Rita Carrotta

Protofibril Formation of Amyloid β-Protein at Low pH via a Non-cooperative Elongation Mechanism

Deposition of the amyloid β-protein (Aβ) in senile or diffuse plaques is a distinctive feature of Alzheimers disease. The role of Aβ aggregates in the etiology of the disease is still controversial. The formation of linear aggregates, known as amyloid fibrils, has been proposed as the onset and the cause of pathological deposition. Yet, recent findings suggest that a more crucial role is played by prefibrillar oligomeric assemblies of Aβ that are highly toxic in the extracellular environment. In the present work, the mechanism of protofibril formation is studied at pH 3.1, starting from a solution of oligomeric precursors. By combining static light scattering and photon correlation spectroscopy, the growth of the mass and the size of aggregates are determined at different temperatures. Analysis and scaling of kinetic data reveal that under the studied conditions protofibrils are formed via a single non-cooperative elongation mechanism, not prompted by nucleation. This process is well described as a linear colloidal aggregation due to diffusion and coalescence of growing aggregates. The rate of elongation follows an Arrhenius law with an activation enthalpy of 15 kcal mol–1. Such a value points to a conformational change of peptides or oligomers being involved in binding to protofibrils or in general to a local reorganization of each aggregate. These results contribute to establishing a clearer relation at the molecular level between the fibrillation mechanism and fibrillar precursors. The observation of a non-cooperative aggregation pathway supports the hypothesis that amyloid formation may represent an escape route from a dangerous condition, induced by the presence of toxic oligomeric species.

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.

Insulin‐activated Akt rescues Aβ oxidative stress‐induced cell death by orchestrating molecular trafficking

Increasing evidence indicates that Alzheimer’s disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid‐β42 (Aβ)‐induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho‐Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro‐apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show that the recovery of the organelle integrity recruits active Akt translocation to the mitochondrion. Here, it plays a role both by maintaining unimpaired the permeability transition pore through increase in HK‐II levels and by blocking apoptosis through phosphorylation of Bad, coming from cytoplasm after Aβ stimulus. Together, these results indicate that the Akt survival signal antagonizes the Aβ cell death process by balancing the presence and modifications of common molecules in specific cellular environments.

Inhibiting effect of αs1-casein on Aβ1–40 fibrillogenesis

BACKGROUND α(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α(s1)-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimers disease. METHODS The aggregation kinetics of β-peptide in presence and absence of α(s1)-casein was followed under shear at 37°C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α(s1)-casein-Aβ(1-40) interaction. RESULTS AND DISCUSSIONS α(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. GENERAL SIGNIFICANCE Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.

Concanavalin A aggregation and toxicity on cell cultures

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which-as a consequence-turns out to be non-toxic.

Different effects of Alzheimer's peptide Aβ(1–40) oligomers and fibrils on supported lipid membranes

Beta-amyloid (1-40) is one of the two most abundant species of amyloid-beta peptides present as fibrils in the extracellular senile plaques in the brain of Alzheimers patients. Recently, the molecular aggregates constituting the early stage of fibril formation, i.e., oligomers and protofibrils, have been investigated as the main responsible for amyloid-beta cytotoxic effect. The molecular mechanism leading to neurodegeneration is still under debate, and it is common opinion that it may reside in the interaction between amyloid species and the neural membrane. In this investigation Atomic Force Microscopy and spectroscopy have been used to understand how structural (and mechanical) properties of POPC/POPS lipid bilayers, simulating the phospholipid composition and negative net charge of neuritic cell membranes, are influenced by the interaction with Aβ(1-40), in different stages of the peptide aggregation. Substantial differences in the damage caused to the lipid bilayers have been observed, confirming the toxic effect exerted especially by Aβ(1-40) prefibrillar oligomers.

Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

Amyloid β-peptide insertion in liposomes containing GM1-cholesterol domains

Neuronal membrane damage is related to the early impairments appearing in Alzheimers disease due to the interaction of the amyloid β-peptide (Aβ) with the phospholipid bilayer. In particular, the ganglioside GM1, present with cholesterol in lipid rafts, seems to be able to initiate Aβ aggregation on membrane. We studied the thermodynamic and structural effects of the presence of GM1 on the interaction between Aβ and liposomes, a good membrane model system. Isothermal Titration Calorimetry highlighted the importance of the presence of GM1 in recruiting monomeric Aβ toward the lipid bilayer. Light and Small Angle X-ray Scattering revealed a different pattern for GM1 containing liposomes, both before and after interaction with Aβ. The results suggest that the interaction with GM1 brings to insertion of Aβ in the bilayer, producing a structural perturbation down to the internal layers of the liposome, as demonstrated by the obtained electron density profiles.

The sea urchin embryo: A model to study Alzheimer’s beta amyloid induced toxicity

Alzheimers disease (AD) is the most common form of dementia. The cause of AD is closely related to the accumulation of amyloid beta peptide in the neuritic plaques. The use of animal model systems represents a good strategy to elucidate the molecular mechanism behind the development of this pathology. Here we use the Paracentrotus lividus embryo to identify molecules and pathways that can be involved in the degenerative process. As a first step, we identified the presence of an antigen related to the human APP, called PlAPP. This antigen, after gastrula stage, is processed producing a polypeptide of about 10kDa. By immunohistochemistry we localized the PlAPP antigen in some serotonin expressing cells. Similarly, after 48 or 96h incubation, a recombinant beta-amyloid peptide, rAbeta42, accumulates around the intestinal tube and oesophagus. In addition, incubation of sea urchin embryos with two different solutions rich in oligomers and fibrillar aggregates of rAbeta42 induce activation of apoptosis as detected by TUNEL assay. Moreover, we demonstrate that aggregates induce apoptosis by extrinsic pathway activation, whereas oligomers induce apoptosis both by extrinsic and intrinsic pathway activation. Utilizing an apoptotic inhibitor, caspases activation was offset and morphological damage rescued. Taken together all these observations suggest that the sea urchin may be a simple and suitable model to characterize the mechanism underlining the cytotoxicity of Abeta42.

α-Casein inhibition mechanism in concanavalin A aggregation process.

The inhibition of the aggregation in protein solutions is currently a subject of great interest in many research fields, from the study of protein-misfolding related diseases to pharmaceutics, biotechnology, and food science. α(s1)-Casein, one of the four types of caseins, which are the largest protein component of bovine milk, has been found to hinder the aggregation process of several proteins, including the amyloid β-peptide, involved in Alzheimers disease. To shed light into the mechanisms by which casein exerts this chaperon-like protective action, we studied its effect on the different steps of the aggregation process of concanavalin A, by means of both static and dynamic light scattering, thioflavin T and ANS fluorescence, circular dichroism, and atomic force microscopy. Our results show that casein has a poor effect on the first step of the process leading to the formation of amyloid-like structures. On the contrary, it has a marked effect on the second step of the process, ascribable to clusters condensation and compaction, up to the formation of very large aggregates. Such an effect requires a molar ratio of casein larger than that necessary to inhibit the fibrillogenesis of the amyloid β-peptide, thus, suggesting a different mechanism of interaction of casein, depending on both conformational properties and relative size of the aggregating molecules.