Biomedical subjects

Mario Encinas

Publications and source records attributed to Mario Encinas.

7 recordsLinked to original sources

PubMed2005

The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis.

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Apoptosis↗

PubMed2004

Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis.

The Ret receptor tyrosine kinase mediates physiological signals of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and is essential for postnatal survival in mice. It is implicated in a number of human diseases and developmental abnormalities. Here, we describe our analyses of mice expressing a Ret mutant (RetDN) with diminished kinase activity that inhibits wild-type Ret activity, including its activation of AKT. All RetDN/+ mice died by 1 month of age and had distal intestinal aganglionosis reminiscent of Hirschsprung disease (HSCR) in humans. The RetDN/+ proximal small intestine also had severe hypoganglionosis and reduction in nerve fiber density, suggesting a potential mechanism for the continued gastric dysmotility in postsurgical HSCR patients. Unlike Ret-null mice, which have abnormalities in the parasympathetic and sympathetic nervous systems, the RetDN/+ mice only had defects in the parasympathetic nervous system. A small proportion of RetDN/+ mice had renal agenesis, and the remainder had hypoplastic kidneys and developed tubulocystic abnormalities postnatally. Postnatal analyses of the testes revealed a decreased number of germ cells, degenerating seminiferous tubules, maturation arrest and apoptosis, indicating a crucial role for Ret in early spermatogenesis.

Alleles↗

PubMed2004

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.

Animals↗

PubMed2004

Trk is a calmodulin-binding protein: implications for receptor processing.

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.

Animals↗

PubMed2004

Basic helix-loop-helix proteins bind to TrkB and p21(Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells.

Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21(Cip1) at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21(Cip1) promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21(Cip1), whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21(Cip1) promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21(Cip1), and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

Antineoplastic Agents↗

PubMed2002

Lipid rafts in neuronal signaling and function.

Lipid rafts are plasma membrane microdomains rich in cholesterol and sphingolipids, which provide a particularly ordered lipid environment. Rafts are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, as well as proteins involved in signal transduction and intracellular trafficking. In neurons, lipid rafts act as platforms for the signal transduction initiated by several classes of neurotrophic factors, including neurotrophins and glial-derived neurotrophic factor (GDNF)-family ligands. Emerging evidence also indicates that such rafts are important for neuronal cell adhesion, axon guidance and synaptic transmission. Thus, lipid rafts are structurally unique components of plasma membranes, crucial for neural development and function.

Animals↗

PubMed2002

The prevention of the staurosporine-induced apoptosis by Bcl-X(L), but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells.

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.

Amino Acid Chloromethyl Ketones↗