Maria Lina Massimino

Alteration in Calcium Handling at the Subcellular Level in mdx Myotubes

In this study, we have tested the hypothesis that augmented [Ca2+] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca2+ homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca2+-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca2+] in the sarcoplasmic reticulum, [Ca2+] sr , show a higher steady state level at rest and a larger drop after KCl-induced depolarization inmdx compared with control myotubes. The peaks in [Ca2+] occurring in the mitochondrial matrix ofmdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca2+ responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca2+ handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca2+ homeostasis in muscular dystrophy.

Multiple phosphorylation of α-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation

The presence of aggregated α‐synuclein molecules is a common denominator in a variety of neurodegenerative disorders. Here, we show that α‐synuclein (α‐syn) is an outstanding substrate for the protein tyrosine kinase p72syk(Syk), which phosphorylates three tyrosyl residues in its C‐terminal domain (Y‐125, Y‐133, and Y‐136), as revealed from experiments with mutants where these residues have been individually or multiply replaced by phenylalanine. In contrast, only Y‐125 is phosphorylated by Lyn and c‐Fgr. Eosin‐induced multimerization is observed with wildtype α‐syn, either phosphorylated or not by Lyn, and with all its Tyr to Phe mutants but not with the protein previously phosphorylated by Syk. Syk‐mediated phosphorylation also counteracts α‐syn assembly into filaments as judged from the disappearance of α‐syn precipitated upon centrifugation at 100,000 × g. We also show that Syk and α‐syn colocalize in the brain, and upon cotransfection in Chinese hamster ovary cells, α‐syn becomes Tyr‐phosphorylated by Syk. Moreover, Syk and α‐syn interact with each other as judged from the mammalian two‐hybrid system approach. These data suggest that Syk or tyrosine kinase(s) with similar specificity may play an antineurodegenerative role by phosphorylating α‐syn, thereby preventing its aggregation.

Involvement of caveolae and caveolae-like domains in signalling, cell survival and angiogenesis

Caveolae, the flask-shaped membrane invaginations abundant in endothelial cells, have acquired a prominent role in signal transduction. Evidence, that events occurring in caveolae participate in cell survival and angiogenesis, has been recently substantiated by the identification of two novel caveolar constituents: prostacyclin synthase (PGIS) and the cellular form of prion protein (PrP(c)). We have shown that PGIS, previously described as an endoplasmic reticulum component, is bound to caveolin-1 (cav-1) and localized in caveolae in human endothelial cells. By generating prostacyclin, PGIS is involved in angiogenesis. Previous observations regarding the localization of PrP(c) in caveolae-like membrane domains (CLDs) have been recently confirmed and extended. It has been demonstrated that PrP(c) is bound to cav-1 and, by recruiting Fyn kinase, can participate in signal transduction events connected to cell survival and differentiation. The new entries of PGIS and PrP(c) in caveolar components place caveolae and CLDs at the centre of a network, where cells decide whether to proliferate or differentiate and whether to survive or to suicide by apoptosis.

Fluorescent silver(I) and gold(I)-N-heterocyclic carbene complexes with cytotoxic properties

Silver(I) and gold(I)-N-heterocyclic carbene (NHC) complexes bearing a fluorescent anthracenyl ligand were examined for cytotoxicity in normal and tumor cells. The silver(I) complex exhibits greater cytotoxicity in tumor cells compared with normal cells. Notably, in cell extracts, this complex determines a more pronounced inhibition of thioredoxin reductase (TrxR), but it is ineffective towards glutathione reductase (GR). Both gold and silver complexes lead to oxidation of the thioredoxin system, the silver(I) derivative being particularly effective. In addition, the dimerization of peroxiredoxin 3 (Prx3) was also observed, demonstrating the ability of these compounds to reach the mitochondrial target. The fluorescence microscopy visualization of the subcellular distribution of the complexes shows a larger diffusion of these molecules in tumor cells with respect to normal cells.

The metabolism and imaging in live cells of the bovine prion protein in its native form or carrying single amino acid substitutions.

Prion diseases are probably caused by an abnormal form of a cellular glycoprotein, the prion protein. Recent evidence suggests that the prion strain causing BSE has been transmitted to humans, thereby provoking a variant form of Creutzfeldt-Jacob disease. In this work, we analyzed the behavior of normal and malformed isoforms of the bovine PrP in transfected mammalian cell lines. Biochemical and immunocytochemical assays were complimented with imaging of live cells expressing fusion constructs between PrP and GFP. Bovine homologues of human E200K and D178N (129M) mutations were used as models of pathogenic isoforms. We show that the GFP does not impair the metabolism of native and mutant bPrPs and is thus a valid marker of PrP cellular distribution. We also show that each amino acid replacement provokes alterations in the cell sorting and processing of bPrP. These are different from those ascribed to both murine mutant homologues. However, human and bovine PrPs carrying the D178N genotype had similar cellular behavior.

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c‐Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis “in situ” of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src‐dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine‐phosphorylated. J. Cell. Biochem. 104: 840–849, 2008.

Direct Monitoring of the Calcium Concentration in the Sarcoplasmic and Endoplasmic Reticulum of Skeletal Muscle Myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100–1000 μm range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 μm, whereas that measured with erAEQ is significantly lower, i.e. around 200 μm. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.

Cellular Prion Protein Promotes Regeneration of Adult Muscle Tissue

ABSTRACT It is now well established that the conversion of the cellular prion protein, PrPC, into its anomalous conformer, PrPSc, is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrPC are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrPC and have been related to PrPC pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrPC. Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrPC significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrPC expression completely rescued the muscle phenotype evidenced in the absence of PrPC.

Gene transfer into satellite cell from regenerating muscle: Bupivacaine allows β-gal transfection and expression in vitro and in vivo

SummaryA large bulk of experimental evidence (15) suggests that myogenic cell transfer can be regarded as a promising therapeutic approach in the cure of inherited pathologies. In particular, it has been shown that primary myoblasts obtained from embryonic or neonatal muscles allows the recovery of the normal phenotype in defective muscle tissues. The utilization of this approach in clinical settings still bears heavy limitations. Apart from the legal and ethical difficulties, the use of muscles obtained from aborted fetus is challenged by a large risk of rejection, due to the incompatibility between donor and recipient. In this context based on the genetic alteration and reimplanting of the patient’s own satellite cells, appears an approach attractive. Myoblasts derived from satellite cells are the obligate candidates for experiments, but the production of sufficient cell numbers is a major problem. Local anesthetics [Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) and related molecules] had been used to induce myofiber damage (and thus satellite cells proliferation) and thereby may represent a tool for increasing the yield of myoblasts from adult muscles (1,9,17). We will show that satellite cells obtained from adult muscles after bupivacaine injection can be transfected in vitro and that the transfected gene is expressed in vitro and in vivo, after reimplantation of the modified myoblasts in recipient muscles.

Thrombin-induced Tyrosine Phosphorylation of HS1 in Human Platelets Is Sequentially Catalyzed by Syk and Lyn Tyrosine Kinases and Associated with the Cellular Migration of the Protein

Thrombin stimulation of platelets triggers Tyr phosphorylation of several signaling proteins, most of which remain unidentified. In this study, we demonstrate for the first time that hematopoietic lineage cell-specific protein 1 (HS1) undergoes a transient Tyr phosphorylation in human platelets stimulated with thrombin. The protein is synergistically phosphorylated by Syk and Lyn tyrosine kinases according to a sequential phosphorylation mechanism. By means of specific inhibitors (PP2, SU6656, and piceatannol) and phosphopeptide-specific antibodies, as well as by coimmunoprecipitation and binding competition experiments, we show that Syk acts as the primary kinase that phosphorylates HS1 at Tyr397 and that Syk phosphorylation is required for HS1 interaction with the Lyn SH2 domain. Upon docking to Syk-phosphorylated HS1, Lyn catalyzes the secondary phosphorylation of the protein at Tyr222. Once the secondary Tyr phosphorylation of HS1 is accomplished the protein dissociates from Lyn and undergoes a dephosphorylation process. HS1 Tyr phosphorylation does not occur when thrombin-induced actin assembly is inhibited by cytochalasin D even under conditions in which Syk and Lyn are still active. Immunofluorescence microscopic analysis shows that the agonist promotes HS1 migration to the plasma membrane and that the inhibition of Lyn-mediated secondary phosphorylation of HS1 abrogates the subcellular translocation of the protein. All together these results indicate that HS1 Tyr phosphorylation catalyzed by Syk and Lyn plays a crucial role in the translocation of the protein to the membrane and is involved in the cytoskeleton rearrangement triggered by thrombin in human platelets.