Amyloid Fibrils Formation - Coursework Example

The Amyloid Fibrils Formation and Amorphous Aggregation in Concanavalin A in Vitro Neurodegenerative diseases like Alzheimer’s disease lead to morbidity in the enhanced, which is a cause for concern given that the elderly population is growing. There is currently no cure for Alzheimer’s disease, which only increases the need for a better understanding of the disease processes, so that the attempts to stem the advance of the disease can be put in place. Theories on the disease processes suggest that protein aggregates as insoluble amyloid fibrils and that this unfolding or aggregation of proteins occurs when they are exposed to destabilizing conditions. Results from studies have also suggested that amyloid aggregation as seen in Alzheimer’s disease and other mental conditions is a generic property of the polypeptide chain, which in all probability is connected to the common structure of the peptide backbone. More recent studies have resulted in theories that suggest that the effects seen in Alzheimer’s disease suggest that prefibrillar aggregates rather than mature fibrils are responsible for the neurotoxicity of amyloid aggregates. In addition it is believed that the high cytotoxicity demonstrated by amyloid aggregates is the result of early prefibrillar assemblies and in some cases the individual misfolded molecules rather than the mature fibrils. In essence the theories on the disease processes involved in the neurodegenerative disease of Alzheimer’s disease is that the disruption of the integrity of the cell membrane by the soluble prefibrillar aggregates and misfolded proteins is the primary phase in the induction of cell death that occurs with AD. The aim of the study is to investigate or observe the amyloid fibrils formation and amorphous aggregation in concanavalin A in vitro. The study involves the investigation or observing of the formation of amyloid fibrils and their amorphous aggregation in concanavalin A in vitro, using the measure of the presence and intensity of tyrosine, tryptophan and Thioflavin T., at three different pH levels, with each of these pH levels further differentiated at three temperature levels and measured over time intervals of 0, 6, 24, 48, 72 and 96 hours. Introduction Neurodegenerative dementia is a cause for concern in the elderly with its potential to disrupt their ability to lead normal lives. Alzheimer’s disease (AD) has been found to be the most frequent causative factor in neurogenerative dementia in the elderly. Estimates for the prevalence of AD in the United States of America show that nearly 4.5 million of the population suffer from AD. Direct and indirect health care costs annually as a result of AD in the United States of America are estimated at more than a hundred billion dollars. The prevalence and cost of health care in AD is only going to rise with growth in the elderly population. By the year 2050 it is expected that the prevalence of AD will rise dramatically to 13.2 million in the United States of America. These factors combined with lack of any known cure in medical science for AD, make it necessary that there is a better understanding of the disease processes in AD, so that the challenge that AD poses can be faced more effectively (Yaari & Coney-Bloom, 2007). Studiers into the mechanisms involved in the neurogenerative diseases like AD, Parkinson’s disease and Huntington’s disease have given to theories of the involvement of amyloid deposits or intracellular occlusions containing abnormal or folded protein fibrils as the cause of these neurogenerative diseases (Irvine et al 2008). Available scientific technology allows the investigation of the amyloid fibrils formation and amorphous aggregation in vitro to assist in the evaluation of theories on the amyloid depoits of abnormal or folded protein fibrils as the basis of neurogenerative diseases. Aim The aim of the study is to investigate or observe the amyloid fibrils formation and amorphous aggregation in concanavalin A in vitro to provide a better understanding in amyloid fibril formation and amorphous aggregation and thereby an insight into disease mechanisms involved in neurogenerative diseases AD, Parkinson’s disease and Huntington’s disease. Theories regarding the mechanism of assembly and toxicity in AD Alzheimer’s Disease Alois Alzheimer was the first to identify the disease in 1906, and hence carries his name. Irvine et al 2008, p.451, define Alzheimer’s disease as “an irreversible, progressive brain disease that slowly destroys memory and cognitive skills.” The factor that poses the greatest risk for AD is advanced age. Alzheimer’s disease afflicts ten percent of all individuals over sixty-five, which rises to fifty percent in those above the age of eighty-five. There is no differentiation in the incidence of AD in terms of race and sex. The disease has an average duration of eight years, though there are instances, when the disease has extended to more than twenty years. Since age is a major criterion for AD, it has been divided into two categories based on age. The first is early onset AD, which occurs between the age of thirty and sixty. Early onset AD is infrequent with its incidence less than two percent of all cases of AD, with genetic causes in more than fifty percent. Late onset AD occurring at the age of over sixty is the common occurrence of the disease. Genetic factors are believed to predispose an individual for AD, but have proven to be difficult to identify as inheritance seems more to modulate the age of onset of the disease than be responsible for the phenotypes of the disease (Irvine et al, 2008). Abnormal Protein Basis of Alzheimer’s Disease Synaptic and neuronal degeneration and the occurrence of intracellular neurofibrillary tangles (NFTs) within the cerebral cortex are considered as the main pathological factor of AD. The NFTs in AD consist of hyperphosphorylated tau protein and extra-cellular deposits of amyloid B-protein (AB) in senile plaques. Such lesions are also found individuals without AD, but a faulty AB homeostasis that encourages the accumulation of AB in the brain is the most commonly accepted first event in the disease processes in AD (Portelius et al, 2008). Such a contention is supported by Irvine et al 2008, who state that many of the neurodegenerative diseases like AD, Parkinson’s disease and Huntington’s disease result from amyloid deposits or intracellular inclusions containing abnormal protein fibrils. The authors go on to add that “burgeoning evidence suggests that accumulation of proteins capable of forming amyloid deposits may represent a common pathological mechanism for all these diverse illnesses” (Irvine et al, 2008, p.451). Amyloid in Alzheimer’s Disease Proteins are made up of small monomers, which are pepetides that demonstrate the ability to chemically bond with other monomers to make up oligomers or polymers. This chemical bonding of protein monomers leads to protein aggregation. Protein aggregation is known to be a common characteristic seen in many of the neurogenerative diseases including AD. It is believed that this protein aggregation has a key role to play in the pathogenesis of these neurogenerative diseases. In the protein aggregation process a monomer of a soluble protein interacts with other monomers of the same protein to form oligomers and polymers. Alterations to the three dimensional structure of the protein, with particular emphasis on the beta strands are a common feature of this process. With the continued aggregation the size of the protein aggregate becomes enhanced, which leads to the precipitation of these protein aggregates as insoluble amyloid fibrils (Irvine et al, 2008). According to Vetri et al 2007, this unfolding or aggregation of proteins occurs when they are exposed to destabilizing conditions. Nearly twenty disparate kinds of peptides and proteins, including the AB peptides, have been found to be the main components of amyloid aggregates in experiments conducted in vivo Over the last decade several natural as well as designed sequences of peptides or proteins that are do not have any association with known mental disorders have been found to be capable of aggregating into fibrils that are in no way different from with the amyloid fibril aggregates associated with mental (Amyloid) diseases. Findings from studies suggest that amyloid aggregation as seen in Alzheimer’s disease and other mental conditions is a generic property of the polypeptide chain, which in all probability is connected to the common structure of the peptide backbone (Bucciantini et al, 2004). Mechanism of Assembly and Toxicity of Amyloids in Alzheimer’s Disease Amyloid fibrils are normally formed by protofilaments. In the protofilaments the molecules of the protein are arranged in B-strands that are perpendicular to the elongation axis. In the formation of amyloid fibrils, these protofilaments may associate or be twisted around each other resulting in fibrils that are of much larger diameter. Initial studies of amyloid aggregates suggested that the toxicity from amyloid aggregates was due to the mature amyloid fibrils. The findings of more recent studies and the better reasoning of the effects seen in Alzheimer’s disease suggest that prefibrillar aggregates rather than mature fibrils are responsible for the neurotoxicity of amyloid aggregates (Vetri et al, 2007). This perspective of amyloid toxicity receive support from Irvine et al, 2008, who reason that in patients dying from Alzheimer’s disease, there is only a weak correlation between the severity of dementia and the density of the fibrillar amyloid. In direct contrast to this there is very strong correlation between the amount of soluble amyloid aggregate and the extent to which synaptic loss is present and the severity of cognitive impairment. Bucciantini et al, 2004 provide further support for the point of view that prefibrillar aggregates rather than the mature fibrils are responsible for cytotoxicity of amyloid. The authors point out that recent studies have shown that prefibrillar aggregates of several disparate peptides and proteins are recognized by polyclonal antibodies. The high cytotoxicity demonstrated by amyloid aggregates is the thus the result of early prefibrillar assemblies and in some cases the individual misfolded molecules rather than the mature fibrils (Bucciantini et al, 2004). The primary phase in the induction of cell death in AD as a result of amyloid aggregates is the disruption of the integrity of the cell membrane that results from the misfolded proteins and peptides and the pore-forming proteins in the amyloid aggregates. The disruption of the integrity of the cell membrane brings about the consequences of imbalance in ion homeostasis and transmembrane electrochemical gradients or damage to the several signalling pathways, which are the main causes of cell damage and death (Bucciantini et al, 2004). Conclusion AD with its potential for death and morbidity in a high proportion of the elderly is a cause for concern. Amyloid aggregation is considered to be pathological mechanism involved in the disease. There is evidence to suggest that the soluble prefibrillar aggregates rather than the mature fibrils are responsible for the neurotoxicity. The disruption of the integrity of the cell membrane by the soluble prefibrillar aggregates and misfolded proteins is the primary phase in the induction of cell death that occurs with AD. Design of the Study of amyloid fibrils formation and amorphous aggregation in concanavalin A in vitro The study involves the investigation or observing of the formation of amyloid fibrils and their amorphous aggregation in concanavalin A in vitro. Fluorescence to test intensity of tyrosine, tryptophan and Thioflavin T and the use of the Transmission Electron Microscope have been taken as adequate for the investigations required in this study. Using fluorescence should help in highlighting and measuring the presence and intensity of tyrosine, tryptophan and Thioflavin T., which can be further measured with the help of the Transmission Electron Microscope and used as the test measures. The study design involves the growing of amyloid fibrils at different pH levels and each of the different pH levels at different temperatures too. The pH levels selected for the study are pH 5, pH 7 and pH 9, while the different temperatures that will be used in the study are 25 degrees C, 37.2 degrees C and 40 degrees C. Amyloid fibrils will be grown from 1mg/ml of Con A with HEPES buffers at each of the prescribed pH levels of 5, 7 and 9 and at the selected temperatures of 25, 37.2 and 40 degrees centigrade. This will give rise to a total of nine separate measures of amyloid fibril growth at differing pH levels and temperatures. The growth of the amyloid fibrils will be measured by testing the intensity of tyrosine, tryptophan and Thioflavin T over a range of time intervals staring with 0 and over the time periods of 1hr, 2hrs, 4hrs, 6hrs, 24hrs, 48hrs, 72hrs and 96hrs. Each of these measures will be done employing fluorescence. The preparation of the Thioflavin T samples for the fluorescence test will involve the following steps. The Thioflavin T samples will be mixed with 3mg/ml deionized water and filtered using a 0.2 uM thin foil and stored at a temperature of four degrees centigrade. Subsequently it will be mixed with 4uM of each of the obtained Con A samples and to arrive at a final concentration of 4uM for each Con A sample, along with 40uM of Thioflavin T for every time a fluorescent reading is to be taken. For the Transmission Electron Microscope measurements a grid for the different pH levels of 5, 7 and 9 and for each of the different temperatures of 25, 37.2 and 40 degrees centigrade and for time intervals starting from 0, 6, 24, 48, 72 and 96 hours will be created. This grid will be then removed from the pack with the help of a forceps applying utmost care. 4 ml of each of the samples would be dropped in and left for sixty second. Then 4ul of 2% uranyl acetate would be dropped in and then left for thirty seconds prior to the use of Transmission Electron Microscope for the investigations. Readings thus received would be collated and tables created to enable the analysis of the data. Appropriate statistical tools would be used to analyse the data where necessary to arrive at the findings of the study. These findings will make up the investigations or observe the amyloid fibrils formation and amorphous aggregation in concanavalin A in vitro. It is assumed that these findings will be in keeping with the results of earlier studies that have given rise to the theories that it is the disruption of the integrity of the cell membrane by the soluble prefibrillar aggregates and misfolded proteins, which is the primary phase in the induction of cell death that occurs with AD. . .   Literary References Bucciantini, M., Calloni, G., Chiti, F., Formigli, L., Nosi, D., Dobson, M. C., Stefani, M. (2004). Prefibrillar Amyloid Protein Aggregates Share Common Features of Cytotoxicity. The Journal of Biological Chemistry, 279(30), 31374-31382. Irvine, B. G., El-Agnaf, M. O., Shankar, M. G. & Walsh, M. D. (2008). Protein Aggregation in the Brain: The Molecular Basis for Alzheimer’s and Parkinson’s Diseases. Molecular Medicine, 14(7-8), 451-464. Portelius, E., Zetterberg, H., Gobom, J., Andreasson, U & Blennow, K. (2008). Targeted Proteomics in Alzheimers Disease: Focus on Amyloid-Beta. Expert Review of Proteomics, 5(2), 225-237. Vetri, V., Canale, C., Relini, A., Librizzi, F., Militello, V., Gliozzi, A. & Leone, M. (2007). Amyloid fibrils formation and amorphous aggregation in concanavlin A. Biophysical Chemistry, 125, 184-190. Yaari, R. & Coney-Bloom, J. (2007). Alzheimer’s Disease. Seminars in Neurology, 27(1), 32-41. Read More