Biomedical subjects

Rafael Linden

Publications and source records attributed to Rafael Linden.

32 records · Page 2Linked to original sources

PubMed2003

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus.

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H(2)O(2) leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H(2)O(2) treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.

Animals↗

PubMed2003

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death.

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.

Animals↗

PubMed2003

Major glycosaminoglycan species in the developing retina: synthesis, tissue distribution and effects upon cell death.

Retinal explants maintained in culture medium retain their histotypic structure and develop similarly to the in vivo condition. Extracellular matrix components, particularly the glycosaminoglycans which are not routinely present in dissociated cell cultures are involved in various cellular events. In this work we characterized and determined the localization of sulfated glycosaminoglycans in the extracellular matrix of rat retinal explants at various stages of normal postnatal development and tested whether disruption of the tissue glycosaminoglycan composition may impose either trophic or toxic effects upon distinct retinal cell populations. Our data show that chondroitin sulfate and heparan sulfate glycosaminoglycan chains are synthesized in different proportions during postnatal retinal development. A peak of synthesis of chondroitin sulfates is evident at around P14. Immunohistochemistry showed chondroitin 6-sulfate in the plexiform layers during the earlier stages while later, intense immunoreactivity was found in the outer retina. Heparan sulfate was found in the neuroblastic layer (NBL) at P1, in both nuclear layers from P5 onwards and in the ganglion cell layer (GCL) at all stages. In contrast to chondroitin 6-sulfate, immunoreactivity to heparan sulfate was absent from the outer retina at both P14 and P21. Treatment with heparitinase modulated the rates of cell death in both the GCL and the NBL in P1 retinal explants. Taken together our data show that among the major sulfated glycosaminoglycans, the developing rat retina synthesizes only heparan sulfate and chondroitin sulfates in a spatiotemporally regulated manner, with a peak of chondroitin sulfates at P14, possibly related to photoreceptor differentiation. In addition, our data suggest a role for heparan sulfate as a modulator of sensitivity to cell death in the retina.

Animals↗

PubMed2002

Cell death in the inner nuclear layer of the retina is modulated by BDNF.

Developing amacrine cells in the vertebrate retina undergo naturally-occurring cell death which is accentuated by the early removal of retinal ganglion cells. We show that providing BDNF or decreasing endogenous BDNF via competitive binding with soluble TrkB receptors in a whole-retina culture assay modulates the frequency of dying cells in the amacrine cell layer. Ganglion cells synthesize BDNF, and amacrine cells express TrkB receptors, suggesting a likely signaling mechanism.

Aging↗

PubMed2002

Structure of laminin substrate modulates cellular signaling for neuritogenesis.

Laminin, a major component of basement membranes, can self-assemble in vitro into a typical mesh-like structure, according to a mass-action-driven process. Previously, we showed that pH acidification dramatically increased the efficiency of laminin self-assembly, practically abolishing the necessity for a minimal protein concentration. Here we have characterized the morphologies of laminin matrices produced in either neutral or acidic conditions and compared their capacities to induce neuritogenesis of rat embryonic cortical neurons. Although laminin matrices formed in neutral buffer presented aggregates of heterogeneous morphology, the acidic matrix consisted of a homogeneous hexagonal sheet-like structure. The latter was comparable to the matrix assembled in vivo at the inner limiting membrane of the retina in newborn rats, shown here, and to matrices secreted by cultivated cells, shown elsewhere. The average neurite length of cortical neurons plated on acidic matrices was 244.9 micro m, whereas on neutral matrices this value dropped to 104.1 micro m. Increased neuritogenesis on the acidic matrix seemed to be associated with a higher degree of neuronal differentiation, since cell proliferation was immediately arrested upon plating, whereas on neutral matrices, the cell number increased six-fold within 24 hours. Investigation of the mechanisms mediating neurite outgrowth on each condition revealed that the extensive neuritogenesis observed on the acidic matrix involved activation of protein kinase A, whereas moderate neuritogenesis on neutral laminin was mediated by activation of protein kinase C and/or myosin light-chain kinase. Explants of cerebral cortex from P2 rats did not grow on the neutral laminin substrate but presented extensive cell migration and neurite outgrowth on the acidic laminin matrix. We propose that laminin can self-assemble independently of cell contact and that the assembling mode differentially modulates neuritogenesis and neuroplasticity.

Animals↗

PubMed2002

Differential effects of cyclin-dependent kinase blockers upon cell death in the developing retina.

Pharmacological blockers of cyclin-dependent kinases (CDKs) can inhibit cell cycle progression. Deferoxamine (DFO) and mimosine (MIMO) arrest cells reversibly at the G1/S transition and olomoucine (OLO) inhibits the cell cycle at both G1/S and G2/M. We investigated the effect of these drugs upon cell death in histotypical explants taken from the retina of neonatal rats. Degeneration of retinal ganglions cells (RGC) induced by axotomy was inhibited by OLO (100 microM) but not by DFO (up to 2 mM) or MIMO (up to 1 mM). On the other hand, after 1 day in vitro, all cell cycle inhibitors induced cell death in the neuroblastic layer (NBL) of the explants. DFO and MIMO induced cell death only of proliferating cells, identified either by their incorporation of bromodeoxyuridine or by immunolabeling the proliferating cell nuclear antigen. In turn, OLO induced cell death of both proliferating and post-mitotic cells. However, the post-mitotic cells were unlabeled with markers of retinal differentiation. Our results indicate that cyclin-dependent kinases are involved in the control of sensitivity to cell death in the retina, and that retinal cells present differentiation-dependent responses to modulation of CDK activity.

Adenylyl Cyclases↗

PubMed2002

Cellular prion protein transduces neuroprotective signals.

To test for a role for the cellular prion protein (PrP(c)) in cell death, we used a PrP(c)-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrP(c)-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106-126, with certain antibodies to PrP(c) or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrP(c) that increased cAMP also induced neuroprotection. Thus, engagement of PrP(c) transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrP(c) may function as a trophic receptor, the activation of which leads to a neuroprotective state.

Animals↗

PubMed2002

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

Animals↗

PubMed2002

Pituitary adenylyl cyclase-activating polypeptide prevents induced cell death in retinal tissue through activation of cyclic AMP-dependent protein kinase.

Multiple neuroactive substances are secreted by neurons and/or glial cells and modulate the sensitivity to cell death. In the developing retina, it has been shown that increased intracellular levels of cAMP protect cells from degeneration. We tested the hypothesis that the neuroactive peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has neuroprotective effects upon the developing rat retina. PACAP38 prevented anisomycin-induced cell death in the neuroblastic layer (NBL) of retinal explants, and complete inhibition of induced cell death was obtained with 1 nm. A similar protective effect was observed with PACAP27 and with the specific PAC1 receptor agonist maxadilan but not with glucagon. Photoreceptor cell death induced by thapsigargin was also prevented by PACAP38. The neuroprotective effect of PACAP38 upon the NBL could be reverted by the competitive PACAP receptor antagonist PACAP6-38 and by the specific PAC1 receptor antagonist Maxd.4. Molecular and immunohistochemical analysis demonstrated PAC1 receptors, and treatment with PACAP38 induced phospho-cAMP-response element-binding protein immunoreactivity in the anisomycin-sensitive undifferentiated postmitotic cells within the NBL. PACAP38 produced an increase in cAMP but not inositol triphosphate, and treatment with the cAMP-dependent protein kinase inhibitor R(p)-cAMPS blocked the protective effect of PACAP38. The results indicate that activation of PAC1 receptors by PACAP38 modulates cell death in the developing retina through the intracellular cAMP/cAMP-dependent protein kinase pathway.

Animals↗

PubMed2002

Cellular prion protein: on the road for functions.

Cellular prion (PrPc) is a plasma membrane glycosyphosphatidylinositol-anchored protein present in neurons but also in other cell types. Protein conservation among species suggests that PrPc may have important physiological roles. Cellular and molecular approaches have established several novel features of the regulation of PrPc expression, cellular trafficking as well as its participation in copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival. It is therefore likely that PrPc plays pleiotropic roles in neuronal and non-neuronal cells, and as such the loss of function of PrPc may be an important component of various diseases.

Models, Biological↗

PubMed2002

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.

Animals↗

PubMed2002

Cytoplasmic c-Jun N-terminal immunoreactivity: a hallmark of retinal apoptosis.

1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA. 2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death. 3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis. 4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis. 5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.

Animals↗

PubMed2002

Apoptosis underlies immunopathogenic mechanisms in acute silicosis.

We investigated immunopathogenic roles for apoptosis in acute murine silicosis. Intratracheal silica instillation induced pulmonary inflammation and enlarged thoracic lymph nodes. Lymphocytes from silica-exposed lymph nodes showed reduced mitogenic responses to T cell receptor (TCR) stimulation, and markedly increased activation-induced cell death, compared with control lymphocytes from saline-exposed lymph nodes. CD4(+) T cell death was mediated by Fas ligand, because CD4(+) T cells from Fas ligand-deficient gld mice did not undergo activation-induced apoptosis. Silica deposition also resulted in increased apoptosis associated with inflammatory infiltrates in lung parenchyma. In vivo treatment with caspase inhibitors reduced neutrophil accumulation, and alleviated inflammation in the lungs of silica-treated mice. These results suggest that silica-induced apoptosis plays an inflammatory role in the lung parenchyma, and creates immunologic abnormalities in regional lymph nodes, with pathogenic implications for the host.

Acute Disease↗

PubMedDate not supplied

Towards cellular receptors for prions.

Transmissible spongiform encephalopathies (TSE) are attributed to the conversion of the cellular prion protein (PrP(c)) into an abnormal isoform (PrP(sc)). This can be caused by the invasion of living organisms by infectious particles, or be inherited due to mutations on the PrP(c) gene. One of the most intriguing problems of prion biology is the inability to generate the infectious agent in vitro. This argues strongly that other cellular proteins besides those added in test tubes or found in cellular preparations are necessary for infection. Despite recent progress in the understanding of prion pathology, the subcellular compartments in which the interaction and conversion of PrP(c) into PrP(sc) take place are still controversial. PrP(c) interacts with various macromolecules at the cell membrane, in endocytic compartments and in the secretory pathway, all of which may play specific roles in the internalisation of PrP(sc) and conversion of PrP(c). A specific interacting protein required for the propagation of prions was originally proposed as a prion receptor, and later referred to as a ligand, a cofactor, protein X, or a partner. However, current studies indicate that PrP(c) associates with multi-molecular complexes, which mediate a variety of functions in distinct cellular compartments. It is proposed that a deeper understanding of the mechanics of such interactions, coupled to a better knowledge of the corresponding signalling pathways and ensuing cellular responses, will have a major impact on the prevention and treatment of TSE.

Humans↗