Alzheimer’s disease (AD) is characterised by two major histological hallmarks; neurofibrillary tangles (NFT) and neuritic plaques. But, unlike plaques, the degree of NFT deposition in the brain correlates to the severity of clinical symptoms of neuronal dysfunction. The major constituent of NFT is hyperphosphorylated tau protein. The involvement of neuropsychological testing, neuroimaging techniques and biochemical measurement of cerebrospinal fluid markers (amyloid beta and tau proteins) have improved the sensitivity and specificity of AD diagnosis. But these diagnostic methods are challenged by individual’s education level, high cost and its invasiveness, hence the need for an alternative analysis platform that overcome these challenges. Peripheral blood fulfils the criteria as a suitable medium to evaluate AD diagnostic markers.
Since NFT deposition correlates with neuronal dysfunction severity and the NFT is constituted mainly of hyperphosphorylated tau protein then, the evaluation of tau in peripheral blood could provide useful information about AD progression. As tau protein in blood is susceptible to thrombin degradation, tau may be enwrapped in a vesicle such as extracellular vesicles (ECVs). The ECVs are formed when a multivesicular body fuses with the plasma membrane and release its contents into the extracellular milieu.
The formation and release of these ECVs are ubiquitous and cell type specific. Besides, ECVs contain several cargo molecules such as proteins, and it can cross the blood brain barrier into peripheral blood. Therefore, blood-based neuron derived extracellular vesicles (ndECVs) could be a dependable source to measure AD-like tau protein.
At present, the significance of tau protein in blood-based ndECVs for the diagnosis of AD remains largely unclear. Hence, this study aimed to quantify tau protein in peripheral blood ndECVs using flow cytometry technique and evaluate the significance of this measured proteins in the diagnosis of AD.
This study set the following objectives: (1) Establish a protocol for the isolation and characterization of ECVs and ndECVs using western blot and flow cytometry techniques, (2). Use the flow cytometry platform to quantify tau protein in the ndECVs of clinical patients who had already undergone clinical diagnostic (such as CSF tau, CSF p-tau, CSF β-amyloid or MMSE evaluation). (3).Evaluate the significance of tau protein in the ndECVs as a potential biomarker for AD.
Using the bead-assisted technique ECVs and ndECVs were isolated from serum and analysed.
This study made attempt to measure tau protein in peripheral blood ndECVs using flow cytometry technique. While quantification of tau protein by flow cytometry platform posed a challenge, ELISA technique was used as alternative to measure the proteins in the ECVs. Tau protein was measured in some of the ndECVs, but study did not observe any significant correlation between measured tau protein in ndECVs and the validated diagnostic markers (CSF tau, CSF p-tau, CSF β-amyloid or MMSE evaluation). Of interest was the trend for a negative correlation between serum tau protein and CSF β-amyloid. Peripheral blood is a relevant source for tau protein measurement and therefore more studies are needed to evaluate the significance of blood serum tau content as a diagnostic biomarker for AD.:TABLE OF CONTENT
LIST OF ABBREVIATION III
LIST OF FIGURES V
1. INTRODUCTION 1
1.1. ALZHEIMER’S DISEASE 1
1.1.1. The neuritic plaque 2
1.1.2. The neurofibrillary tangles 2
1.1.3. Pathophysiology of NFT 3
1.2. EPIDEMIOLOGY AND SOCIO-ECONOMIC BURDEN OF ALZHEIMER’S DISEASE 4
1.3. CLINICAL DIAGNOSIS OF ALZHEIMER’S DISEASE 5
1.4. CHALLENGES ASSOCIATED WITH THE CURRENT DIAGNOSIS OF AD 7
1.5. EXTRACELLULAR VESICLES 8
1.5.1. Formation of ECVs (ectosomes and exosomes) 9
1.5.2. Size, density and composition of ECVs 9
1.5.3. Functions of central nervous system ECVs 10
1.6. TAU PROTEIN, ECVS AND PERIPHERAL BLOOD 11
1.7. AIM OF THE STUDY 13
1.8. OBJECTIVES 13
2. METHODOLOGY 15
2.1. STUDY DESIGN 15
2.2. ETHICAL ISSUES 15
2.3. MATERIALS 15
2.4. METHODS 21
2.4.1. Blood sampling 21
2.4.2. SH-SY5Y Cell culture processing 21
2.4.3. Isolation of ECVs 22
2.4.4. Detection, characterization and quantification of ECVs and the cargo tau protein 26
2.5. STATISTICAL ANALYSIS 31
3. RESULTS 32
3.1. ESTABLISHMENT OF PROTOCOL FOR THE ISOLATION AND CHARACTERISATION OF ECVS 32
3.1.1. Precipitation versus ultracentrifugation techniques of isolating ECVs 32
3.1.2. Purification of ECVs using iodixanol or sucrose density gradient 33
3.1.3. Demonstration of other marker proteins of ECVs 34
3.1.4. Analysis of reproducibility 36
3.1.5. ECVs’ density determination 37
3.1.6. Comparing serum and plasma as ideal starting material 41
3.1.7. Optimal minimum starting volume of serum 43
3.1.8. Isolation of ndECVs from ultracentrifuge or pre-cleaned serum 44
3.2. DETECTION AND QUANTIFICATION OF ECVS’ CARGO PROTEINS, HSP70 AND TAU 45
3.2.1. western blot technique 46
3.2.2. Flow cytometry 47
3.2.3. ELISA as an alternative approach to quantify of tau protein in ndECVs 49
4. DISCUSSION 56
4.1. BASIS FOR CONSIDERING BLOOD-BASED MARKERS FOR THE DIAGNOSIS OF AD 56
4.2. BASIS FOR TARGETING TAU PROTEIN IN PERIPHERAL BLOOD 57
4.3. POTENTIAL ROLE OF NDECVS IN THE TRANSPORTATION OF TAU PROTEIN 58
4.4. ESTABLISHMENT OF PROTOCOL FOR ISOLATION AND CHARACTERISATION OF ECVS 59
4.4.1. Precipitation versus ultracentrifugation 60
4.4.2. Purification of ECVs by density gradient technique 61
4.4.3. Detection of ECVs’ cargo protein using western blot technique 63
4.4.4. ECVs isolation using the Bead-assisted technique 64
4.4.5. Isolation of cell type specific ECVs (ndECVs) 65
4.4.6. Comparison of plasma and serum as a source of ndECVs 66
4.5. DETECTION AND QUANTIFICATION OF ECVS’ CARGO PROTEINS (HSP70 & TAU) 67
4.5.1. Flow cytometry analysis 67
4.5.2. ELISA analysis 68
4.5.3. Tau protein in the ECVs of hibernating animals 71
5. CONCLUSION 73
6. REFERENCES 74
7. APPENDICES 100
7.1. CURRICULUM VITAE 100
7.2. PUBLICATIONS 101
7.3. DECLARATION OF THE INDEPENDENT WRITING OF THIS THESIS 103
7.4. ACKNOWLEDGEMENT 104
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:38586 |
| Date | 02 March 2020 |
| Creators | Simpong, David Larbi |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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