Mutations That Replace Aromatic Side Chains Promote Aggregation of the Alzheimerâ€™s đ?›źđ?›˝ Peptide. Mandeep Takhar Copyright 2013 Alzheimerâ€™s disease is considered to be a leading cause of dementia in the elderly. It was first diagnosed nearly 100 years ago, but treatment has not been found even to this day. The name Alzheimerâ€™s was given because of the individual who first described symptoms of the disease, psychiatrists Alois Alzheimer. Alzheimerâ€™s is caused by an accumulation of folded amyloid beta and amyloid tau proteins in the brain. Beta amyloids are broken down by apolipoproteins; however, some isoforms (APOE4) are not as effective, which can lead to amyloid buildup in the brain leading to Alzheimerâ€™s. Betaamyloid is a fragment of amyloid precursor protein. This protein plays a crucial role in neuron growth and repair. On the other hand, tau protein aids in stabilization of microtubules, but once it becomes chemically altered it can cause tangles in the neuronâ€™s transport system. One drug that is currently undergoing clinical trials to treat Alzheimerâ€™s is called bapineuzumab (Kerchner, 2010). Bapineuzumab is a humanized monoclonal IgG1 antibody, which is used against the beta-amyloid N-terminus. An excess amount of beta-amyloid is thought of as the cause of Alzheimerâ€™s disease2. Bapineuzumabâ€™s purpose is to clear that plaque buildup in the brain. Patients who were treated with bapineuzumab were investigated for amyloidrelated imaging abnormalities (Sperling, 2012). Bapineuzumab was used in order to reduce cerebral amyloid-beta. As these clinical trials were on going with treatment with bapineuzumab, amyloid-related imaging abnormalities were becoming apparent. A flow
chart was presented with two studies that were conducted along with the number of patients in each study. A total of 144 patients were assigned to bapineuzumab and 118 to placebo. From those two categories only 210 patients were eligible for risk factor analysis. A risk factor analysis is a chart that presents patients pre-existing health conditions and then lists the risk percentage of developing a specific condition, such as amyloid-related imaging abnormalities in this case. Of the 210 patients only 36 developed amyloid-related imaging abnormalities with suggestive of sulcal effusions and vasogenic oedema during the treatment with bapineuzumab (Sperling, 2012). Sulcus is a fissure in the surface of the brain and effusion is merely the movement of molecules through small pores. Vasogenic edema is when fluid penetrates into the parenchymal extracellular space due to a breakdown of tight endothelial junctions, which make up the blood-brain barrier. Amyloid-related brain-imaging abnormalities were thought to be the swelling of the brain caused by accumulation of fluid in the brain (vasogenic oedema) near a ridge on the cerebral cortex (sulcal effusion). A figure was also presented which contained MRI brain scans showing amyloid-related imaging abnormalities. In order to better understand the mechanism of Alzheimerâ€™s different aspects of it must be analyzed and understood. The đ?œ‹-stacking of aromatic residues has been suggested as a key feature promoting the assembly of polypeptides into amyloid fibrils (Armstrong et al., 2011). The đ?›źđ?›˝ sequence contains tyrosine at position 10 and phenylalanine at positions: 4, 19, and 20. To investigate whether or not đ?œ‹-stacking plays a key role in amyloid formation and aggregation, phenylalanine was replaced by leucine and isoleucine at positions, 19 and 20. Both leucine and isoleucine are similar to phenylalanine in size and
hydrophobicity, but they are not aromatic (Vagenende, 2013). Thus, if đ?œ‹-stacking plays a vital role in aggregation then removing aromaticity should reduce aggregation. Two figures were presented which tested whether the replacement of phenylalanine (removal of aromaticity) had any effect on aggregation. In the first figure, fluorescence was labeled on the y-axis and the time in days on the x-axis. A benzothiazole compound thioflavin-T (ThT), which binds to beta sheet structures and its dye displays enhanced fluorescence, was used to monitor the amount of aggregation in leucine and isoleucine. ThT assays were prepared by 20uM peptide solutions dissolved in 0.5 mg of purified peptide in 300uL of DMSO and diluted with a solution of 5mL of 8mM of NaOH and 300uL 20x PBS buffer. These samples were then incubated at 37 degrees Celsius and then removed at specific time periods (different days) to be read. When the samples were removed, they were mixed with a 100 uL of a solution of ThT to measure fluorescence. Fluorescence was measured on a play reader at excitation wavelength of 450 nm and emission wavelength 483 nm. The first table showed that as the days went on both the leucine and isoleucine showed more aggregation (more fluorescence through ThT) than the wild-type đ?›źđ?›˝ 42. More fluorescence is provided by a shift in the excited state charge from the dimethylaminobenzyl portion (blue) of ThT to the benzothiazole portion (red). This positive signal is provided by the binding to highly ordered and rigid amyloid aggregates. The soluble proteins were separated from the aggregated proteins and then analyzed by gel electrophoresis. Gel electrophoresis was used to acquire a more quantitative result, which the ThT is unable to provide due to its high background fluorescence. The results after 6 days showed that isoleucine showed a reduced concentration of soluble peptide relative to the wild-type đ?›źđ?›˝ 42. After 14 days both isoleucine and leucine showed a
reduced concentration of soluble peptide compared to the wild-type đ?›źđ?›˝ 42. These results proved that both isoleucine and leucine aggregate more rapidly. Transmission electron microscopy (TEM) was also used to monitor the aggregation of both 42LL and 42II. After one day, the 42II showed the more formation of fibrils, while wild-type đ?›źđ?›˝ 42 and 42LL both displayed smaller aggregates. After 14 days the wild-type đ?›źđ?›˝ 42, 42II, and 42LL all showed a considerable amount of aggregation. Thus, in agreeance with the prior experiments, fluorescence and gel electrophoresis, the removal of aromaticity did not prevent amyloid formation. After observing the outcome of đ?›źđ?›˝ 42, similar experiments were performed on đ?›źđ?›˝ 40. The number following the amyloid beta Greek symbols represent the number of amino acids it contains, yet both are generated from proteolytic cleavage of the amyloid precursor protein by đ?›˝ and đ?›ž secretases (Zhai et al., 2012). The two forms of đ?›źđ?›˝ with different lengths depend on the cleavage site. Phenylalanine was replaced by leucine and isoleucine at positions, 19 and 20. All the same steps are performed with đ?›źđ?›˝ 40, as they were described for đ?›źđ?›˝ 42. A similar figure was presented with the fluorescence labeled on the y-axis and the time in days on the x-axis. This figure presented findings similar to those with the đ?›źđ?›˝ 42. As the days increased both leucine and isoleucine displayed an increased amount of aggregation. Gel electrophoresis was also performed and the results were presented in two figures, one for 6 days and another for 14 days. After 6 days 40II showed an enhanced propensity to aggregated compared to the đ?›źđ?›˝ 40. After 12 days 40LL showed that its propensity to aggregate was enhanced relative to the đ?›źđ?›˝ 40. Transmission electron microscopy (TEM) was also used to monitor the aggregation of both 42LL and 42II compared to that of the wild-type đ?›źđ?›˝ 42. Images of each were taken
after 7 and 21 days. After 7 days the 40II aggregated considerably, 40LL contained some fibrils, and wild-type đ?›źđ?›˝ 42 was blank for the most part with the occasional long fibril. After 21 days, all three peptides showed a considerable amount of fibril formation. Compounds known to inhibit đ?›źđ?›˝ aggregation: Curcumin, Tannic Acid, Triazine E2, and a non-aromatic control, MTMA were investigated. Curcumin and Tannic Acid have both been shown to prevent đ?›źđ?›˝ fibrillation and aggregation. Triazine E2 has been shown to be an aggregation inhibitor. MTMA (myristyltrimethylammonium bromide) has also shown to reduce đ?›źđ?›˝ aggregation. These compounds were incubated at 37 degrees Celsius along with 20 uM of the peptide solution for 7 days. These samples were then measured for ThT fluorescence at the emission wavelength of 483 nm. Representative TEM images after 7 days were also illustrated. Both results were similar in showing that when no compound was added, aggregation occurred. When the compounds (Curcumin, Tannic Acid, Triazine E2, and MTMA) were added, a drastic decrease in aggregation of fibrils was observed in đ?›źđ?›˝ 42, 42II, and 42LL. After these experiments were performed it was evident that đ?œ‹-stacking of aromatic residues does not play a key role in promoting aggregation of fibrils. The outcome of these experiments showed that not only does the elimination of aromaticity not reduce aggregation, but yields higher levels of aggregation over time. Although the outcome of this experiment does not narrow down the exact mechanism behind aggregation of fibrils, it does eliminate one possibility. Removing the đ?œ‹-stacking does not reduce aggregation in đ?›źđ?›˝ 40, nor đ?›źđ?›˝ 42. Â Â
Work Cited Arimon, Muriel, Fausto Sanz, Ernest Giralt and Natalia Carulla. Template-Assisted Lateral Growth of Amyloid-beat42 Fibrils Studied by Differential Labeling with Gold Nanoparticles. Bioconjugate Chemistry. 2012. (23), pp 27-32. Armstrong, Anne, Jermont Chen, Angela Fortner McKoy, and Michael H. Hecht. Mutations That Replace Aromatic Side Chains Promote Aggregation of the Alzheimerâ€™s alpha-beta Peptide. Biochemistry including biophysical chemistry & molecular biology. 2011. (50), pp 4058-4067. Kerchner, Geoffrey. "Bapineuzumab." NIH Public Access. (2010): 1121-1130. Print. Sperling, Reisa. "Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with abpineuzumab: a retrospective anaylsis." Center for Alzheimer Research and Treatment. 11. (2012): Vagenende, Vincint, Alvin Han, Monika Mueller, and Bernhardt L. Trout. ProteinAssociated Cation Clusters in Aqueous Arginine Solutions and Their Effects on Protein Stability and Size. Chemical Biology. 2013. (8), pp 416-422. Zhai, Jiali, Tzong-Hsien Lee, David Small, and Marie-Isabel Aguiar. Characterization of Early Stage Intermediates in the Nucleation Phase of alpha-beta Aggregation. Biochemistry including biophysical chemistry & molecular biology. 2012. (51), pp1070-1078.