Elena Del Favero

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.

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.

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+.

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.

Gerstmann-Sträussler-Scheinker Disease Amyloid Protein Polymerizes According to the “Dock-and-Lock” Model

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues ∼82–146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82–146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82–146 small oligomers (1–5-mers, flowing species) onto soluble prefibrillar PrP82–146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82–146 flowed onto the scrambled sequence of PrP82–146 or onto prefibrillar Aβ42 aggregates. As previously found with Aβ40, SPR data could be adequately fitted by equations modeling the “dock-and-lock” mechanism, in which the “locking” step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82–146 than with Aβ40. Such differences suggest that PrP82–146 has a greater propensity to polymerize and greater stability of the aggregates.

Experimental evidence of a temperature-related conformational change of the hydrophilic portion of gangliosides.

The present paper reports the experimental observation of an interesting thermodynamic process which could be biologically important: such behaviour, shown by some gangliosides, is likely to be peculiar of these glycosidic compounds as it has never been observed for other membrane-type amphiphilic molecules. In water solution, gangliosides have been found to present a bistable behaviour between two stable states (called A and B) which does not involve any change in the primary structure of the molecule. The interconversion between state A and state B, and vice versa, does not occur spontaneously, but has to be triggered by some external agent, which makes this system a potentially regulated process with important biological correlations. In the present experiments, state B is reached from state A with a temperature rise in the range 30-55 degrees C. The new state is stable regardless of any possible temperature cycle. The initial state A is then regained when the ganglioside solution is dried and the solute is redissolved. The two states are believed to correspond to two different conformations of the hydrophilic portion of the molecule. The bistable behaviour is shown by the gangliosides GM2, GM1, GD1a, GD1b and Fuc-GD1b, GT1b, however, does not show such an effect.

Gangliosides and the multiscale modulation of membrane structure

Cellular membranes are highly organized structures with multiple and multi-dimensional levels of order where lipid components are active players. The lipid role is especially evident in rafts, where lipid-driven collective interaction dictates the local structure of a membrane. However, lipids play as well other roles in many aspects of membrane mechanics and function. In this review, we would like to re-focus the attention of the readers on the importance of gangliosides in organizing the fine structure of cellular membranes, in lateral and transverse directions. Important biological events are likely to be affected such as the dynamic control of the shape of specialized plasma membrane areas and of the intracellular organelles, the in- and outward budding and fusion of membrane vesicles, the physical and functional coupling of the outer and the inner plasma membrane leaflet, involved in the transduction of signals across the membrane.

Structural aspects of ganglioside-containing membranes

The demand for understanding the physical role of gangliosides in membranes is pressing, due to the high number of diverse and crucial biological functions in which they are involved, needing a unifying thread. To this purpose, model systems including gangliosides have been subject of extensive structural studies. Although showing different levels of complication, all models share the need for simplicity, in order to allow for physico-chemical clarity, so they keep far from the extreme complexity of the true biological systems. Nonetheless, as widely agreed, they provide a basic hint on the structural contribution specific molecules can pay to the complex aggregate. This topic we address in the present review. Gangliosides are likely to play their physical role through metamorphism, cooperativity and demixing, that is, they tend to segregate and identify regions where they can dictate and modulate the geometry and the topology of the structure, and its mechanical properties. Strong three-dimensional organisation and cooperativity are exploited to scale up the local arrangement hierarchically from the nano- to the mesoscale, influencing the overall morphology of the structure.

Different mutations at V363 MAPT codon are associated with atypical clinical phenotypes and show unusual structural and functional features.

Microtubule-associated protein tau gene (MAPT) is one of the major genes linked to frontotemporal lobar degeneration, a group of neurodegenerative diseases clinically, pathologically, and genetically heterogeneous. In particular, MAPT mutations give rise to the subgroup of tauopathies. The pathogenetic mechanisms underlying the MAPT mutations so far described are the decreased ability of tau protein to promote microtubule polymerization (missense mutations) or the altered ratio of tau isoforms (splicing mutations), both leading to accumulation of hyperphosphorylated filamentous tau protein. Following a genetic screening of patients affected by frontotemporal lobar degeneration, we identified 2 MAPT mutations, V363I and V363A, leading to atypical clinical phenotypes, such as posterior cortical atrophy. We investigated in vitro features of the recombinant mutated tau isoforms and revealed unusual functional and structural characteristics such as an increased ability to promote microtubule polymerization and a tendency to form oligomeric instead of filamentous aggregates. Thus, we disclosed a greater than expected complexity of abnormal features of mutated tau isoforms. Overall our findings suggest a high probability that these mutations are pathogenic.

Dependence of the form factor of ganglioside micelles on a conformational change with temperature

Abstract The gangliosides GM2, GM1 and GD1b, biological amphiphiles with a double tail hydrophobic part and an oligosaccharide chain headgroup, form micelles in solution. Light scattering experiments have shown that ganglioside micelles which have gone through a temperature cycle have a smaller molecular mass and hydrodynamic radius than those which have been kept at room temperature. This fact has been interpreted with the hypothesis that, with temperature, the ganglioside molecules undergo a conformational change which affects their micellar properties appreciably. Careful small angle X-ray experiments, aimed to confirm the light scattering data and to evidence differences in the micellar internal structure are presented. Ganglioside micelles are quite inhomogeneous particles with respect to X-ray scattering, since there is a large contrast variation between the inner lipid part and the external hydrated sugar layer. Experimental form factors are fitted with a double-shell oblate-ellipsoid model.