Laura Cantù

A Recessive Mutation in the APP Gene with Dominant-Negative Effect on Amyloidogenesis

β-Amyloid precursor protein (APP) mutations cause familial Alzheimers disease with nearly complete penetrance. We found an APP mutation [alanine-673→valine-673 (A673V)] that causes disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing, resulting in enhanced β-amyloid (Aβ) production and formation of amyloid fibrils in vitro. Co-incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the autosomal recessive pattern of inheritance and have implications for genetic screening and the potential treatment of Alzheimers disease.

Aggregative properties of gangliosides in solution

The aggregative properties of gangliosides in diluted aqueous solutions are discussed on the basis of simple and well-established thermodynamic concepts. Theoretical assumptions are compared with experimental data obtained, mainly by scattering techniques, on GM3, GM2, GM1, GD1a, GalNAc-GD1a, GD1b, GD1b lactone and GT1b gangliosides, all containing ceramide portions of similar composition, and on GM1 molecular species containing different well-defined ceramide structures. We also report on mixed aggregates with amphiphilic compounds and on the ganglioside aggregate-soluble protein interaction effects which give rise to very stable lipoproteic complexes of well-defined ganglioside-protein composition.

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.

Nonionic micelles in mixed water-glycerol solvent

Abstract The aggregation properties of the nonionic amphiphile C 12 E 8 in water-glycerol mixtures are investigated at various glycerol volume fractions X by surface tension, light scattering, refractometry, and viscosimetry measurements. In the range 0 X X , and the aggregation number m decreases with X . For X > 0.4, the CMC grows considerably, and the Krafft temperature becomes larger than room temperature. The minimum of the cloud point curve decreases from 74°C at X = 0 to 43°C at X = 0.6.

Aggregation properties of GM3 ganglioside (II3Neu5AcLacCer) in aqueous solutions

The aggregative properties of highly pure GM3 ganglioside in aqueous solution have been studied by static and dynamic laser light scattering measurements and fluorescence spectroscopy experiments performed by the use of a GM3 derivative containing the pyrene group at the end of the fatty acid moiety. GM3 ganglioside spontaneously aggregates as unilamellar vesicles, down to a concentration of 1.25 x 10(-8) M, showing molecular weight and hydrodynamic radius ranging from 15,000 to 30,000 kDa and from 350 to 470 A, respectively. GM3 vesicles are stable with dilution and can be stored at room temperature for some weeks without appreciable change.

The Peculiar Role of the A2V Mutation in Amyloid-β (Aβ) 1–42 Molecular Assembly

Background: A2V mutation is associated with early onset AD-type dementia in homozygous individuals. Results: A2V mutation leads to a peculiar kinetics of Aβ oligomerization. Conclusion: The Aβ N-terminal region plays an important role in the molecular assembly. Significance: in the homozygous condition the A2V mutation led to aggregation, whereas in the heterozygous state the evolution and kinetics of the aggregation process was hindered. We recently reported a novel Aβ precursor protein mutation (A673V), corresponding to position 2 of Aβ1–42 peptides (Aβ1–42A2V), that caused an early onset AD-type dementia in a homozygous individual. The heterozygous relatives were not affected as an indication of autosomal recessive inheritance of this mutation. We investigated the folding kinetics of native unfolded Aβ1–42A2V in comparison with the wild type sequence (Aβ1–42WT) and the equimolar solution of both peptides (Aβ1–42MIX) to characterize the oligomers that are produced in the early phases. We carried out the structural characterization of the three preparations using electron and atomic force microscopy, fluorescence emission, and x-ray diffraction and described the soluble oligomer formation kinetics by laser light scattering. The mutation promoted a peculiar pathway of oligomerization, forming a connected system similar to a polymer network with hydrophobic residues on the external surface. Aβ1–42MIX generated assemblies very similar to those produced by Aβ1–42WT, albeit with slower kinetics due to the difficulties of Aβ1–42WT and Aβ1–42A2V peptides in building up of stable intermolecular interaction.

Structure of self-organized multilayer nanoparticles for drug delivery.

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency.

Casein phosphopeptide promotion of calcium uptake in HT‐29 cells − relationship between biological activity and supramolecular structure

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT‐29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs β‐CN(1–25)4P and αs1‐CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated β‐CN(1–25)4P, [β‐CN(1–25)0P]. The biological effect was found to be: (a) maximal with β‐CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol·L−1 Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by β‐CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+‐free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with β‐CN(1–25)0P. With both β‐CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol·L−1 Ca2+.

Evidence for spontaneous segregation phenomena in mixed micelles of gangliosides.

A light scattering study of the effect of mixing in aqueous solution two gangliosides, GM2 and GT1b, having different hydrophilic headgroups and similar lipid moieties is presented. Mixed micelle formation with spatial segregation of one ganglioside with respect to the other was observed. It is also shown that segregation is a spontaneous phenomenon which is explainable only in terms of simple geometrical arguments, that is by the fact that the large headgroup of GT1b provides the lipidic core of the aggregate with a better shielding from water in the highly curved regions than the smaller headgroup of GM2 can do. This finding may be of help in understanding the behaviour of gangliosides in artificial and natural membranes.

Specific recognition of biologically active amyloid-β oligomers by a new Surface Plasmon Resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans

Background: Aβ oligomers are major players in Alzheimer disease, but the tools for their detection are not satisfactory. Results: We developed a SPR-based immunoassay and a test in C. elegans, specifically identifying toxic oligomers. Conclusion: These methods allow study of the effects of mutations or drugs on Aβ oligomerization. Significance: The SPR-based immunoassay provides new opportunities for the detection of toxic oligomers in biological samples. Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimers disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ1–42, discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.