The Effect of Down Regulating the AB-Degrading Protease Neoprilsyn - Research Paper Example

 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 suffers 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 (1). 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 (2). 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 (2). 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 (3). 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” (2). Figure – 1 (4). Figure 1 shows a high-power picture of a section taken from the cerebral cortex of an AD patient, and developed with the purpose of the showing neurofibrillary tangles. The shape of the tangle seen arises from the shape of the neuron from which it was formed. A dense mass of thread-like material is seen and constitutes the neurophil threads, which are hyperphosphorylated tau in the broken axons and dendrites of neurons that are with tangles (4). Figure – 2 (4). Figure 2 shows a high power picture of a section taken from the cerebral cortex of an AD patient, and developed with the purpose of the showing high density of plaques and visualization of the amyloid beta peptide (ABP), which is the main component of plaques. The picture clearly shows differing morphological appearances of plaques, whereby some plaques are darkly stained with clearly defined borders, while others show lower staining and borders that are not clearly seen. Amyloid in Alzheimer’s Disease Proteins are made up of small monomers, which are peptides that demonstrate the ability to chemically bond with other monomers to make up bloomers 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 bloomers 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 (2). 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 (6). 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 (5). 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 (6). 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 7signaling pathways, which are the main causes of cell damage and death (6). Extensive research has gone into the mechanisms of protein aggregation and amyloid fibrils formation. The general acceptance is that formation of amyloid fibrils stems from partially unfolded structures. This makes conformational flexibility and solvent accessibility to the critical regions in protein structure very important factors that induce amyloid fibril formation (7). Amyloid Beta Precursor Protein Proteolysis According to Nelson and Alkon, 2005, p. 7378, amyloid precursor protein (APP) is “is a large membrane-bound copper-binding protein that is essential in maintaining synaptic function and may play a role in synaptogenesis”. ABP is a product of ectodomain cleaved APP through the intramembranous proteolysis by beta secretase and gamma-secretase. Gamma-secretase is a complex enzyme made up of presenilin and a few other peptides (8). Cleavage of APP through the enzyme beta secretase produces about 100-kDa soluble N-terminal fragment and a 12-kDa C-terminal fragment (C99). The C99 fragment may be further cleaved by the gamma secretase enzyme to produce the APP intracellular domain (AICD) and 40 and 42 amino acid long ABPs of AB40 and AB42. AB42 is the ABP that has been identified with the greatest tendency to form oligomers and plaques, which has been termed as the amyloidogenic pathway. Another enzyme activity occurs through alpha-secretase, which cuts APP approximately in the middle of the AB region and along with the action of gamma-secretase producing the AB 17-42 fragment known as p3. This alternate metabolic pathway of APP may be termed as the non-amyloidogenic pathway. During normal metabolism of APP, AB levels are kept tightly regulated through amyloid-degrading enzymes, like insulin-degrading enzyme and neprilysyn (9). Figure – 3 (3) Figure 3 shows the metabolism of the largest isoform of amyloid precursor protein (APP770) leading to generation of AB and p3. The initial 28 residues of the AB domain can be seen outside the membrane, while the remaining 12-14 residues are in the transmembrane domain of APP. The figure also shows the nonamyloidal pathway (a) and the amyloidogeneic pathway (b). Figure – 4 (3) Figure – 4 shows the amyloidogenic pathway with generation of AB in detail. A – Angiotension-converting enzyme, B – beta-site amyloid precursor protein cleaving enzyme, C- Cathepsin B, E – Endothelin-converting enzyme, I – Insulin-degrading enzyme, M2 – MMP-2, M9 – MMP-9, MMP – Matrix metalloprotease, N – Neprilsyn, P – Plasmin, B1 – BACE1, B2 – BACE2, y – gamma-secretase. Figure – 5 (3) Figure 5 is a MALDI time-of-flight mass spectrum that displays 17 C-terminally truncated AB peptides immunoprecipitated from the cerebrospinal fluid with the assistance of antibody 6E10. The bold residues seen in the AB42 amino acid sequence reflects the epitope of 6E10. The most common peptide seen is AB40. AB- Beta amyloid, m/z – Mass-charge ration. Beta Amyloid and Pathogenesis of Alzheimer’s disease Beta-secretease and gamma secretease clip APP at the N-and C-terminal ends of the AB sequence respectively and through that contribute to amyloidogenesis through the production of whole AB. AB so formed can be found in the cells and fluids of the body. It appears that for deposition of AB as plaques, there is the requirement of a physical transformation of the peptide into an aggregated from and subsequently into a fibrilar form. Oxygen insult is known to create the required conditions for such aggregation of AB. Fibrilar AB is known to occur in compact AB deposits. Such a presence suggests the end stage of plaque maturation and is associated with microglia and dystrophic neuritis (4). Nalivaeya et al 2004, suggest that ischemia and hypoxia contribute to the proteolysis of APP along the amyloidogenic pathway leading to the formation of neuro-toxic AB peptide. Furthermore many metallopeptides have a role to play in the degradation of AB peptide and the presence of any inhibitory factors to this role played by the metallopeptides contribute to the pathogenesis of AB in Alzheimer’s disease. Animal based studies on prenatal hypoxia and acute hypoxia have demonstrated that oxygen insults have the effect of not just increasing the amyloid precursor protein expression in the brain, but also contributing to a decrease in activity of alpha secretase or the non-amyloidogenic pathway. Hypoxia is also seen to contribute to a reduction in AB degrading enzymes of neprilsyn, endothelin-converting enzyme and metalloproteases (10). The brain has its own immune system that is coordinated by immuno- competent cells like microglia and is vulnerable to some of the defense mechanism activities like inflammation. Inflammation as associated with the brain is not the same as with peripheral inflammation, since there is no edema and neutrophil invasion. However tissue levels of the inflammatory mediators like cytokines, chemokines, oxygen free radicals and nitrogen species get changed (11). A very large component of senile plaques is the abnormally aggregated AB protein. Deposition of AB outside of the cell can result in a broad range of pathological processes that include gila activation, neuro-inflammation, and neuronal apoptosis. There is sufficient evidence to link the accumulation of AB outside the cell and the pathological responses with the neuropathological changes that occur in AD (12) Animal studies reveal that inflammatory cytokines like Interleukin (IL) -1B, IL – 6, Tumor necrosis factor or Transforming growth factor can enhance the expression of APP and lead to augmented AB formation. Cytokines are also known to transcriptionally enhance the b-secretase mRNA, protein and enzymatic activity and through that increased AB formation (11). APP also plays a role as a pro-inflammatory receptor on monocytic lineage cells and beta integrin-mediated pro-inflammatory activation of monocytes depends on APP. Such activation through APP results in increases in pro-inflammatory cytokine release and a tyrosine kinase-independent enhancement in the production of AB 1-42 (13) In addition to the action of AB degrading enzymes of neprilsyn, endothelin-converting enzyme, and metalloproteases in regulating AB, endoplasmic reticulum (ER) chaperones like glucose-regulated protein 78 (GRP78 ) contribute to the quality control of proteins in the ER. Over expression of ER chaperones decrease the expression of AB. In the case of GRP78 it acts by inhibiting the maturation of APP. Such action is believed to occur through GRP78 binding itself directly to inhibit the maturation of APP and through that suppress the proteolysis of APP (14). Deposition of AB aggregates in the brain, which is a characteristic feature of AD. Is thus an imbalance in the rates at which AB is generated and the rate at which it is cleared from the brain. Such an imbalance can occur through factors that cause an increase in AB generation factors, while acting to reduce the AB expression controlling factors, like ischemia or hypoxia. There is a naturally occurring factor that contributes to a reduction in AB controlling factors and that is normal aging. Normal aging has the effect of down regulating the AB-degrading protease neoprilsyn and thereby increasing the risk for AD with advancing age (15). Works Cited 1. Yaari, R. & Coney-Bloom, J. “Alzheimer’s Disease”. Seminars in Neurology 27.1. (2007): 32-4. 2. Irvine, B. G., El-Agnaf, M. O., Shankar, M. G. & Walsh, M. D. “Protein Aggregation in the Brain: The Molecular Basis for Alzheimer’s and Parkinson’s Diseases”. Molecular Medicine 14.7-8 (2008): 451-464. 3. Portelius, E., Zetterberg, H., Gobom, J., Andreasson, U & Blennow, K. “Targeted Proteomics in Alzheimer's Disease: Focus on Amyloid-Beta”. Expert Review of Proteomics 5.2 (2008): 225-237. 4. Geula, Gengiz. “Pathogenesis of Alzheimer's Disease”. 2000. Medscape Today 1 March 2008. . 5. Vetri, V., Canale, C., Relini, A., Librizzi, F., Militello, V., Gliozzi, A. & Leone, M. “Amyloid fibrils formation and amorphous aggregation in concanavlin A. Biophysical Chemistry, 125 (2007): 184-190. 6. 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 (2004): 31374-31382. 7. Vetri, V., Librizzi, F., Militello, V. & Leone, M. “Effects of succinylation on thermal induced amyloid formation in Concanavalin A”. European Biophysics Journal 36(2007): 733-741. 8. “Nelson, T. J. & Alkon, D. L. “Oxidation of cholesterol by amyloid precursor protein and beta-amyloid peptide”. The Journal of biological chemistry 280.8 (2005): 1377-7387. 9. “Vetri, V., Librizzi, F., Militello, V. & Leone, M. “Effects of succinylation on thermal induced amyloid formation in Concanavalin A”. European Biophysics Journal 36(2007): 733-741. 10. “Nalivaeya, N. N., Fisk, L., Kochkina, E. G., Plesneva, S. A., Zhuravin, I. A., Debrota, D., & Turner, A. J. “Effect of hypoxia/ischemia and hypoxic preconditioning/reperfusion on expression of some amyloid-degrading enzymes”. Annals of the New York Academy of Sciences 1035 (2004): 21-33. 11. “Lee, J. W., Lee, Y. K., Yuk, D. Y., Choi, D. Y., Ban, S. B., Oh, K. W. & Hong, J. T. “Neuro-Inflammation Induced By Lipopolysaccharide Causes Cognitive Impairment Through Enhancement Of Beta-Amyloid Generation”. Journal of Neuroinflammation 5.37 (2008) 1 March 2008. . 12. “Baranowska- Bik, A., Bik, W., Wolinska-Witort, E., Martynska, L., Chmielowska, M., Barcikowska, M. & Baranowska, B. “Plasma beta amyloid and cytokine profile in women with Alzheimer's disease”. Neuro endocrinology letters 29.1 (2008): 75-79. 13. “Sondag, C. M. & Combs, C. K. “Amyloid precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells”. Journal of Neurochemistry 97.2 (2006): 449-461. 14. “Hoshino, T., Nakaya, T., Araki, W., Suzuki, K., Suzuki, W. & Mizushima, T. “Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides”. The Biochemical journal 402.3 (2007): 581-589. 15. Farris, W. Schutz, S. G., Cirrito, J. R., Shankar, G. M., Sun, X., George, A. Leissring, M. A>, Walsh, D. M., Qiu, W. Q., Holtzman, D. M. & Selkoe, D. J. “Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy”. The American journal of pathology 171.1 (2007): 241-251. Read More