Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

REACTIVE OXYGEN SPECIES AND THE STRUCTURAL REMODELING OF THE VISUAL SYSTEM AFTER OCULAR ENUCLEATION

Redox processes associated with controlled generation of reactive oxygen species (ROS) by NADPH oxidase (Nox) add an essential level of regulation to signaling pathways underlying physiological processes. We evaluated the ROS generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation in adult rats. Dihydroethidium (DHE) oxidation revealed increased ROS generation in SC and DLG between 1 and 30 days postlesion. ROS generation was decreased by the Nox inhibitors diphenyleneiodonium chloride (DPI) and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment. Taken together, the findings of the present study suggest a novel role for Nox-induced ROS signaling in mediating neuronal remodeling in visual areas after ocular enucleation. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Susceptibility of neuron-like cells derived from bovine Wharton's jelly to bovine herpesvirus type 5 infections

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Different protocols of physical exercise produce different effects on synaptic and structural proteins in motor areas of the rat brain

The plastic brain responses generated by the training with acrobatic exercise (AE) and with treadmill exercise (TE) may be different. We evaluated the protein expression of synapsin I (SYS), synaptophysin (SYP), microtubule-associated protein 2 (MAP2) and neurofilaments (NF) by immunohistochemistry and Western blotting in the motor cortex, striatum and cerebellum of rats subjected to TE and AE. Young adult male Wistar rats were divided into 3 groups: sedentary (Sed) (n=15), TE (n=20) and AE (n=20). The rats were trained 3 days/week for 4 weeks on a treadmill at 0.6 km/h, 40 min/day (TE), or moved through a circuit of obstacles 5 times/day (AE). The rats from the TE group exhibited a significant increase of SYS and SYP in the motor cortex, of NF68, SYS and SYP in the striatum, and of MAP2, NF and SYS in the cerebellum, whereas NF was decreased in the motor cortex and the molecular layer of the cerebellar cortex. On the other hand, the rats from the AE group showed a significant increase of MAP2 and SYP in the motor cortex, of all four proteins in the striatum, and of SYS in the cerebellum. In conclusion, AE induced changes in the expression of synaptic and structural proteins mainly in the motor cortex and striatum, which may underlie part of the learning of complex motor tasks. TE, on the other hand, promoted more robust changes of structural proteins in all three regions, especially in the cerebellum, which is involved in learned and automatic tasks. (C) 2012 Elsevier B.V. All rights reserved.

Neurons derived from P19 embryonic carcinoma cells as a platform for biosensor applications - optimisation and characterisation

P19 is a mouse-derived embryonal carcinoma cell line capable of differentiation toward ectodermal, mesodermal and endodermal lineages and could thus be differentiated into neurons. Different culture conditions were tested to optimise and increase the efficiency of neuronal differentiation since the population of P19-derived neurons was reported to be heterogeneous with respect to the morphology and neurotransmitters they synthesise. P19-derived neurons were cultured on microelectrode arrays as cell aggregates and as dissociated cells. Improved neuronal maturation was shown by the presence of microtubule associated protein 2, neurofilament and synaptophysin formation when initiation of neuronal differentiation was prolonged. High initial cell density cultures and coating of surfaces with polyethylenimine-laminin further improved neuronal maturation of differentiated P19 cells. Increased spontaneous activities of the P19-derived neurons were correspondingly recorded. Two to three hours recordings were performed between 17 and 25 days when extracellular signals were stabilised. It was found that P19-derived neurons developed network properties as partially synchronised network activities. P19-derived neurons appeared to give inhomogenous response to the 2 major neurotransmitters, -aminobutyric acid (GABA) and glutamate. The P19-derived neuronal networks obtained from optimised protocol in this thesis were predominantly GABAergic. The reproducible long term extracellular recordings performed showed that neurons derived from P19 embryonal carcinoma cells could be applied as a model for cell based biosensor in corporation with microelectrode arrays.

The Effects of Altered Prenatal Melatonin Signaling on Adult Behavior and Hippocampal Gene Expression of the Male Rat: A Circadioneuroendocrine-Axis Hypothesis of Psychopathology

Disturbances in melatonin - the neurohormone that signals environmental darkness as part of the circadian circuit of mammals - have been implicated in various psychopathologies in humans. At present, experimental evidence linking prenatal melatonin signaling to adult physiology, behavior, and gene expression is lacking. We hypothesized that administration of melatonin (5 mg/kg) or the melatonin receptor antagonist luzindole (5 mg/kg) to rats in utero would permanently alter the circadian circuit to produce differential growth, adult behavior, and hippocampal gene expressionin the male rat. Prenatal treatment was found to increase growth in melatonin-treated animals. In addition, subjects exposed to melatonin prenatally displayed increased rearing in the open field test and an increased right turn preference in the elevated plusmaze. Rats administered luzindole prenatally, however, displayed greater freezing and grooming behavior in the open field test and improved learning in the Morris water maze. Analysis of relative adult hippocampal gene expression with RT-PCR revealed increasedexpression of brain-derived neurotrophic factor (BDNF) with a trend toward increased expression of melatonin 1A (MEL1A) receptors in melatonin-exposed animals whereas overall prenatal treatment had a significant effect on microtubule-associated protein 2(MAP2) expression. Our data support the conclusion that the manipulation of maternal melatonin levels alters brain development and leads to physiological and behavioral abnormalities in adult offspring. We designate the term circadioneuroendocrine (CNE)axis and propose the CNE-axis hypothesis of psychopathology.

Stem Cells From Umbilical Cord Wharton's Jelly From Preterm Birth Have Neuroglial Differentiation Potential

Objective:The aim of the study is to determine the neuroglial differentiation potential of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from preterm birth when compared to term delivery.Study Design:The WJ-MSCs from umbilical cords of preterm birth and term controls were isolated and induced into neural progenitors. The cells were analyzed for neuroglial markers by flow cytometry, real-time polymerase chain reaction, and immunocytochemistry. Results:Independent of gestational age, a subset of WJ-MSC displayed the neural progenitor cell markers Nestin and Musashi-1 and the mature neural markers microtubule-associated protein 2, glial fibrillary acidic protein, and myelin basic protein. Neuroglial induction of WJ-MSCs from term and preterm birth resulted in the enhanced transcription of Nestin and Musashi-1.Conclusions:Undifferentiated WJ-MSCs from preterm birth express neuroglial markers and can be successfully induced into neural progenitors similar to term controls. Their potential use as cellular graft in neuroregenerative therapy for peripartum brain injury in preterm birth has to be tested.

Dehydroepiandrosterone: A potential signalling molecule for neocortical organization during development

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) are the most abundant steroids produced by the human adrenal, but no receptors have been identified for these steroids, and no function for them has been established, other than as precursors for sex steroid synthesis. DHEA and DHEAS are found in brains from many species, and we have shown that enzymes crucial for their synthesis, especially P450c17 (17α-hydroxylase/c17,20 lyase), are expressed in a developmentally regulated, region-specific fashion in the developing rodent brain. One region of embryonic expression of P450c17, the neocortical subplate, has been postulated to play a role in guiding cortical projections to their appropriate targets. We therefore determined if products of P450c17 activity, DHEA and DHEAS, regulated the motility and/or growth of neocortical neurons. In primary cultures of mouse embryonic neocortical neurons, DHEA increased the length of neurites containing the axonal marker Tau-1, and the incidence of varicosities and basket-like process formations in a dose-dependent fashion. These effects could be seen at concentrations normally found in the brain. By contrast, DHEAS had no effect on Tau-1 axonal neurites but increased the length of neurites containing the dendritic marker microtubule-associated protein-2. DHEA rapidly increased free intracellular calcium via activation of N-methyl-d-aspartate (NMDA) receptors. These studies provide evidence of mechanisms by which DHEA and DHEAS exert biological actions, show that they have specific functions other than as sex steroid precursors, mediate their effects via non-classic steroid hormone receptors, and suggest that their developmentally regulated synthesis in vivo may play crucial and different roles in organizing the neocortex.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-β, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix–loop–helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Cytoskeletal microdifferentiation: A mechanism for organizing morphological plasticity in dendrites

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys.

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.

Selective neuronal toxicity of cocaine in embryonic mouse brain cocultures.

Cocaine exposure in utero causes severe alterations in the development of the central nervous system. To study the basis of these teratogenic effects in vitro, we have used cocultures of neurons and glial cells from mouse embryonic brain. Cocaine selectively affected embryonic neuronal cells, causing first a dramatic reduction of both number and length of neurites and then extensive neuronal death. Scanning electron microscopy demonstrated a shift from a multipolar neuronal pattern towards bi- and unipolarity prior to the rounding up and eventual disappearance of the neurons. Selective toxicity of cocaine on neurons was paralleled by a concomitant decrease of the culture content in microtubule-associated protein 2 (MAP2), a neuronal marker measured by solid-phase immunoassay. These effects on neurons were reversible when cocaine was removed from the culture medium. In contrast, cocaine did not affect astroglial cells and their glial fibrillary acidic protein (GFAP) content. Thus, in embryonic neuronal-glial cell cocultures, cocaine induces major neurite perturbations followed by neuronal death without affecting the survival of glial cells. Provided similar neuronal alterations are produced in the developing human brain, they could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following in utero exposure to cocaine.

Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/ threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK There are now more than 50 proteins shown to be substrates for JNK These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.