Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H 2 O 2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H 2 O 2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1μM H 2 O 2 , a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H 2 O 2 concentrations. However, at 50 μM H 2 O 2 , an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H 2 O 2 ]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1μM H 2 O 2 . This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.Fil: Helfenberger, Katia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Villalba, Nerina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Buchholz, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Boveris, Alberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Poderoso, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Gelpi, Ricardo Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Poderoso, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

Anxious Behavior of Adult CD1 Mice Perinatally Exposed to Low Concentrations of Ethanol Correlates With Morphological Changes in Cingulate Cortex and Amygdala

Perinatal ethanol (EtOH) exposure is associated with high incidence of behavioral disorders such as depression and anxiety. The cerebral areas related with these consequences involve the corticolimbic system, in particular the prefrontal cortex, hippocampus, amygdala, and cingulate cortex, although the latter has not been thoroughly studied yet. Different animal models of prenatal or perinatal EtOH exposure have reported morphofunctional alterations in the central nervous system, which could explain behavioral disorders along life; these results focus on youth and adolescents and are still controversial. In the light of these inconclusive results, the aim of this work was to analyze adult behavior in CD1 mice perinatally exposed to low concentrations of EtOH (PEE) during gestation and lactation, and describe the morphology of the cingulate cortex and amygdala with a view to establishing structure/function/behavior correlations. Primiparous CD1 female mice were exposed to EtOH 6% v/v for 20 days prior to mating and continued drinking EtOH 6% v/v during pregnancy and lactation. After weaning, male pups were fed food and water ad libitum until 77 days of age, when behavioral and morphological studies were performed. Mouse behavior was analyzed through light–dark box and open field tests. Parameters related to anxious behavior and locomotor activity revealed anxiogenic behavior in PEE mice. After behavioral studies, mice were perfused and neurons, axons, serotonin transporter, 5HT, CB1 receptor (CB1R) and 5HT1A receptor (5HT1AR) were studied by immunofluorescence and immunohistochemistry in brain sections containing cingulate cortex and amygdala. Cingulate cortex and amygdala cytoarchitecture were preserved in adult PEE mice, although a smaller number of neurons was detected in the amygdala. Cingulate cortex axons demonstrated disorganized radial distribution and reduced area. Serotonergic and endocannabinoid systems, both involved in anxious behavior, showed differential expression. Serotonergic afferents were lower in both brain areas of PEE animals, while 5HT1AR expression was lower in the cingulate cortex and higher in the amygdala. The expression of CB1R was lower only in the amygdala. In sum, EtOH exposure during early brain development induces morphological changes in structures of the limbic system and its neuromodulation, which persist into adulthood and may be responsible for anxious behavior.Fil: Madarnas, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Villalba, Nerina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Soriano, Delia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Brusco, Herminia Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Nerve spectroscopy: understanding peripheral nerve autofluorescence through photodynamics

Background: Being able to accurately identify sensory and motor nerves is crucial during surgical procedures to prevent nerve injury. We aimed to (1) evaluate the feasibility of performing peripheral human nerve visualization utilizing nerves’ own autofluorescence in an ex-vivo model; (2) compare the effect of three different nerve fiber fixation methods on the intensity of fluorescence, indicated as the intensity ratio; and (3) similarly compare three different excitation ranges. Methods: Samples from various human peripheral nerves were selected postoperatively. Nerve fibers were divided into three groups: Group A nerve fibers were washed with a physiologic solution; Group B nerve fibers were fixated with formaldehyde for 6 h first, and then washed with a physiologic solution; Group C nerve fibers were fixated with formaldehyde for six hours, but not washed afterwards. An Olympus IX83 inverted microscope was used for close-up image evaluation. Nerve fibers were exposed to white-light wavelength spectrums for a specific time frame prior to visualization under three different filters—Filter 1—LF405-B-OMF Semrock; Filter 2—U-MGFP; Filter 3—U-MRFPHQ Olympus, with excitation ranges of 390–440, 460–480, and 535–555, respectively. The fluorescence intensity of all images was subsequently analyzed using Image-J Software, and results compared by analysis of variance (ANOVA). Results: The intensity ratios observed with Filter 1 failed to distinguish the different nerve fiber groups (p = 0.39). Conversely, the intensity ratios seen under Filters 2 and 3 varied significantly between the three nerve-fiber groups (p = 0.021, p = 0.030, respectively). The overall intensity of measurements was greater with Filter 1 than Filter 3 (p < 0.05); however, all nerves were well visualized by all filters. Conclusion: The current results on ex vivo peripheral nerve fiber autofluorescence suggest that peripheral nerve fiber autofluorescence intensity does not greatly depend upon the excitation wavelength or fixation methods used in an ex vivo setting. Implications for future nerve-sparing surgery are discussed.Fil: Dip, Fernando. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Aleman, Rene. Cleveland Clinic Florida; Estados UnidosFil: Socolovsky, Mariano. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Villalba, Nerina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Falcone, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Lo Menzo, Emanuele. Cleveland Clinic Florida; Estados UnidosFil: White, Kevin P.. Scienceright Research Consulting; CanadáFil: Rosenthal, Raul J.. Cleveland Clinic Florida; Estados Unido

p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain

Programmed and damage aging theories have traditionally been conceived as stand-alone schools of thought. However, the p66Shc adaptor protein has demonstrated that aging-regulating genes and reactive oxygen species (ROS) are closely interconnected, since its absence modifies metabolic homeostasis by providing oxidative stress resistance and promoting longevity. p66Shc(−/−) mice are a unique opportunity to further comprehend the bidirectional relationship between redox homeostasis and the imbalance of mitochondrial biogenesis and dynamics during aging. This study shows that brain mitochondria of p66Shc(−/−) aged mice exhibit a reduced alteration of redox balance with a decrease in both ROS generation and its detoxification activity. We also demonstrate a strong link between reactive nitrogen species (RNS) and mitochondrial function, morphology, and biogenesis, where low levels of ONOO− formation present in aged p66Shc(−/−) mouse brain prevent protein nitration, delaying the loss of biological functions characteristic of the aging process. Sirt3 modulates age-associated mitochondrial biology and function via lysine deacetylation of target proteins, and we show that its regulation depends on its nitration status and is benefited by the improved NAD+/NADH ratio in aged p66Shc(−/−) brain mitochondria. Low levels of protein nitration and acetylation could cause the metabolic homeostasis maintenance observed during aging in this group, thus increasing its lifespan

Characterization of Tunneling Nanotubes in Wharton's jelly Mesenchymal Stem Cells : An Intercellular Exchange of Components between Neighboring Cells

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.Facultad de Ciencias Médica

Characterization of Tunneling Nanotubes in Wharton’s jelly Mesenchymal Stem Cells. An Intercellular Exchange of Components between Neighboring Cells

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton^s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.Fil: Sanchez, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Villalba, Nerina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Fiore, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Luzzani, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; ArgentinaFil: Boveris, Alberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gelpi, Ricardo Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Brusco, Herminia Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Poderoso, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentin

Mitochondrial ERK localization depends on differential phosphorylation.

<p>Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H₂O₂ during 0–30 min. From top to bottom of the panels images show increasing H₂O₂ incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H₂O₂ arbitrarily defined as 1. Bar = 10 μm.</p

Summary of the proposed mechanistic design for double ERK phosphorylation in cytosol and mitochondria under different redox conditions.

<p>After EGF or 1 μM H₂O₂ stimulation, which exert a well described and robust proliferative action, ERK2 is rapidly phosphorylated by cytosolic MEK in Tyr185 (pYERK2), allowing a further association with mitochondria and phosphorylation of Thr183, by a mitochondrial MEK pool. This second and compartmentalized phosphorylation drives fully active-ERK2 (2pERK2) translocation to the nucleus to promote gene transcription and cellular division (A). Panel B shows mitochondrial pThr ERK2 kinetics in response to 1 and 50 μM H₂O₂. At 1 μM H₂O₂ Thr183 is rapidly phosphorylated in mitochondria with a transient localization and fast translocation to the nucleus (white circles); on the other hand, in the presence of 50 μM H₂O₂ arresting cell cycle, Thr183 phosphorylated signal is retained in the mitochondrial milieu and ERK translocation to the nucleus is impeded.</p

Redox status modulates ERK cellular traffic through differential phosphorylation.

<p>LP07 cells were incubated in the presence or absence of 1 μM or 50 μM H₂O₂ for the indicated times. Then, mitochondrial, nucleus and cytosolic fractions were obtained and the detection of pTyr (pYERK) and Thr/Tyr phosphorylated ERK1/2 (2pERK) was analyzed by Western blot. Representative images for the cytosolic (Panel A), mitochondrial (Panel B) and nuclear fraction (Panel C) of three independent experiments. Actin, complex III (CIII) and TFIID protein antibodies were used as loading control markers to obtain relative phosphorylated ERK levels. Levels of pYERK and 2pERK relative to actin (Panel A), CIII (Panel B) and TFDII (Panel C) expressed in arbitrary units (A.U.). Panel D shows representative immunoblots with specific antibodies for each subcellular fraction. Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p

Double phosphorylation of ERK by MEK relies almost completely on mitochondrial phospho-Thr183.

<p>LP07 cells were transiently transfected with wild type ERK2-V5 (WT), Y183A or T183A ERK mutants, using Lipofectamine 2000 reagent. Forty-eight hours post-transfection LP07 were stimulated with 1 μM H₂O₂ during 0–30 min and then incubated in the presence or absence of 1 μM H₂O₂ for the indicated times. Then, the cytosolic, mitochondrial and nuclear fractions were obtained and the detection of ERK2 was analyzed by western blot using anti-V5 antibody. Representative images of cytosolic and mitochondrial fractions ERK2-WT (Panel A) and nuclear fraction ERK2-WT (panel D) distribution and representative images of Y185A and T185A distribution in the cytosolic (Panel B), mitochondrial (Panel C) and nuclear (Panel D) fractions, from three independent experiments. Relative levels of ERK mutants in cytosol and mitochondria relative to basal conditions, arbitrarily defined as 1 (Panels B and C). Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p