Giovanna Montana

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid beta-protein (Abeta) in the brain is a hallmark of Alzheimers disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Abeta oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Abeta small oligomers and fibrillar aggregates to simulate the action of diffusible Abeta oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Abeta. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Abeta peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.

Ferulic acid inhibits oxidative stress and cell death induced by Ab oligomers: Improved delivery by solid lipid nanoparticles

Oxidative stress and dysfunctional mitochondria are among the earliest events in AD, triggering neurodegeneration. The use of natural antioxidants could be a neuroprotective strategy for blocking cell death. Here, the antioxidant action of ferulic acid (FA) on different paths leading to degeneration of recombinant β-amyloid peptide (rAβ42) treated cells was investigated. Further, to improve its delivery, a novel drug delivery system (DDS) was used. Solid lipid nanoparticles (SLNs), empty or containing ferulic acid (FA-SNL), were developed as DDS. The resulting particles had small colloidal size and highly negative surface charge in water. Using neuroblastoma cells and rAβ42 oligomers, it was demonstrated that free and SLNs-loaded FA recover cell viability. FA treatment, in particular if loaded into SLNs, decreased ROS generation, restored mitochondrial membrane potential (Δψm) and reduced cytochrome c release and intrinsic pathway apoptosis activation. Further, FA modulated the expression of Peroxiredoxin, an anti-oxidative protein, and attenuated phosphorylation of ERK1/2 activated by Aβ oligomers.

Toxicity of recombinant β-amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus

A distinctive feature of Alzheimers disease is the deposition of amyloid β‐protein (Aβ) in senile or diffuse plaques. The 42 residue β‐peptide (Aβ42) is the predominant form found in plaques. In the present work we report a high‐yield expression and purification method of production of a recombinant Aβ42. The purified recombinant peptide shows characteristics similar to the synthetic human peptide. Different size aggregates, either small oligomers or larger aggregates, were obtained upon dissolving the recombinant Aβ42 peptide under different conditions at pH 7.2 or pH 3, respectively. We report a new toxicity assay on the morphogenic development of the sea urchin Paracentrotus lividus and study the toxicity of the two kinds of aggregates. Despite the difference between the ionic strength of human extracellular fluid (0.154 mol/l) and artificial sea water (0.48 mol/l), toxicity data collected in this system have an intrinsic relevance. The different ionic strength, in fact, could change the kinetics of oligomer formation, but the effect of morphogenic development reported here is related to the final oligomer sizes. Results of the toxicity assay of Aβ42 on sea urchin development also show a dose‐dependent effect. After only 4 h of embryo development, one can note morphological defects in the cell membrane. Retardation of the embryos development, along with cellular disorders visible inside the blastocoele, can be observed after 1 day of development. Cellular degeneration in two different pathological phenotypes—the occluded blastulae and the occluded prism—is present after 48 h of development. Results show that a greater effect on cell death is induced by the small oligomers stabilized under physiological conditions than at acid pH. In this case only occluded blastulae are found after 48 h of development.—Carrotta, R., Di Carlo, M., Manno, M., Montana, G., Picone, P., Romancino, D., San Biagio, P. L. Toxicity of recombinant β‐amyloid prefibrillar oligomers on the morphogenesis of the sea urchin Paracentrotus lividus. FASEB J. 20, E1301–E1308 (2006)

Cloning, expression, and localization of a new member of a Paracentrotus lividus cell surface multigene family

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16‐cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso‐macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed.

Characterization of a Par j 1/Par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy.

Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens.

An allergen-polymeric nanoaggregate as a new tool for allergy vaccination

A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E. coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, zeta potential, electrophoretic mobility and atom force microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity suggesting its employment as potential vaccine against Parietaria-induced allergies.

Folding and binding activity of the 3′UTRs of Paracentrotus lividus bep messengers

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3′UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lenghts of these regions required to bind the 54‐kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3′UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.

Lipid nanoparticles as delivery vehicles for the Parietaria judaica major allergen Par j 2.

Parietaria pollen is one of the major causes of allergic reaction in southern Europe, affecting about 30% of all allergic patients in this area. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease by restoring a normal immunity against allergens. The preparation of allergen-solid lipid nanoparticles as delivery vehicles for therapeutic proteins, P. judaica major allergen Par j 2, was investigated. The Par j 2 allergen was expressed in a large amount in Escherichia coli and purified to homogeneity. Its immunological properties were studied by western blotting and enzyme-linked immunosorbent assay inhibition. Solid lipid nanoparticles were obtained by water-in-oil-in-water multiple emulsion method and characterized in terms of mean size and surface charge. These systems (approximately 250 nm diameter and negative surface charge) incorporated recombinant Par j 2 with 40% or greater efficiency. Moreover, the endotoxin level and anaphylactic activity of the empty solid lipid nanoparticles and recombinant Par j 2-loaded solid lipid nanoparticles were evaluated by looking at the overexpression of CD203c marker on human basophils. These results demonstrate that recombinant Par j 2-nanoparticles could be proposed as safe compositions for the development of new therapeutic dosage forms to cure allergic reactions.

Cloning and sequencing of a cell surface protein-encoding gene conserved in sea urchin species.

We report the nucleotide sequence of a fragment of DNA derived from a sea urchin genomic clone containing the cell surface Bep4 (butanol-extracted protein 4)-encoding gene. The structural gene is interrupted by four introns and the promoter region contains TATA and CAAT consensus motifs. The transcription start point (tsp) was also determined. Remarkable homologies, between Bep4 and other proteins known to be involved in cell interactions, were observed regarding two potential Ca(2+)-binding sites and the corresponding DNA consensus sequences. We also report the conservation of the bep4 gene and its corresponding Bep4 protein between various sea urchin species by way of Southern and Western blotting.

EGFR signalling is required for Paracentrotus lividus endomesoderm specification

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-alpha, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axis was also detected, after AG1478 treatment, by the extended distribution of HE and decreased nuclearization of beta-catenin in vegetal cells. Moreover, inhibition of EGFR-like reduced ERK phosphorylation, necessary for cell fate specification in the micromeres and their derivates. Taken together these results indicate that EGFR-like activity is required both for A/V axis formation and endomesoderm differentiation.