Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

Increased autophagy/mitophagy levels in primary tumours of patients with pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS: We assessed the levels of autophagy and mitophagy, that are linked to cancer development and drug resistance, in well differentiated pancreatic neuroendocrine neoplasms (PanNENs) and correlated them with clinico-pathological parameters. METHODS: Fluorescent immunostaining for the autophagy markers LC3Β and p62/or LAMP1 was performed on 22 PanNENs and 11 controls of normal pancreatic tissues and validated through Western blotting. Autophagy quantitative scoring was generated for LC3B-positive puncta and analysed in relation to clinico-pathological parameters. TOMM20/LC3B qualitative assessment of mitophagy levels was undertaken by fluorescent immunostaining. The presence of autophagy/mitophagy was validated by transmission electron microscopy. RESULTS: Autophagy levels (LC3B-positive puncta/cell) were discriminative for normal vs. NEN pancreatic tissue (p = 0.007). A significant association was observed between autophagy levels and tumour grade (Ki67 < 3% vs. Ki67 ≥ 3%; p = 0.021), but not functionality (p = 0.266) size (cut-off of 20 mm; p = 0.808), local invasion (p = 0.481), lymph node- (p = 0.849) and distant metastases (p = 0.699). Qualitative assessment of TOMM20/LC3B demonstrated strong mitophagy levels in PanNENs by fluorescent immunostaining as compared with normal tissue. Transmission electron microscopy revealed enhanced autophagy and mitophagy in PanNEN tissue. Response to molecular targeted therapies in metastatic cases (n = 4) did not reveal any patterns of association to autophagy levels. CONCLUSIONS: Increased autophagy levels are present in primary tumours of patients with PanNENs and are partially attributed to upregulated mitophagy. Grade was the only clinico-pathological parameter associated with autophagy scores.

Silicon scaffolds promoting three-dimensional neuronal web of cytoplasmic processes.

Primary neurons were grown on structured silicon (Si) substrates, in the absence of chemotropic factors or synthetic extracellular matrix. The Si substrates used for the study comprise hierarchical structures in the micro- and nanolength scales. The substrates were structured via femtosecond laser irradiation of the Si wafer, in a reactive SF(6) environment. Electron microscopy revealed that the neurons formed an elaborate web of cytoplasmic processes in the absence of glial elements. The neuronal cytoplasm autografted the depth of the spikes, and the neurite sprouting took place over the spikes surface. Here we demonstrate how microfabrication of a Si surface provides an excellent platform for multifaceted studies of neuronal specimens.

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival.

Cardiac contractility is regulated through the activity of various key Ca(2+)-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca(2+) transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca(2+) by SR membranes during relaxation. Recently, the antiapoptotic HS-1-associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575-594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca(2+) levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca(2+) stores.

Role of T and NK cells and IL7/IL7r interactions during neonatal maturation of lymph nodes.

Lymph node (LN) development depends on prenatal interactions occurring between LN inducer and LN organizer cells. We have distinguished defects in LN formation due to failure in embryonic development (aly/aly) from defects in postnatal maturation (Il2rgamma(-/-)Rag2(-/-)). Both mutant strains form normal primordial LNs with differing fate. In aly/aly mice, the LN primordium dissipates irreversibly late in gestation; in contrast, Il2rgamma(-/-)Rag2(-/-) LN anlage persists for a week after birth but disperses subsequently, a process reversible by neonatal transfer of WT IL7r(+) TCR(+) T or natural killer (NK) cells, suggesting a role for IL7/IL7r interactions. Thus, we reveal a unique stage of postnatal LN development during which mature lymphocytes and IL7/IL7r interactions may play an important role.

Lipid rafts: cell surface platforms for T cell signaling.

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.

Vav1 transduces TCR signals required for LFA-1 function and cell polarization at the immunological synapse.

Activation of T lineage cells through the TCR by peptide-MHC complexes on APC is critically dependent on rearrangement of the actin cytoskeleton. Vav1 is a guanine nucleotide exchange factor for members of the Rho/Rac family of GTPases which is activated following TCR stimulation, suggesting that it may transduce TCR signals to the activation of some or all actin-controlled processes. We show that Vav1-deficient double-positive thymocytes are less efficient at forming conjugates with APC presenting agonist peptide than wild-type cells are. Furthermore we demonstrate that Vav1 is required for TCR-induced activation of the integrin LFA-1, which is likely to explain the defect in conjugate formation. However, once Vav1-deficient cells form a conjugate, the assembly of proteins into an immunological synapse at the conjugate interface is normal. In contrast, thymocyte polarization is defective in the absence of Vav1, as judged by the relocalization of the microtubule-organizing center. These data demonstrate that Vav1 transduces signals to only a subset of cytoskeleton-dependent events at the immunological synapse.

Modulation of heterochromatin protein 1 dynamics in primary Mammalian cells.

Heterochromatin protein 1 (HP1beta), a key component of condensed DNA, is strongly implicated in gene silencing and centromeric cohesion. Heterochromatin has been considered a static structure, stabilizing crucial aspects of nuclear organization and prohibiting access to transcription factors. We demonstrate here, by fluorescence recovery after photobleaching, that a green fluorescent protein-HP1beta fusion protein is highly mobile within both the euchromatin and heterochromatin of ex vivo resting murine T cells. Moreover, T cell activation greatly increased this mobility, indicating that such a process may facilitate (hetero)chromatin remodeling and permit access of epigenetic modifiers and transcription factors to the many genes that are consequently derepressed.

Lipid rafts: cell surface platforms for T cel signaling

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways (AU)#S