Biomedical subjects

Vincenzo Coppola

Publications and source records attributed to Vincenzo Coppola.

14 recordsLinked to original sources

PubMed2006

A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin.

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1-tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.

ADP-Ribosylation Factor 6↗

PubMed2006

The scaffold protein Cybr is required for cytokine-modulated trafficking of leukocytes in vivo.

Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

Animals↗

PubMed2004

5-Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependent p53 activation.

5-Lipoxygenase (5LO) is involved in the production of leukotrienes and reactive oxygen species (ROS) from arachidonic acid. Its strong activation has been associated with several diseases like cancer and neurodegeneration. Here we show that 5LO activity increases during senescence-like growth arrest induced by oncogenic ras or culture history in both human and mouse embryo fibroblasts. Overexpression of 5LO promotes senescence-like growth arrest via a p53/p21-dependent pathway, and this occurs independently of telomerase activity. 5LO stabilizes p53 through phosphorylation at Ser15 and increases expression of the p53-transcriptional target p21. This is achieved by regulating ROS production. Indeed, ROS are increased in 5LO-arrested cells. Antioxidants and a low oxygen environment prevent 5LO-induced growth arrest. Finally, 5LO inhibition reduces the growth arrest induced by oncogenic ras or culture history and these effects are neutralized by the addition of exogenous ROS. These data link the 5LO pathway to oxidative crises of primary fibroblast and suggest that the ability of 5LO to induce senescence-like growth arrest may be important in the pathogenesis of 5LO-associated disorders.

Animals↗

PubMed2004

Control of hyperphagia prevents obesity in BDNF heterozygous mice.

Reduced levels of BDNF in mice cause obesity and behavioral abnormalities including increased aggression and hyperactivity. While it has been shown that the obesity is in part caused by increased food consumption it is still not clear whether defects in other mechanisms involved in the control of body weight homeostasis can also affect this phenotype. Here we report that mice with reduced levels of BDNF do not develop obesity and have normal blood glucose levels if fed over a prolonged period of time the amount of food that control mice usually consume. Thus, hyperphagia appears to be the primary cause of obesity development rather than changes in mechanisms controlling metabolism.

Age Factors↗

PubMed2004

Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water.

The "comet-tail" is an ultrasound sign detectable with ultrasound chest instruments; this sign consists of multiple comet-tails fanning out from the lung surface. They originate from water-thickened interlobular septa and would be ideal for nonradiologic bedside assessment of extravascular lung water. To assess the feasibility and value of ultrasonic comet signs, we studied 121 consecutive hospitalized patients (43 women and 78 men; aged 67 +/- 12 years) admitted to our combined cardiology-pneumology department (including cardiac intensive care unit); the study was conducted with commercially available echocardiographic systems including a portable unit. Transducer frequencies (range 2.5 to 3.5 MHz) were used. In each patient, the right and left chest was scanned by examining predefined locations in multiple intercostal spaces. Examiners blinded to clinical diagnoses noted the presence and numbers of lung comets at each examining site. A patient lung comet score was obtained by summing the number of comets in each of the scanning spaces. Within a few minutes, patients underwent chest x-ray, with specific assessment of extravascular lung water score by 2 pneumologist-radiologists blinded to clinical and echo findings. The chest ultrasound scan was obtained in all patients (feasibility 100%). The imaging time per examination was always <3 minutes. There was a linear correlation between echocardiographic comet score and radiologic lung water score (r = 0.78, p <0.01). Intrapatient variations (n = 15) showed an even stronger correlation between changes in echocardiographic lung comet and radiologic lung water scores (r = 0.89; p <0.01). In 121 consecutive hospitalized patients, we found a linear correlation between echocardiographic comet scores and radiologic extravascular lung water scores. Thus, the comet-tail is a simple, non-time-consuming, and reasonably accurate chest ultrasound sign of extravascular lung water that can be obtained at bedside (also with portable echocardiographic equipment) and is not restricted by cardiac acoustic window limitations.

Aged↗

PubMed2004

NT-3 replacement with brain-derived neurotrophic factor redirects vestibular nerve fibers to the cochlea.

Survival of inner ear sensory neurons depends on two neurotrophins, BDNF and NT-3, and their respective receptors, TrkB and TrkC. Because both receptors are present in the same neuron, it has been suggested that BDNF and NT-3 are functionally redundant in promoting neuronal survival. Knock-in of one ligand into the locus of the other one confirmed this hypothesis for the cochlea, leaving open the question of why two neurotrophins are required for proper innervation of the mammalian ear. Here, we show that the precise spatiotemporal pattern of expression of the two neurotrophins is essential for proper patterning of the inner ear innervation. Mice expressing BDNF under the control of the NT-3 promoter develop exuberant projections of vestibular sensory neurons to the basal turn of the cochlea. This projection can be enhanced by combining the transgene with a null mutation of BDNF. However, vestibular fibers rerouted into the cochlea do not reach hair cells and remain outside the organ of Corti, suggesting a chemotactic role for neurotrophins on these fibers. Our data provide genetic evidence that neurotrophins in the ear exert both survival and axon guidance roles.

Animals↗

PubMed2004

Ablation of TrkA function in the immune system causes B cell abnormalities.

The nerve growth factor (NGF) receptor TrkA is widely expressed in non-neural tissues suggesting pleiotropic functions outside the nervous system. Based on pharmacological and immuno-depletion experiments, it has been hypothesized that NGF plays an important role in the normal development and function of the immune system. However, attempts to unravel these functions by conventional gene targeting in mice have been hampered by the early postnatal lethality caused by null mutations. We have developed a novel 'reverse conditional' gene targeting strategy by which TrkA function is restored specifically in the nervous system. Mice lacking TrkA in non-neuronal tissues are viable and appear grossly normal. All major immune system cell populations are present in normal numbers and distributions. However, mutant mice have elevated serum levels of certain immunoglobulin classes and accumulate B1 cells with aging. These data, confirmed in a classical reconstitution model using embryonic fetal liver from TrkA-null mice, demonstrate that endogenous NGF modulates B cell development through TrkA in vivo. Furthermore, they demonstrate that many of the dramatic effects previously reported by pharmacological or immuno-depletion approaches do not reflect physiological developmental roles of TrkA in the immune system.

Animals↗

PubMed2004

[Risk stratification after acute myocardial infarction: limitations of dobutamine stress echocardiography in females].

BACKGROUND: Dobutamine stress echocardiography (DSE) is an imaging test widely used for risk stratification of patients after acute myocardial infarction. METHODS: We evaluated the sensitivity of DSE with respect to the gender and the stenotic coronary artery in patients who survived a myocardial infarction and with angiographic evidence of single-vessel coronary artery disease. RESULTS: The sensitivity of DSE was generally low. In particular, it was significantly lower in the presence of stenosis of the left circumflex and right coronary arteries with respect to the left anterior descending coronary artery. In females it was lower, especially when the stenosis involved the right coronary and left circumflex arteries. CONCLUSIONS: Our data suggest that in female gender the use of other imaging tests and particularly of coronarography should be strongly recommended for risk stratification after acute myocardial infarction.

Coronary Angiography↗

PubMed2003

BRCA2 deficiency in mice leads to meiotic impairment and infertility.

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.

Animals↗

PubMed2003

Cdk2 knockout mice are viable.

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.

Animals↗

PubMed2002

Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease.

Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.

Alleles↗

PubMed2002

Brain-derived neurotrophic factor is essential for opiate-induced plasticity of noradrenergic neurons.

Chronic opiate exposure induces numerous neurochemical adaptations in the noradrenergic system, including upregulation of the cAMP-signaling pathway and increased expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. These adaptations are thought to compensate for opiate-mediated neuronal inhibition but also contribute to physical dependence, including withdrawal after abrupt cessation of drug exposure. Little is known about molecules that regulate the noradrenergic response to opiates. Here we report that noradrenergic locus ceruleus (LC) neurons of mice with a conditional deletion of BDNF in postnatal brain respond to chronic morphine treatment with a paradoxical downregulation of cAMP-mediated excitation and lack of dynamic regulation of TH expression. This was accompanied by a threefold reduction in opiate withdrawal symptoms despite normal antinociceptive tolerance in the BDNF-deficient mice. Although expression of TrkB, the receptor for BDNF, was high in the LC, endogenous BDNF expression was absent there and in the large majority of other noradrenergic neurons. Therefore, a BDNF-signaling pathway originating from non-noradrenergic sources is essential for opiate-induced molecular adaptations of the noradrenergic system.

Animals↗

PubMed2002

Genomic instability in mice lacking histone H2AX.

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.

Amino Acid Sequence↗

PubMedDate not supplied

Encapsulated cells producing retroviral vectors for in vivo gene transfer.

BACKGROUND: Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated. METHODS: Vector (pBAG)-producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X-gal staining, as well as for the release of particles by performing RT-PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for beta-galactosidase activity and by RT-PCR. RESULTS: In culture, the virus-producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self-limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus-producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC-identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co-implanted capsules. CONCLUSION: The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept.

3T3 Cells↗