Abnormal Biophysical Properties in Aβ-induced Neurodegeneration for Disease Diagnosis, Pathogenesis and Treatment

在Aβ誘導的神經退行性疾病中異常的生物物理特性以用於疾病的診斷、發病機理研究和治療

Student thesis: Doctoral Thesis

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Award date16 Jan 2019

Abstract

Amyloid peptide beta (Aβ), derived from amyloid precursor protein (APP), was found to deposit primarily in brains of advanced Alzheimer’s disease (AD) patients. It is a group of peptides with 39 to 43 amino acid residues and shows dramatic toxicity to neural cells. This thesis aims to study abnormal biophysical properties in Aβ-induced neurodegeneration and investigate approaches for disease diagnosis, pathogenesis, and treatment. The main contributions of this thesis are summarized as follows:

First, we examined the interactions between the transcription factor Sp1-f3 and DNA fragment from an APP promoter in different ionic solutions using atomic force microscopy (AFM). Sp1-f3 molecules immobilized on the Si substrate were collected by the APP promoter, which was linked to AFM tips via covalent bond. As Pb(II) substituted Zn(II) in specificity protein 1 (Sp1) and provided a higher binding capability to APP promoter and facilitated APP expression, the interactions were strongly influenced by Pb(II). The results revealed that the enhanced interaction force facilitated APP expression and that APP overexpression could increase the risk of neurodegeneration incidence. The increased interaction force in Pb(II) was consistent with the low binding free energy obtained from our computer-assisted studies. AFM also detected the impacts of Pb(II) on cell morphology and mechanical properties. APP overexpression was beneficial to actin reorganization, which resulted in increased Young’s modulus and viscosity. Moreover, significant Sp1-APP promoter interaction caused alternation of cell activities. We also applied AFM to investigate the interference of other heavy metal ions with DNA transcription by detecting energy landscape parameters. Results suggested that the binding affinity of this complex in Pb(II) solution was stronger than that of the natural situation and weaker than the normal condition when present in Cd(II). Therefore, we considered that the alternation of energy landscape was associated with and could be used as a predictive indicator of heavy metal poisoning. With a high affinity for Cys residue, the Au compound showed the ability to coordinate with Sp1. Data analysis demonstrated that Au(I) helped restore the natural binding affinity between Sp1-f3 and DNA.

Second, the toxic effect of Aβ oligomer was investigated in live SH-SY5Y neuroblastoma cells by characterizing cell morphology and cell mechanics using high-resolution AFM scanning. Aβ1-40 oligomer-induced cytoskeleton reorganization was also observed under confocal microscopy and accounted for the reduction in Young’s modulus of cells. Meanwhile, tau phosphorylation increased after Aβ1-40 oligomer treatment, possibly resulting in microtubule disassembly. Results demonstrate the link between cellular mechanical changes and neurodegeneration mediated by Aβ1-40. To explore the dynamic effect of Aβ on cell mechanics, we monitored dynamic changes of cell mechanics in Aβ1-42 oligomer at different concentrations and different times. Result demonstrated a two-stage dynamic change of cell mechanics during this neurodegeneration process. An increased Young’s modulus was observed in treated cells during the short-time period, due to the alteration of surface tension, osmotic pressure, and actin polymerization. Rough cellular membranes were observed from AFM measurement in cells treated with Aβ1-42. However, cellular Young’s modulus gradually decreased with continuous exposure to Aβ1-42 or when presented to a high concentration of Aβ1-42. The primary cause of the decreased Young’s modulus is due to microtubule disassembly.

Third, we detected Aβ toxicity on cell membrane and mitochondria. The presence of Aβ in the cell membrane caused membrane damage and was associated with neurological impairments in patients. The calcium-permeable channel formed on a membrane by Aβ accelerated ion dysregulation. AFM-based penetration experiments demonstrated that cholesterol in the membrane modulate the incorporation of Aβ. To verify the configuration of Aβ ion channels, a graphene-based biosensor was applied to detect Ca2+ ion passing through the lipid bilayer.

Finally, SSD Mobilenet model, a convolutional neural network (CNN), was fine-tuned with our AFM results to detect biophysical signal from force-distance (FD) curve automatically. We evaluated the performance by F1 score. The results showed that the network can detect and identify the unbinding force in FD curve. We found progressively increased levels of voltage-dependent anion channel 1 protein (VDAC1), which caused mitochondrial dysfunction after Aβ treatment. Manual analysis and CNN detection were applied to detect the interaction force and the expression of VDAC1 on the mitochondria. Increased expression of VDAC1 demonstrated that VDAC1 could act as a therapeutic target for Aβ-induced neurodegeneration.

In summary, we investigated the alternation of biophysical properties in Aβ-induced neurodegeneration. Molecular interaction allows us to quantify kinetic parameters in the dissociation process. This indicates that the unbinding force is an indicator for heavy metal-related disease. Cell mechanics was detected by AFM nanoindentation. Characteristic changes in cell mechanics enabled us to describe dynamic neurodegeneration process of cells induced by Aβ. Aβ toxicity on the cell membrane and mitochondrial protein was investigated. Computer-assisted analysis of interaction force could be utilized to screen drugs for Aβ-related disease. We believed that this study provides deep insights into the pathogenesis of Aβ-induced neurodegeneration and effective methods for diagnosis and treatment.