Light scattering as an easy tool to measure vesicles weight concentration

Over the last few decades, liposomes have emerged as promising drug delivery systems and effective membrane models for studying biophysical and biological processes. For all applications, knowing their concentration after preparation is crucial. Thus, the development of methods for easily controlling vesicles concentration would be of great utility. A new assay is presented here, based on a suitable analysis of light scattering intensity from liposome dispersions. The method, tested for extrusion preparations, is precise, easy, fast, non-destructive and uses a tiny amount of sample. Furthermore, the scattering intensity can be measured indifferently at different angles, or even by using the elastic band obtained from a standard spectrofluorimeter. To validate the method, the measured concentrations of vesicles of different matrix compositions and sizes, measured by light scattering with different angles and instruments, were compared to the data obtained by the standard Stewart assay. Consistent results were obtained. The light scattering assay is based on the assessment of the mass fraction lost in the preparation, and can be applied for methods such as extrusion, homogenization, French press and other microfluidic procedures

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

Deposition of the amyloid beta-protein (Abeta) in senile or diffuse plaques is a distinctive feature of Alzheimer's disease. The role of Abeta 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 Abeta 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

Insulin Promotes Survival of Amyloid-Beta Oligomers Neuroblastoma Damaged Cells via Caspase 9 Inhibition and Hsp70 Upregulation

Alzheimer's disease (AD) and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta) in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD

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

Alzheimer’s 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 10 kDa. By immunohistochemistry we localized the PlAPP antigen in some serotonin expressing cells. Similarly, after 48 or 96 h incubation, a recombinant b-amyloid peptide, rAb42, 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 rAb42 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 Ab42

Early events in insulin fibrillization studied by time-lapse atomic force microscopy

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils relies not only on its medical consequences, but also on its more basic properties of self--organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In the present work, we address the early events preceeding amyloid fibril formation in solutions of zinc--free human insulin incubated at low pH and high temperature. Aside from being a easy--to--handle model for protein fibrillation, subcutaneous aggregation of insulin after injection is a nuisance which affects patients with diabetes. Here, we show by time--lapse atomic force microscopy (AFM) that a steady-state distribution of protein oligomers with an exponential tail is reached within few minutes after heating. This metastable phase lasts for few hours until aggregation into fibrils suddenly occurs. A theoretical explanation of the oligomer pre--fibrillar distribution is given in terms of a simple coagulation--evaporation kinetic model, in which concentration plays the role of a critical parameter. Due to high resolution and sensitivity of AFM technique, the observation of a long-lasting latency time should be considered an actual feature of the aggregation process, and not simply ascribed to instrumental inefficency. These experimental facts, along with the kinetic model used, claim for a critical role of thermal concentration fluctuations in the process of fibril nucleation

Investigation on MMACHC-R161Q pathological mutant from cblC disease

The cblC disease is a rare inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by combined methylmalonic aciduria and homocystinuria. The clinical consequences are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The molecular genetic cause of the disease was found in the mutations of the gene coding for MMACHC, a 282 amino acid protein that transports and processes the various forms of Cbl. Here we present the biophysical characterization of wild type MMACHC and a variant, p.R161Q, resulting from the most common missense pathological mutation found in cblC patients. By using a biophysical approach we investigated the stability of the two proteins and their ability to bind and transform the vitamin B12, and to assemble in a dimeric structure. Moreover, interesting indications about the behaviour of the proteins resulted from the Molecular Dynamics (MD) simulations. Overall, our results reveal how a biophysical approach based on the complementarity of computational and experimental methods can offer new insights in the study of the specific effects of the pathological cblC mutation and help prospecting new routes for the cblC treatment

The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway