6, IL-8, and TNF-α gene expression levels in IL-1ß-stimulated IVDs. CONCLUSION: The present study provides evidence for an immunomodulatory paracrine effect of MSCs in degenerated IVD without an apparent effect in ECM remodeling, and suggest an MSCs mechanism-of-action dependent on a cytokine feedback loop. LEVEL OF EVIDENCE: 5.