an-efficient-protocol-to-assess-erk-activity-modulation-early

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Ospedale Pediatrico Bambino Gesù, IRCCS

Glutathione provides means of regulating protein function by the process of glutathionylation. Despite the role of oxidative stress biomarkers assumed recently by glutathionylated proteins in human diseases, so far no information is available on the intracellular distribution of glutathionylated proteins in human cell lines. In this study, we combined the specificity of monoclonal antibody labeling for protein-bound glutathione (GS-Pro) with the ability of confocal microscopy to localize molecules with high spatial resolution. We performed immunofluorescence analysis on dermal fibroblasts, both in steady state than in proliferative conditions, and on in situ extracted matrix samples. For the first time, we report the compartmentalization of constitutively glutathionylated proteins in different subcellular districts and we found a tight association between glutathione, nuclear lamina, and cytoskeleton. In proliferating cells, total GS-Pro fluorescence increases in the early phases of growth and significantly drops when cells reach confluence. Interestingly, a nuclear shift of GS-Pro was observed between 6 and 48 hours after plating, becoming homogeneous with the cytoplasm when growth slows. The ability to visualize a detailed intracellular distribution of this critical marker of protein oxidation may provide an additional tool to highlight pathways in turns 'redox-activated' and to identify new pathogenic pathways in human diseases.

Protein glutathionylation in cellular compartments: a constitutive redox signal.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells.

Monocytes and macrophages as biomarkers for the diagnosis of megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: relevance to MLC disease pathogenesis.

S-glutathionylation is a mechanism of signal transduction by which cells respond effectively and reversibly to redox inputs. The glutathionylation regulates most cellular pathways. It is involved in oxidative cellular response to insult by modulating the transcription factor Nrf2 and inducing the expression of antioxidant genes (ARE); it contributes to cell survival through nuclear translocation of NFkB and activation of survival genes, and to cell death by modulating the activity of caspase 3. It is involved in mitotic spindle formation during cell division by binding cytoskeletal proteins thus contributing to cell proliferation and differentiation. Glutathionylation also interfaces with the mechanism of phosphorylation by modulating several kinases (PKA, CK) and phosphatases (PP2A, PTEN), thus allowing a cross talk between the two processes of signal transduction. Glutathionylation of proteins may also act on cell metabolism by modulating enzymes involved in glycosylation, in the Krebs cycle and in mitochondrial oxidative phosphorylation. Perturbations in protein glutathionylation status may contribute to the etiology of many diseases, thus it is clear the importance to visualize the distribution of glutathionylated proteins in subcellular compartments. This chapter describes the immunofluorescence technique that permits simultaneous detection of glutathionylated proteins and their localization in cellular compartments, using multiple stained cell samples. By confocal laser microscopy analysis of the immunofluorescent cells it is possible to obtain detailed information of submicroscopic structures inside cells and tissues, and to perform correct co-localization analysis between two proteins. The association between glutathione, nuclear lamina, and cytoskeleton has been investigated by employing a helpful assay consisting on the in situ extraction of the cellular matrix from cultured dermal fibroblasts followed by multiple stainings with several primary antibodies. This protocol can be used for the detection of the intracellular distribution and expression of interest proteins and can be customized for a large variety of cells and tissues.

Intracellular distribution of glutathionylated proteins in cultured dermal fibroblasts by immunofluorescence.

Cardiac hypertrophy occurs in response to different stimuli and is mainly characterized by an enlargement of cardiomyocyte size. During hypertrophy, cardiomyocytes undergo not only radical changes of the cellular architecture but also activation of signaling cascades that counteract the atrophy genes. Experimental studies highlighted that chronic low concentrations of H2O2, induce a hypertrophic phenotype, while higher levels of H2O2 promote apoptosis. In this study, we explored the early and long-term hypertrophic effects of high concentrations of H2O2 on H9c2 rat cardiomyocytes. We found that 2-h stimulation with 200μM H2O2 caused an early dramatic reduction of cell viability, accompanied, 5-days later, by increased cell size and up-regulation of atrial natriuretic peptide transcription. This hypertrophic phenotype is associated to increased Akt phosphorylation and a consequent reduction of the FOXO3a and atrogin-1 gene expression. Moreover, we observed that H2O2 caused the overexpression of miR-212/miR-132 cluster concomitantly to a down-regulation of PTEN transcript without changes in its protein expression. Noteworthy, we found that the treatment of cardiomyocytes with H2O2 further led to an increase of oxidized glutathione and glutathionylation of proteins, including PTEN. In conclusion, our results permit to reconstruct the molecular cascade triggering the cardiomyocyte hypertrophy upon high concentrations of H2O2.

High concentrations of H2O2 trigger hypertrophic cascade and phosphatase and tensin homologue (PTEN) glutathionylation in H9c2 cardiomyocytes.

Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1; enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.

Stefania Petrini

Microscopy facility,
Research laboratories,
Microscopy facility, Research laboratories

Ospedale Pediatrico Bambino Gesù, IRCCS

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Stefania Petrini

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Microscopy facility,Research laboratories,Microscopy facility, Research laboratories

Ospedale Pediatrico Bambino Gesù, IRCCS

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Microscopy facility,
Research laboratories,
Microscopy facility, Research laboratories