1 Overexpression, characterisation, and encapsulation of LRP/LR for treatment of neurodegenerative disorders by Chandni Madhav (1348622) Dissertation Submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of Witwatersrand, Johannesburg, South Africa Supervisor: Dr Eloise van der Merwe Co-supervisors: Dr Tyrone Otgaar and Prof. Stefan FT Weiss (in memoriam) June 2023 2 Declaration I, Chandni Madhav (1348622), declare that this dissertation is my own work, unaided work. It is being submitted for the degree of Master of Science at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other university. 05th day of June, 2023 at Johannesburg 3 Abstract The neurodegenerative diseases of Parkinson's disease (PD) and Alzheimer’s disease (AD) are debilitating conditions affecting millions of people worldwide. Both diseases pose a significant economic burden and require therapeutic strategies. AD is identified by intracellular neurofibrillary tangles and the accumulation of amyloid beta (Aβ) plaques, while PD is characterised by the loss of dopamine-producing neurons and mutations in PINK1, parkin, and α-synuclein proteins. The 37 kDa/67 kDa laminin receptor (LRP/LR) is a multifunctional receptor found to impede the progression of AD and PD. In-vitro studies have revealed that the LRP::FLAG overexpression increases hTERT expression and telomerase activity while rescuing cells from Aβ-mediated cytotoxicity. Cuttler et al. 2019 also revealed that LRP::FLAG overexpression decreased phosphorylated tau levels and tauopathy-related proteins. Within PD, in vitro studies demonstrated that LRP::FLAG overexpression plays a protective role, through the degradation of α-synuclein and rescuing cells from MPP+ cytotoxicity. Thus, the current study comprised of three parts. The first part aimed at investigating the effect of overexpressing LRP::FLAG in HEK293 cells by assessing LRP/LR and PINK1 protein levels, as well as cell viability after treatment with MPP+, TBHP, and Aβ!", in vitro AD and PD models. The results demonstrated that the overexpression of LRP::FLAG rescued cells from MPP+, TBHP, and Aβ!" induced cytotoxicity, and increased LRP/LR and PINK1 protein levels. The second part of the study focussed on overexpressing, purifying, and structurally characterizing the 37 kDa LRP protein. The protein was successfully overexpressed and purified through Co2+-IMAC while structural characterisation indicated that protein was correctly folded and predominantly α- helical, as expected. The purified LRP protein was then encapsulated in PLGA nanoparticles to develop an efficient in vivo delivery system. Thus, the third part of the study investigated the effect of empty and LRP-encapsulated PLGA nanoparticles on cell viability, LRP levels, and telomerase activity in SH-SY5Y and HEK 293 cells. The results demonstrated that LRP- encapsulated PLGA nanoparticles successfully delivered the therapeutic protein and increased exogenous LRP protein levels. Additionally, SH-SY5Y cells treated with LRP- encapsulated PLGA nanoparticles exhibited increased cellular viability and telomerase activity. Therefore, the LRP-encapsulated PLGA nanoparticles could be a possible therapeutic for conditions such as ageing and age-related diseases including AD and PD. 4 Dedications ⋅ 𝑻𝒐 𝒎𝒚 𝒔𝒖𝒑𝒑𝒐𝒓𝒕 ⋅ Mom, Papa, Didi & Akshay Chhikas Chotus Madhavs Maharajs Mika, Mocha & Hugo 5 Acknowledgments I would like to show appreciation to; My supervisor, Dr Eloise van de Merwe, for providing unconditional guidance, support and opportunities. Thank you for always believing in me and pushing me to achieve more than I ever imagined possible. I will be eternally grateful for the loving environment you created in the lab. My co-supervisor, Dr Tyrone Otgaar, thank you for pushing me when I felt I wasn't accomplishing enough, thank you for checking in with me every day, and thank you for your support not only as a supervisor but as a friend. My co-supervisor, the late Prof. Stefan FT Weiss (in memoriam - 2023), for providing me with support and opportunities. My colleagues of Weiss and Van de Merwe labs, thank you for providing a positive work environment in which to share ideas around and for assisting on days I felt overwhelmed. Thank you for the enjoyable moments of shared laughter and heartfelt conversations Riyadh Mayet and members of the PSFRU unit, thank you for being patient and for welcoming us into your working space. Thank you for all the advice, assistance and mentorship during our protein purification. My family, thank you for your endless support and for allowing me the freedom to pursue my dreams. Financial Aid The National Research Foundation (NRF) – Postgraduate Scholarships (2021-2022) Wits Seed Fund (2021-2023) Research outputs Bignoux, M.J., Bernert, M., Otgaar, T.C., Madhav, C., Weiss, S.F.T., & Ferreira, E. PLGA nanocapsules as a delivery mechanism for LRP/LR targeting molecules. In preparation 6 Table of Contents Declaration ........................................................................................................................ 2 Abstract............................................................................................................................. 3 Dedications ....................................................................................................................... 4 Acknowledgments ............................................................................................................. 5 List of abbreviations .......................................................................................................... 8 List of figures ................................................................................................................... 11 List of tables .................................................................................................................... 12 1. Introduction ................................................................................................................. 13 1.1. Epidemiology of Alzheimer’s disease and Parkinson’s disease ........................................... 13 1.2. Alzheimer’s disease pathology and molecular mechanisms ............................................... 15 1.3. Parkinson’s disease pathology and molecular mechanisms ............................................... 17 1.4. 37 kDa Laminin Receptor Precursor/ 67 kDa high affinity Laminin Receptor (LRP/LR) ........ 19 1.4.1. Protein structure of LRP/LR .................................................................................. 20 1.4.2. LRP/LR role in diseases ......................................................................................... 22 1.5. Telomerase in Alzheimer’s and Parkinson’s Disease .......................................................... 23 1.6. PLGA nanoparticles as a drug delivery system ................................................................... 26 2. Rationale ..................................................................................................................... 28 3. Aims and Objectives ..................................................................................................... 29 3.1. Aims .................................................................................................................................. 29 3.2. Objectives ......................................................................................................................... 30 3.2.1. Aim 1 ..................................................................................................................... 30 3.2.2. Aim 2 ..................................................................................................................... 30 3.2.3. Aim 3 ..................................................................................................................... 30 4. Methods and Materials ............................................................................................... 31 4.1. LRP Protein Production and Purification ........................................................................... 31 4.1.1. Plasmid and Protein construct ................................................................................ 31 4.1.2. Transformation ....................................................................................................... 32 4.1.3. Protein expression .................................................................................................. 33 4.1.4. Purification ............................................................................................................. 34 4.1.5. Protein concentration and purity ............................................................................ 35 4.1.6. Protein characterisation .......................................................................................... 37 4.2. LRP encapsulated nanoparticle production and in vitro studies ......................................... 39 4.2.1. LRP encapsulated PLGA Nanoparticle production ............................................... 39 4.2.2. Cell culture ............................................................................................................. 41 4.2.3. pCIneo-moLRP::FLAG stable transfection ........................................................... 41 7 4.2.4. Extraction of protein .............................................................................................. 42 4.2.5. BCA™ Protein Assay ............................................................................................ 42 4.2.6. SDS PAGE ............................................................................................................. 43 4.2.7. Western Blotting .................................................................................................... 43 4.2.8. MTT Assay ............................................................................................................ 44 4.2.9. Telomerase activity detection using quantitative polymerase chain reaction (qPCR) ............................................................................................................................. 45 4.2.10. Statistical Analysis ............................................................................................... 46 5. Results ......................................................................................................................... 47 5.1. LRP::FLAG plasmid in vitro study ....................................................................................... 47 5.2.1. Confirmation of transfection with LRP::FLAG ..................................................... 47 5.1.2. LRP::FLAG overexpression in HEK293 cells increase LRP/LR and PINK1 protein levels .................................................................................................................... 48 5.1.3. LRP::FLAG overexpression in HEK293 cells increases cell viability after MPP+ treatment .......................................................................................................................... 49 5.1.4. LRP::FLAG overexpression in HEK293 cells increases cell viability after TBHP and Aβ42 treatment ......................................................................................................... 51 5.2. LRP Protein Purification ..................................................................................................... 54 5.2.1. Overexpression trials for the optimization of LRP expression .............................. 55 5.2.2. LRP solubilization using Triton X-100 .................................................................. 57 5.2.3. Nickel Immobilised Metal-Ion Affinity Chromatography (IMAC) ....................... 59 5.2.4. Cobalt Immobilised Metal-Ion Affinity Chromatography (IMAC) ...................... 61 5.2.5. LRP purity and concentration through absorbance spectroscopy .......................... 63 5.2.6. Intrinsic Tryptophan Fluorescence ........................................................................ 64 5.2.7. Circular Dichroism Spectropolarimetry ................................................................. 66 5.3. Encapsulation of the 37 kDa LRP in PLGA nanoparticles as a delivery system ..................... 66 5.3.1. LRP PLGA nanoparticle treated cells exhibit an increase in LRP protein levels in HEK293 cells ................................................................................................................... 67 5.3.2. LRP PLGA nanoparticles exhibit a higher cell viability than empty PLGA nanoparticles in treated SH-SY5Y cells .......................................................................... 68 5.3.3. Telomerase activity significantly increases in SH-SY5Y cells after LRP PLGA nanoparticles treatment .................................................................................................... 70 6. Discussion .................................................................................................................... 72 6.1. LRP::FLAG in-vitro study .................................................................................................... 72 6.1.1. LRP::FLAG overexpression increases LRP and PINK1 protein levels ................ 72 6.1.2. LRP::FLAG overexpression rescues cells from MPP+ induced cytotoxicity ........ 73 6.1.3. Overexpression of LRP::FLAG rescues HEK293 cells from Aβ42 and ROS- induced cytotoxicity ......................................................................................................... 74 6.2. The structure of 37 kDa LRP protein .................................................................................. 76 6.2.1. 37 kDa LRP expression and purification ............................................................... 77 8 6.2.2. 37 kDa LRP structural determination .................................................................... 80 6.3. Assessment of LRP PLGA nanoparticles ............................................................................. 81 6.3.1. LRP PLGA nanoparticle treated cells exhibit an increase in LRP protein levels in HEK293 cells ................................................................................................................... 81 6.3.2. LRP PLGA nanoparticles increases viability of SH-SY5Y cells .......................... 82 6.3.3. LRP PLGA nanoparticles significantly increase telomerase activity in SH-SY5Y cells .................................................................................................................................. 83 7. Conclusion ................................................................................................................... 84 8. References ................................................................................................................... 86 List of abbreviations Aß Amyloid Beta AD Alzheimer's Disease Aß40 40 amino acid isoform of amyloid beta Aß42 42 amino acid isoform of amyloid beta APP Amyloid precursor protein BCA Bicinchoninic acid assay BSA Bovine serum albumin DNA Deoxyribonucleic acid CD Circular dichroism CHAPS 3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate CNS Central nervous system CTD C-terminal domain CTRD C-terminal ring domain DMEM Dulbecco's modified eagle medium DMSO Dimethyl sulfoxide 9 EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent assay FBS Foetal bovine serum FT Flow through HRP Horseradish peroxide HEK293 Human embryonic kidney cell line hTERT Human Telomerase Reverse Transcription hTERC Human telomerase RNA component IgG1-iS18 High affinity LRP/LR-specific antibody IMAC Immobilized metal ion affinity chromatography IPTG isopropyl ß-D-1-thiogalactopyranoside ITF Intrinsic tryptophan fluorescence LB Lewy body LRP/LR 37-kDA/67-kDA laminin receptor precursor/high affinity laminin receptor MRE Mean residue ellipticity Mt-DNA Mitochondrial deoxyribonucleic acid MMP Mitochondrial membrane potential MPP Mitochondrial processing peptide MPP+ 1-Methyl-4-phenylpyridinium iodide MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MWM Molecular weight marker 10 OMM Outer mitochondrial membrane PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction PD Parkinson’s disease PMSF Phenylmethylsulphonyl fluoride qPCR Quantitative Polymerase Chain reaction PBS Phosphate buffered saline PBST Phosphate buffered saline with Tween-20 PCA Protocatechuic acid PD Parkinson's disease Pink-1 PTEN induced putative kinase 1 PTEN Phosphatase and tensin homolog PVDF Polyvinylidene fluoride RIPA Radioimmunoprecipitation assay lysis buffer ROS Reactive oxygen species RNA Ribonucleic-acid ROS Reactive oxygen species RPM Revolutions/minute SDS Sodium dodecyl sulphate SNPc Substantia nigra pars compacta 11 SH-SY5Y Neuroblastoma cell line SW Salt wash TEMED Tetramethyl ethylenediamine TERC Telomerase reverse transcriptase RNA component TERT Telomerase reverse transcriptase UBL Ubiquitin-like domain UPS Ubiquitin proteasome system UV Ultraviolet WB Western Blotting List of figures 1. Figure 1: Worldwide projections of Alzheimer’s disease prevalence 2. Figure 2: The two pathways of APP 3. Figure 3: The potential factors which can attribute to the progression of Parkinson’s disease 4. Figure 4: A schematic representation LRP/LR 5. Figure 5: 37 kDa laminin receptor (full length) 6. Figure 6: A schematic representation of the enzyme, telomerase, and two essential domains hTERC & hTERT 7. Figure 7: A schematic representation of the double emulsion technique 8. Figure 8. Transfection of HEK293 with pCIneo-LRP::FLAG plasmid was successful 9. Figure 9. Western blot analysis reveals that LRP::FLAG overexpression increases total LRP/LR and PINK1 protein levels in HEK293 cell 10. Figure 10. LRP::FLAG overexpression rescues HEK293 cells at 500 μM and 750 μM 𝑀𝑃𝑃#treatment 11. Figure 11. LRP::FLAG overexpression enhances HEK293 cell viability at 50 μM, 100 μM and 200 μM TBHP 12 12. Figure 12. LRP::FLAG overexpression enhances HEK293 cell viability after 𝐴𝛽!" induced cytotoxicity 13. Figure 13. Overexpression trials of LRP to identify optimal conditions for soluble protein production 14. Figure 14. Solubilization of overexpressed LRP from the insoluble pellet with Triton X-100 15. Figure 15. Immobilized nickel metal ion affinity chromatography purification of LRP 16. Figure 16. Immobilized cobalt metal ion affinity chromatography purification of LRP 17. Figure 17. Western blot analysis reveals the presence of LRP protein 18. Figure 18. Absorbance spectrum of LRP 19. Figure 19. Intrinsic tryptophane fluorescence spectrum of LRP in the presence and absence of a denaturant 20. Figure 20. Circular dichroism spectrum of LRP 21. Figure 21. Determination of total LRP/LR levels in HEK293 cells after treatment with empty and LRP-encapsulated PLGA nanoparticles 22. Figure 22. LRP nanoparticle treatments increase SH-SY5Y cell viability as compared to empty nanoparticle treatments 23. Figure 23. LRP nanoparticle treatments increases telomerase activity in SH-SY5Y cells List of tables 1. Table 1: Antibodies (primary and secondary) used for western blotting 13 1. Introduction 1.1. Epidemiology of Alzheimer’s disease and Parkinson’s disease Alzheimer’s and Parkinson’s disease are classified as progressive neurodegenerative disorders which are categorized as continuing degeneration of the function and structure of the central and peripheral nervous system (Cummings et al., 2002). In particular, Alzheimer's disease (AD) is considered an incurable and advancing type of cognitive disorder that leads to a decline in memory and cognitive abilities while on the other hand, Parkinson's disease (PD) is identified as a chronic, degenerative nervous system disorder that impacts the motor system, resulting in impaired movement (Bekris et al., 2011; Nussbaum et al., 2003). While AD and PD share mutual causes and effects, the diseases each have different pathological mechanisms which impacts the brain (Nussbaum et al., 2003). Individually, Alzheimer’s disease is known as an age-related disease and the leading form of progressive neurodegenerative diseases. AD is a common form of dementia, making up over 80% of all dementia cases (Goedert et al., 2006). Worldwide, the prevalence of AD is estimated to affect over 55 million patients, with over 10 million patients diagnosed yearly (Li et al., 2022). According to the world Alzheimer’s report there is an estimation of approximately 187 000 patients within South Africa living with dementia, mostly those who are over the age of 60 (Alzheimer’s Disease International, 2015). Additionally, given that both developing and developed countries are rapidly aging, it is estimated that the frequency will double every 20 years (Mayeux et al., 2012). According to the worldwide projections of AD, it is estimated by 2050 that there will be over 139 million AD cases (World Health Organisation, 2022). The rising prevalence of dementia, which causes cognitive decline, imposes a significant physical and economic burden on society (Reitz et al., 2011). Presently, the only available treatments for AD simply assist with symptoms of cognitive changes including memory loss and behaviour changes. One of those treatments are drugs such as cholinesterase inhibitors, these drugs are administered to help improve the neuropsychiatric symptoms of AD such as depression and agitation, however it has been found that these drugs have adverse side effects such as nausea, loss of appetite, and cardiac arrhythmia (Neugroschl et al., 2011). Additionally, treatment plans such as adaptive living and routine habits are used to create a safe and supportive environment for the patients, to assist with the AD sufferers’ well-being and ability to function (Alzheimer’s Disease International, 2015). Palliative treatments have only been used due to poor understanding of the disease's molecular mechanisms. In essence, this highlights the significance of creating new and targeted treatments that can slow down 14 the advancement of AD, ultimately decreasing the demand for care and expense associated with it. Figure 1: The worldwide projections of AD prevalence. Currently there is over 40 million cases of Alzheimer’s disease worldwide, and it is predicted that by 2050 over 100 million cases will be prevalent (Adapted from Brookmeyer et al., 2007). PD, which affects more than 10 million people worldwide, is the second most prevalent neurodegenerative illness (Parkinson’s Foundation, 2019). Worldwide, the prevalence of PD is known to increase with age, affecting 1 in 100 people over the age of 60, and 4 in 100 over the age of 80 (Reeve et al., 2014). Unfortunately, South African and African PD epidemiology and statistics are limited; but even so, PD has been found to be most common in people of Northern European origin (De Lau et al., 2006). Symptoms of PD progress gradually, starting with slight tremors in the hand, eventually leading to the onset of body stiffness and slow movement (Sveinbjornsdottir et al., 2016). As of now, there are palliative treatments available for PD, including care homes and occupational therapy, since a curative treatment has not yet been established (Singh et al., 2007). Furthermore, a commonly used drug which assists in symptom control of PD is levodopa, which is a dopamine precursor, known as an effective dopamine enhancer agent. Studies have found that levodopa does in fact improve symptoms associated with the disease; however it has adverse side effects from 15 prolonged usage such nausea and vomiting (Tarakad & Jankovic, 2017). Additionally, non- pharmacological treatments are available which aims at treating the symptoms which arise from PD, these include functional stereotaxic neurosurgery, physiotherapy, and dietary changes (Oertel & Schultz, 2016). These treatment options are not curative as it can only alleviate symptoms of the disease or impede the pathogenesis of the disease. In conclusion, there is a pressing demand for cost-effective and non-palliative therapies that can impede the advancement of AD and PD by preventing the degeneration of neurons with minimal or no adverse effects. 1.2. Alzheimer’s disease pathology and molecular mechanisms There are two fundamental neuropathological features distinguish AD; (a) the neurotoxic amyloid beta-42 (Aβ!") peptide accumulation which results in the A𝛽 plaque formation, extracellularly (Xiao et al., 2015) and (b) the hyper-phosphorylated tau aggregation of which produces intracellular neurofibrillary tangles (NFT), each of which result in the degradation of neural networks and cerebral tissue (Choi et al., 2014). Collectively, these factors cause the characteristic impairments of the behavioural and cognitive functions in those patients who suffer from AD (Serrano-Pozo et al., 2011). Amyloid plaques are composed of the 4 kDa Aβ peptides, more specifically an isoform Aβ!" (42 amino acid (aa)) product of proteolytic cleavage (Caetano et al., 2011). The Aβ peptide is generated through the cascade of the amyloidogenic pathway where the amyloid protein precursor (APP) is cleaved by 𝛾-secretase and β-secretase (Choi et al., 2011). Firstly, the cleavage of APP is by β-secretase at the N-terminus releasing the secreted APPβ (sAPPβ) fragment. The process of Aβ shedding involves the cleavage of the C-terminal region of APP by γ-secretase, which results Aβ fragments being released into the extracellular space (Caetano et al., 2011) (Figure 2). This process results in the formation of the Aβ oligomers, such as the isomers Aβ!$ and Aβ!", each which are produced according to a specific cleavage pattern of APP (Selkoe, 1996). In normal physiological circumstances, the processing of APP occurs via the non-amyloidogenic pathway. In this pathway, α-secretase initiates cleavage of APP, producing APPsα (which inhibits the formation of Aβ). This is followed by γ-secretase cleavage, resulting in the creation of AICD (Figure 2). While the exact physiological roles of these substances are not entirely clear, some evidence suggests that APPsα functions in growth promotion and brain development. Furthermore, research has indicated that APPsα 16 may facilitate neurite growth, contribute to pro-survival pathways and enhance memory formation (Chow et al., 2010). In contrast, the advancement of AD happens when the non- amyloidogenic pathway is disturbed, or when the degradation of Aβ!" is reduced, leading to the formation of clumps that create neurotoxic plaques (Jarrett et al., 1993). The accumulation of the Aβ!" peptides are favoured because they have a higher propensity to aggregate and is known to be more cytotoxic than the Aβ!" isomer (Verdier & Penke, 2004). The accumulation of Aβ plaques enhances lipid oxidation and thereby oxidative stress which impairs the mitochondrial DNA (mtDNA) and induces mitochondrial dysfunction (Choi et al., 2014). Furthermore, the accumulation of the Aβ!" peptides lead to the production of ROS initiating the intracellular oxidative damage potential (Sayre et al., 2000; Serrano-Pozo et al., 2011). This leads to the ROS mediated mtDNA damage, mitochondrial dysfunction, and cytotoxicity (Mattson, 1997). Consequently, the formation of Aβ plaques induces oxidative stress thereby increasing cytotoxicity, senescence (permeant cell cycle arrest) and cell apoptosis (Stebbins et al., 2009). Figure 2: The two pathways of APP. APP can be metabolised by the non-amyloidogenic and the amyloidogenic pathway. Under normal circumstances, APP is cleaved by α-secretase and by γ-secretase releasing sAPPα and p3. In the amyloidogenic pathway, the APP is cleaved by β-secretase and γ-secretase producing APPβ and AICD (Adapted from Menting and Claassen, 2014). On the other hand, the accumulation of NFTs is also associated with AD pathology, which consists of hyperphosphorylated and misfolded tau proteins (Choi et al., 2014). These 17 proteins have been found to increase the production of ROS, which results in neuronal dysfunction (Caetano et al., 2011). Essentially, it is this relationship between Aβ, NFTs and ROS which mitigate mitochondrial dysfunction and ROS production (Avlia, 2006). Ultimately, these hallmarks enhance the accumulation and thus the oxidative damage potential of ROS which consequently induces senescence and apoptosis, thereby promoting the formation of cerebral lesions that further the progression of AD (Avlia, 2006). 1.3. Parkinson’s disease pathology and molecular mechanisms AD and PD are both defined as protein misfolding diseases and are characterised by the aggregates of protein deposits in the brain (Pimentel et al., 2012). Specifically, PD is defined through the impairment of the dopamine-producing neurons which are found in the substantia nigra pars compacta (SNPc). The loss of these neurons are ultimately responsible for the altered motor behaviour of PD patients, which are resultant of organ shrinkage and the accumulation of Lewy bodies (Dauer & Przedborski, 2003). The progression of SNPc neural death is yet to be fully understood however is thought to be compromised by oxidative stress (OS), mitochondrial dysfunction, neuro-inflammation, and genetic alterations (Paillard et al., 2015) (Figure 3). 18 Figure 3: The potential factors which can attribute to the progression of Parkinson’s disease. Potential causes which may cause PD is environmental factors inducing oxidative stress, mitochondrial dysfunction and genetic mutations which contribute to the neural death of the SNPc dopaminergic neurons. (Paillard et al., 2015) Although the majority of PD cases occur intermittently, there are several cases which have linked gene mutations to the disease. It has been established that familial PD is caused by mutations in PD related genes such as α-synuclein and parkin (Yasuda & Mochizuki, 2010). Accordingly, α-synuclein is a presynaptic neuronal protein encoded by SNCA, and mutations in this gene form aggregations of α-synuclein which is a constituent of Lewy bodies (Moraitou et al., 2011). Furthermore, α-synuclein is known to be intrinsically unstable and can natively unfold in aqueous solution, supporting that the protein has a high propensity for aggregation (Meade et al., 2019). The α-synuclein protein is composed of 140 amino acids and can be divided into three distinct domains the N-terminal domain (NTD), the hydrophobic core region - NAC (non Aβ-component) and the C-terminal domain (CTD) (Rodriguez et al., 2015; Emamzadeh, 2016). The hydrophobic core domain of α-synuclein generates cross-β structures, which promote the formation of fibril aggregation (Meade et al., 19 2019). Conversely, the acidic C-terminal end of α-synuclein interacts with the NAC region and is responsible for suppressing α-synuclein fibril aggregation (Emamzadeh, 2016). Mutations in the α-synuclein gene are known to generate the fibrillar form of α-synuclein, which is the primary component of Lewy bodies (LB). It is the fibrillar aggregation of Lewy bodies which disrupt the neurotransmitter transmission of dopamine which allows the onset of parkinsonism (Poulopoulos et al., 2012). Another protein which furthers the pathogenesis of PD is parkin which is a 465-residue ligase (Mizuno et al., 2001). In normal circumstances, parkin plays a role in the covalent labelling of molecules with ubiquitin, which then undergo degradation in lysosomes and proteasomes (Song et al., 2016; Seirafi, 2015). The parkin protein can be divided into three domains, the N-terminal ubiquitin like domain (UBL), the in between RING finger domain (IBR), and the C-terminal RING domain (Van Coelln et al, 2004). Furthermore, parkin is responsible for the ubiquitin-mediated degradation of O-glycosylated forms of α-synuclein, and mutations in parkin have been linked to the aggregation of α-synuclein and the formation of Lewy bodies, which eventually contributes to the progression of Parkinson's disease (Shimura et al., 2000). Furthermore, the overexpression of parkin has shown to protect cells from α-synuclein induced aggregation. Furthermore, parkin has been shown to regulate mitochondrial integrity by interacting with the PTEN-induced putative kinase 1 (PINK1) protein (Bingol et al., 2016). PINK1 is found on the mitochondrial surface, where it recognizes mitochondrial dysfunction and recruits’ parkin from the cytoplasm to the mitochondria, thereby inducing mitophagy (Miklya et al., 2014). As a result, parkin is an important protein with several neuroprotective functions, two of which are the regulation of dysfunctional mitochondria and the prevention of α-synuclein aggregation (Dawson & Dawson, 2010) 1.4. 37 kDa Laminin Receptor Precursor/ 67 kDa high affinity Laminin Receptor (LRP/LR) Neurodegenerative diseases and cancer have been found to associated with the 37 kDa laminin receptor precursor/67 kDa high affinity laminin receptor (LRP/LR) (Da Costa Dias et al., 2013). LRP/LR or namely, LamR is a type II transmembrane receptor containing two functional domains; extracellular C terminal domain and the globular intracellular N terminal (Zidane et al., 2012). It is mostly located in the area of the plasma membrane known as the lipid raft permitting a considerable number of interactions with the proteins found in the 20 extracellular matrix, but also has been found within the nucleus and cytoplasm (Mbazima et al., 2010). As a result, due to the interactions between LRP/LR receptors in the areas surrounding cells, it attaches to various molecules, including laminin-1, elastin, heparin sulphate proteoglycans (HSPGs), and cellular prion proteins (PrP% and PrP&%) (Gauzynski et al., 2006), and the AD-causing agent Aβ!" (Jovanovic et al., 2013). Specifically, composed of 295 amino acid residues, the receptor contains multiple binding sites including two high affinity binding sites for laminin 1 (Otgaar et al., 2017) (Fig. 4). Under normal conditions, LRP/LR plays part in cell growth, differentiation, migration, and adhesion which are vital for survival of cells (DiGiacomo & Meruelo, 2015; Omar et al., 2012). Figure 4: A schematic representation LRP/LR. There are three sections to the receptor, namely the cytosolic N-terminal domain (coloured blue), the transmembrane domain (coloured red), and the ECM C-terminal domain (coloured purple). The C-terminus region of the receptor comprises binding sites for various substrates, including 𝑃𝑟𝑃' and a peptide G- binding site. The latter serves as a binding location for laminin-1 and heparin. Both laminin- 1 and 𝑃𝑟𝑃' bind to the 209-229 aa site, while the IgG binding domain is found between the 272-280 aa region. (Image adopted from Jonanovic et al., 2015) 1.4.1. Protein structure of LRP/LR The first identified receptor of laminin was the non-integrin 67 kDa laminin receptor (LR) identified through laminin-1 binding (Lesot et al. 1983). The 67 kDa LR is assumed to arise 21 from the 37 kDa LR via post translational modifications, however, this is still poorly understood (Landowski et al., 1995). Previous efforts to sequence the 37 kDa LRP protein has been met with limited success attributed to the restricted capability to extract the protein in adequate amounts and with a high degree of purity (Wewer et al., 1986). Furthermore, the amino acid composition of purified 67 kDa LR has been resolved, but efforts to further sequence the protein is absent from literature. LRP has been discovered to be targeted to the membrane at the cell surface by fatty acid acylation, and exists as both a monomer (37 kDa) and a dimer (67 kDa) (Butò et al., 1998; Fatehullah et al., 2010). Though they are both reported to bind laminin, the homodimeric and heterodimeric states of LRP have not yet been thoroughly resolved (Jamieson et al., 2008). Despite the fact that the characterization of the interactions between LRP and laminin has not been definitively determined, some regions of the protein, such as residues 161-180 (peptide G) and residues 205-229, have been suggested as binding sites for laminin- (Castronovo et al., 1991; Landowski et al., 1995). Figure 5: 37 kDa laminin receptor (full length). Model showing the laminin binding sites: Peptide-G binding site (green), peptide 205-229 (blue) and transmembrane (TM) region (red) (Adapted from DiGiacomo et al., 2015) Analysing the structure of LRP/LR will help comprehend how the receptor associates with its binding partners, and it will support the development of treatments that can imitate or prevent 22 LRP/LR interactions in situations related to viral infections, neurodegenerative diseases, and cancer. The aa 9–205 were resolved in the crystal structure of the 37 kDa human LR (LAMR, 1-220) at resolution 2.15 Å (Jamieson et al., 2008). According to the study, the globular NTD (aa 1-209) and composed of a central β-sheet surrounded by 𝛼-helices, whereas the CTD (aa 210-295) has been found to be intrinsically disordered as seen in far UV CD (Jameison et al., 2008; Schlessinger et al., 2009). The CTD consists of aspartic acid and glutamic residues, giving it a highly negative charge, particularly found in the 205-229 amino acid region which is assumed to be a laminin binding site (Jamieson et al., 2008). Taken all together, the function and structure of LRP plays a definitive role in various disease pathogenesis, including AD. 1.4.2. LRP/LR role in diseases It is especially important to note that LRP/LR interactions are linked many forms of diseases, including neurodegenerative diseases (prion disorders and AD), viral infections and various forms of cancer (Leucht et al., 2003; Jovanovic et al., 2013; Jovanovic et al., 2014; Naidoo et al., 2015; Weiss et al., 2017). LRP/LR has been discovered to be significantly overexpressed in cancer, which results in increased adhesion and invasion leading to metastasis. Additionally, it inhibits apoptosis and triggers angiogenesis, as evidenced by various studies (Zuber et al., 2008; Omar et al., 2012; Moodley and Weiss., 2013; Khumalo et al., 2013; Khusal et al., 2013; Chetty et al., 2014; Rebelo et al., 2016; Vania et al., 2016). Moreover, additional research has shown that LRP/LR plays a role in the aging process by interacting with telomerase, an enzyme that is recognized for hindering the aging process (Otgaar et al., 2017). Subsequently, studies have revealed overexpression LRP/LR overexpression enhances telomerase activity and thus, decrease senescence markers (Otgaar et al., 2017). Initially, LRP/LR is found to be involved in AD, through cell surface interaction with Aβ as well as the enhancement of the Aβ shedding, due to the interaction with APP, γ-secretase and β-secretase which are AD-related proteins (Jovanovic et al., 2014). Studies have exhibited that LRP/LR and Aβ co-localise and that a receptor for Aβ!" peptides could be LRP/LR (Da Costa Dias et al., 2013). Studies have revealed that when IgG1-iS18 antibody and short hairpin RNA (shRNA) technologies were used to mediated LRP/LR blockade and downregulation, respectively, it was found that there was a significant reduction in Aβ shedding and cytotoxicity (Jovanovic et al., 2013; Da Costa Dias et al., 2013). Therefore, it 23 was established that LRP/LR is involved in the pathogenesis of AD which endorsed further study. Additionally, it was noticed that the interaction between LRP/LR and Aβ!" on the surface of cells suppressed cell growth and caused apoptosis. This was demonstrated by the detection of apoptotic fragments after exposure to significant quantities of Aβ!" (Da Costa Dias et al., 2014). Subsequently, it was due to the involvement of LRP/LR in the Aβ!" internalization which led to the accumulation of intercellular Aβ!" and ultimately Aβ!"- related cytotoxicity (Jovanovic et al., 2014; Da Costa Dias et al., 2013). Furthermore, observations indicated that LRP/LR co-localises with AD related proteins (APP, γ-secretase and β-secretase) both inside and at the cell surface. However, treatment with the shRNA and IgG1-iS18 did not change APP, γ-secretase and β-secretase expression but rather reduced sAPPβ levels and release of the Aβ extracellularly. Interestingly, there was an indirect interaction between LRP/LR and β-secretase and a direct interaction with the catalytic subunit of 𝛾-secretase which initiated APP cleavage thus releasing sAPPβ. Therefore, the downregulation of LRP/LR demonstrated β- and γ-secretase activity impediment thereby reducing sAPPβ and ultimately reducing Aβ shedding (Jovanovic et al, 2013). Upon further investigation, Aβ shedding and intracellular Aβ!" levels were significantly reduced in cells overexpressing LRP::FLAG and thus rescues cells from cytotoxicity mediated by Aβ (Bignoux et al., 2019) The study showed that the overexpression of LRP::FLAG increased hTERT expression which increased telomerase activity while decreasing intracellular Aβ!" levels and shedding (Bignoux et al., 2019). Cuttler et al demonstrated that LRP/LR and tau coexist in the perinuclear section of cells and verified that LRP/LR interacts directly with tau. The study also showed that overexpressing LRP::FLAG caused a reduction in phosphorylated tau levels, as well as a reduced PrP% levels, a tauopathy linked protein (Cuttler et al., 2020). 1.5. Telomerase in Alzheimer’s and Parkinson’s Disease As mentioned earlier, LRP/LR plays a crucial role in age-related illnesses, and recent studies demonstrate that LRP/LR supports telomerase activity in both cancer and aging (Naidoo et al., 2015; Otgaar et al., 2017). Extensive research has established that cell viability is preserved through the telomerase-mediated maintenance of telomeres (Shay et al., 2004). Telomeres are a segment of genetic material consisting of repeating sequence (TTAGGG) that are found at the ends of chromosomes. Their primary function is to protect the DNA 24 against fusion and deterioration of chromosomes. Telomeres are sustained by telomerase, a ribonucleoprotein enzyme that adds telomeric repeats to the ends of telomeres, enabling their maintenance (Cong et al., 2002). The human telomerase comprises two units, the telomerase RNA component (TERC) subunit and the telomerase reverse transcriptase (TERT) subunit, that work together to produce telomeres in a 3' to 5' direction. (Nakamura & Cech, 1998) (Fig. 6). The activity of telomerase is crucial in both cellular senescence and the process of immortalization. Therefore, it holds great importance in both the aging process and the cancerous state (Shay & Wright, 2005). Cellular senescence occurs from cellular stresses such as telomere shortening, instability of the genome and mitochondrial disruption, which consequently disrupt tissue due to the replicative and regenerative limit of the cells (Molofksy et al., 2006). While telomerase primarily functions in extending telomeres, its catalytic subunit, hTERT, possesses additional cellular roles that are essential for maintaining cell viability (Ahmed et al., 2008). Firstly, proteins which are involved in the Wnt pathway are activated by hTERT, regulating transcription and promoting cell replication (Cong & Shay, 2008). Additionally, hTERT plays a crucial role in protecting mitochondria (Ahmed et al., 2008). It has been found that hTERT contains a signal peptide (20 aa) on its N terminal, which facilitates its mitochondrial translocation in response to increased ROS levels (Haendeler et al., 2009). Once in the mitochondria, the role that hTERT has is preventing cytochrome C release (inhibition of apoptosis) and prevents oxidative damage in mtDNA (Santos et al., 2006; Zhang et al., 2003). 25 Figure 6: A schematic representation of the enzyme, telomerase, and two essential domains hTERC & hTERT. Telomere elongation occurs when the TERT and TERC subunits catalyse the addition of TTAGGG repeats to the 3' end of the chromosome. (Adopted from Townley et al., 2014) Supporting evidence suggests that AD pathogenesis may be implicated by telomerase, which consequently results in the presence of short telomere lengths in the neural and T cells of AD sufferers (Franco et al., 2006). Furthermore, it has been shown that ROS preferably binds to the guanine rich region of telomeric DNA resulting in telomere instability and reducing the binding of telomerase to telomeres ultimately leading to telomere shortening (Thomas et al., 2008; Lee et al., 2017). Additionally, in vitro studies have shown that Aβ!" inhibits telomerase activity, through direct binding of Aβ-peptides to the telomeric complex of DNA- RNA (Wang et al., 2015). Therefore, this suggests that there is a conflicting association between Aβ and telomerase in nerve cells, and that telomere reduction plays a role in the development of Alzheimer's disease in individuals. Interestingly, it has been discovered that hTERT and LRP/LR interact for allowing cell survival. LRP/LR and hTERT have been observed to interact and co-localize in perinuclear and at the cell surface. It has been found that downregulation of LRP/LR using small interfering RNAs (siRNAs) leads to a decrease in telomerase activity, and on the other hand, overexpression of LRP::FLAG is associated with an increase in hTERT expression, telomerase activity, and telomere lengthening, which ultimately protects cells from Aβ-induced apoptosis (Otgaar et 26 al., 2017). These findings suggest that increasing TERT and telomerase activity levels through LRP::FLAG overexpression could be a potential therapeutic strategy for AD. The extent to which TERT is involved in PD has not been extensively researched, however, a recent study by Wan et al investigated the role of TERT expression in transgenic PD mice models. The results showed that when treated with telomerase activators, the total aggregated α-synuclein levels were significantly reduced in the hippocampus and neocortex of mice. This reduction was attributed to improved markers of autophagy, indicating better degradation of toxic α-synuclein. The study also concluded that increased TERT expression was linked to decreased α-synuclein protein levels, through activating autophagy thereby reducing the impairment degradation mechanisms during progression of the disease (Wan et al., 2021). Furthermore, it was discovered that when cells were treated with a PD inducer, MPP, the overexpression of LRP::FLAG rescued cells from the MPP+ induced cytotoxicity (Burns et al., in preparation) 1.6. PLGA nanoparticles as a drug delivery system As previously stated, numerous studies have shown that the use of LRP::FLAG, specifically the in vitro overexpression of LRP/LR, impedes the pathogenesis of neurodegenerative diseases. For that reason, it is required to assess the effect of LRP/LR overexpression in vivo. Although the mammalian expression system offers suitable protein folding and post translational modifications, there are major limitations to using this method, including the high cost of production due to the slow cell growth, expensive media and transfection reagents, but more specifically delivery systems inability to cross the blood brain barrier (BBB) (Pardridge et al., 2012). The BBB protects the central nervous system (CNS) from substances and most biologics which contributes to the lack of progress in the treatment of CNS diseases including AD and PD (Li et al., 2013). Therefore, a promising alternative is the use of polymeric nanoparticles as a delivery vehicle for drugs to the target tissue while regulating the rate and location of drug release. A promising alternative to the regular drug delivery systems is the use of polymeric encapsulated nanoparticles. These serve as nanocarriers for macromolecular drugs. In recent years, nanoparticles have gained popularity for their applications in the field of medicine and molecular biology as a delivery system for small hydrophilic, hydrophobic drugs, and 27 biological macromolecules (Danhier et al., 2012). These nanoparticles have four main benefits. First of all, cell membranes and the BBB can be bypassed by using nanoparticles with diameters between 1 to 1000 nm (Irving, 2007). Secondly, encapsulation allows for the protection from chemical and enzymatic breakdown, thus, extending their in vivo half-life (Mudshinge et al., 2011). The particles’ biodegradability, which enables a controlled release of the drug, is the third benefit. The fourth benefit is that the particles’ surfaces can be modified to enable cell, tissue, and organ specificity, such as the addition of cell-specific ligands (Abhilash, 2010). Thus, a potential option for the encapsulation of the LRP protein are the biodegradable polymer matrix nanoparticles made up of polylactic-co-glycolic-acid (PLGA). PLGA encompass all of the advantages mentioned above as they are FDA approved, highly stable and allow for controlled drug release to a disease localised site (Basarkar & Singh, 2007). PLGA has become increasingly popular due to its ability to break down into lactic acid and glycolic acid, which are natural metabolites that can be easily processed by the body via the tricarboxylic acid (TCA) cycle (Krebs cycle) and released water and carbon dioxide, therefore resulting in a minimal systemic toxicity (Semete et al., 2010). There are many methods of nanoparticle preparation, and depending on the method the structural organisation differs. The drug is either entrapped within the core of the nano capsule or entrapped on the surface of the nanosphere matrix (Barratt, 2000; Couvrar et al., 1995). The primary technique employed to create PLGA nanoparticles is the process of single or double-emulsion-solvent evaporation. In the single-emulsion method, an oil in water (o/w) emulsion is used for water insoluble (hydrophobic) drugs such as steroids, whereas, in the double emulsion method a water in oil in water (w/o/w) method is used and suited for water soluble (hydrophilic) drugs such as proteins (Garti et al.,1998) (Figure 7). Briefly, PLGA is added into an organic phase which is emulsified with water, the drug is either directly added to the oil phase (hydrophobic drugs) or can be initially emulsified with the polymer (hydrophilic drugs) before the formation of nanoparticles (Perez-Moral et al., 2014). 28 Figure 7: A schematic representation of the double emulsion technique (Adapted from Iqbal et al., 2015) Ultimately, the PLGA nanoparticles used as a delivery vehicle for LRP, would be prepared by the double emulsion method and would require nanoparticles to be smooth, round, hollow and within 250-500 nm in size. 2. Rationale AD and PD progressive neurodegenerative disorders affecting globally over 50 million and 10 million patients, respectively. Due to the high occurrence and economic burden of these diseases there is a need for non-palliative therapeutic strategies, as only palliative treatments are available. It has been discovered that LRP/LR contributes to the prevention of AD and PD. In vitro studies have demonstrated that the overexpression of LRP::FLAG impedes Aβ shedding, reduces intracellular Aβ!" levels thus rescuing cells from Aβ-mediated cytotoxicity. Furthermore, the overexpression of LRP::FLAG has been found to increase hTERT expression which concurrently decreased telomerase activity while decreasing intracellular Aβ!" levels and shedding. Cuttler et al. demonstrated that LRP/LR and tau co- localize and interact directly. They also found that overexpression of LRP:FLAG led to a decrease in total and phosphorylated tau levels, as well as a decrease in PrP% levels, a protein related to tauopathies. Within PD, in vitro studies demonstrated that LRP/LR plays a protective role in PD, likely through the degradation of α-synuclein and by increasing cell viability and the MMP of cells treated with the PD-inducer, MPP+. As demonstrated, the use of LRP::FLAG, specifically the in vitro overexpression of LRP/LR, impedes the progression 29 of these neurodegenerative diseases. For that reason, it is required to assess the effect of LRP/LR overexpression in vivo. Therefore, the present study was divided into three parts, with the initial part focusing on exploring the impact of LRP::FLAG overexpression in HEK293 cells. This was done to determine whether the overexpression of LRP plays an important role in both AD and PD, and if targeting LRP/LR can provide a novel and potentially effective strategy for these diseases. The overexpression of LRP::FLAG assessed LRP and PINK1 protein levels by western blotting, as well as the resultant effect on cell viability after treatment with MPP+, TBHP, and Aβ!" by MTT analysis. However, the translation of this research to an in vivo investigation required isolated 37 kDa LRP and an effective delivery system. Thus, the second part of the study focused on successfully overexpressing and purifying the 37 kDa (1- 295 residue) LR protein, to produce large amount of the isolated therapeutic protein for PLGA nanoparticle encapsulation. This also involved the structural characterisation of the 37 kDa LRP through ITF and far UV CD which assessed the structural integrity of the protein. Lastly, the third part of the study focused on assessing the use of PLGA nanoparticles as a potential delivery system for the LRP-encapsulated nanoparticles. This involved investigating the effect of the empty and LRP encapsulated nanoparticles on cell viability, LRP levels, and telomerase activity in HEK293 and SH-SY5Y cells. 3. Aims and Objectives 3.1. Aims 3.1.1. Aim 1: To assess the effect of LRP::FLAG in Parkinson's and Alzheimer’s disease cell culture models 3.1.2. Aim 2: To purify and characterise LRP protein for the downstream application of encapsulating in PLGA nanoparticles 3.1.3. Aim 3: To investigate the effect of treatment with exogenous LRP encapsulated in PLGA nanoparticles in Parkinson’s and Alzheimer’s disease cell culture models 30 3.2. Objectives 3.2.1. Aim 1 a. To overexpress LRP::FLAG and to determine whether LRP::FLAG overexpression affects LRP and PINK-1 protein levels in HEK293 cells b. To investigate the effect of LRP::FLAG overexpression on cell viability in Alzheimer’s like-state Aβ42 and TBHP treated HEK293 cells c. To investigate the effect of LRP::FLAG overexpression on cell viability in Parkinson’s-like state MPP+ treated HEK293 cells 3.2.2. Aim 2 a. To optimise the overexpression of hLRP protein in T7 Express pLysS competent E.coli cells b. To scale-up hLRP solubilisation using the 0.5% Triton X-100 detergent c. To purify the hLRP protein using immobilized metal ion affinity chromatography d. To characterise the structure of hLRP through intrinsic tryptophan fluorescence and far UV circular dichroism 3.2.3. Aim 3 a. To optimise the production of empty and LRP encapsulated PLGA nanoparticles b. To assess the effect of empty and LRP (1-5% (v/v)) encapsulated PLGA nanoparticles on cytotoxicity and LRP protein levels in SH-SY5Y cells c. To investigate the effect of treatment with empty and LRP (1-10% (v/v)) encapsulated nanoparticles on cell viability in SH-SY5Y cells d. To assess the effect of empty and LRP (1-5% (v/v)) encapsulated PLGA nanoparticles on telomerase activity in SH-SY5Y cells 31 4. Methods and Materials 4.1. LRP Protein Production and Purification This study was performed using the LRP protein (295 amino acid residues) containing both laminin binding sites. A pure and soluble form of the protein was required to perform the downstream experiments of the study. 4.1.1. Plasmid and Protein construct The gene sequence of the full length of the LRP protein (residues 1-295) was constructed and inserted into a pET-11a plasmid (GenScript, USA). The LRP gene sequence contained a Hexahistidine-tag (His-tag). The His-tag were used to facilitate the downstream purification by immobilized metal-ion affinity chromatography (IMAC). Plasmids are circular, dsDNA molecules found naturally in yeast and bacteria that replicate independently from the cell's DNA. Multiple copies of the plasmid are produced during DNA replication, which occurs prior to each cell division, ensuring continuous propagation through successive host cell generations (Esser et al., 1986). Plasmids are inexpensive, easy to use, and stable, making them ideal for producing a large amount of protein in a short period of time. Plasmids’ circular nature may limit insert size and make them unsuitable for longer sequences (Rosano and Ceccarelli, 2014). The pET-11a plasmid used was 5.7 kb long, while the gene that was inserted was 295 bp long, which made it appropriate for the study. The T7 Express pLysS Competent E.coli cells (New England Biolabs, USA) were transformed using the pET-11a LRP plasmid. Using the T7 lac promoter in bacteria, this vector expression system generates a high level of heterologous protein (Briand et al., 2016). The T7lac promoter cassette includes a T7 promoter and a T7 terminator, as well as the lac operon and a ribosomal binding site. The T7 promoter initiates a high level of gene expression if the T7 RNA is present. The pET-11a plasmid also includes a pBR322 replication origin, which allows for a high plasmid copy number while also reducing leaky protein expression in the absence of an inducer. The AmpR gene provides bacteria with ampicillin resistance by encoding the 𝛽-lactamase enzyme, which is required to degrade the 𝛽-lactam ring in ampicillin, allowing for bacterial selection after transformation (Neu, 1969). 32 Plasmids can replicate independently within the host cell; however, the host cell replication machinery, polymerases, are required to promote plasmid replication. Bacteria are frequently used as plasmid expression vectors because they replicate quickly, resulting in the production of many plasmids and thus increased protein production (Rosano et al., 2016). Furthermore, the T7 RNA polymerase required to initiate transcription of the T7 promoter is coded for in the host cell by the T7 RNA polymerase gene under the control of the LacUV6 promoter, which is induced by a non-hydrolysable allolactose analogue, isopropyl 𝛽-d-1- thiogalactopyranoside (IPTG) (Gomes, 2020). Additionally, the lac repressor protein is encoded by the Lacl gene, and functions to bind and block expression of the lacO gene, preventing leaky basal expression of LRP in the absence of IPTG. Furthermore, the lac repressor protein binds to and inhibits the lacUV5 promoter in the host-cell thereby blocking the transcription of the T7 RNA polymerase in the absence of IPTG. Once IPTG is added, it aids in blocking the inhibitory action of the lac repressor, thereby promoting the expression of the T7 RNA polymerase in the host-cell, as well as preventing lac repressor inhibition in the gene of interest within the plasmid (Tan et al., 2012). 4.1.2. Transformation Using the heat shock method, the pET-11a plasmid, containing the LRP insert was used to transform the T7 Express Competent E.coli cells. Initially, the competent E.coli cells was thawed for 15 minutes on ice. Transformation was initiated by adding 2 µl of the plasmid (50 ng/µl) which was incubated with 50 µl of cells and was placed on ice for 30 minutes allowing for stabilization of the lipid membranes in the cells. Thereafter, cells were heat shocked for at 42°𝐶 for 45 seconds and incubated for 5 minutes on ice. When cells are exposed to higher temperatures, the kinetic energy of the molecules in the fluid membrane increases, destabilizing it. This makes the cells more permeable to the plasmid. Therefore, lowering the temperature after the cells were heat shocked allows for the cells to recover. The pellet was resuspended in 1ml of prewarmed (37°𝐶) sterile Super Optimal broth with Catabolite repression (SOC) media (0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 10 mM MgCl", 20 mM glucose, and 2.5 mM KCl). SOC media allows for cell recovery following the heat shock-induced stress. Thereafter, the transformed cells were grown for 1 hour at 37°𝐶, while shaking at 230 rpm for aeration. 33 Following cell growth, the cells were spread onto freshly prepared sterile Luria Broth (LB)- agar (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1.5% (w/v) agar, and 1% (w/v) NaCl) plates, supplemented with 0.1 mg/ml ampicillin (Melford, UK) used to select for a successful transformation. Incubation of the LB agar plates were at 37°𝐶 for 19 hours. To ensure there were no contamination, spread plates of SOC medium, LB agar and untransformed E.coli cells were prepared. Once cells were transformed, an isolated colony was picked and used to inoculate into 100 ml of fresh sterile 2x Yeast extract-Tryptone (YT) media (1% (w/v) yeast extract, 1.6% (w/v) tryptone, and 0.5% (w/v) NaCl) containing 0.1 mg/mL ampicillin. Supplementation of ampicillin is added for the prevention of growth of any untransformed cells, as well as for contamination prevention. Additionally, an isolated small colony was picked to ensure cells were genetically identical. The cell culture was then grown at 37°𝐶 for 16 hours, while shaking at 180-190 rpm to ensure aeration. Glycerol stocks (1 ml) were prepared by mixing this culture with 80% sterile glycerol (v/v) at 1:1 ratio. The stocks were snap frozen in liquid nitrogen and placed at -80°𝐶 for storage. The culture was also used to isolate the pET-11a plasmid, containing the LRP insert using GeneJET plasmid mini-prep kit (Thermo Fisher Scientific, USA), as per suggested protocol. The purpose of this kit is to efficiently and affordably extract plasmid DNA from recombinant E.coli cultures in a small quantity. The kit utilizes a unique spin column, which is composed of a silica-based membrane technology. The plasmids (100 ng/µl) were sent for sequencing (Inqaba Biotechnical Industries, RSA) to ensure LRP gene integrity. 4.1.3. Protein expression Following bacterial transformation with the pET-11a plasmid containing the LRP insert, the 37 kDa LRP protein was produced by heterologous protein overexpression in T7 Express pLysS competent E.coli cells. This was performed to ensure that enough protein was produced for the downstream applications of this project. Glycerol stocks (1 ml ) were used in a 1 in 1000 dilution to inoculate freshly prepared, sterile 2x YT media which contained 0.1 mg/mL ampicillin. The prepared culture was grown at 37°𝐶 overnight, while shaking at 180- 195 rpm which allowed for aeration and growth. Thereafter, a large volume of freshly prepared 2x YT media with 0.1 mg/ml ampicillin was inoculated in a 1 in 50 dilution of the overnight culture. Thereafter, cells were grown until an 𝑂𝐷)$$ of 0.6 AU was reached, which 34 indicated that the E.coli cells were in the exponential phase of growth, which is optimal for the induction of heterologous expression. Once the 𝑂𝐷)$$ was obtained, the culture was cold- shocked on ice for 30 minutes. Thereafter, overexpression was induced using 0.3 mM IPTG (Melford, UK). IPTG effectively turns the lacUV5 promoter on which induces protein expression. Cold shocking the cells allowed for the rate of protein synthesis to be lowered, resulting in soluble and functionally folded protein. The cells were then incubated at 20°𝐶 for 20 hours, while shaking a 230 rpm. The temperature was reduced to 20°𝐶 as it decreased the rate of protein folding, thereby reducing the possibility of obtaining nonfunctional and misfolded protein. Following incubation, cells were pelleted by centrifugation (5000 xg for 30 minutes at 4°𝐶) and resuspended in 50 ml of equilibration buffer. Resuspended cell lysates were stored at -20°𝐶. 4.1.4. Purification Cell lysis was performed to successfully isolate protein from the cell lysate. The cell lysates were thawed up at 20°𝐶, after which, 0.1 mg/ml lysozyme (Sigma-Aldrich, USA) and 1 mM phenylmethylsulphonyl fluoride (PMSF) (Roche, Germany) were added. Lysozyme was used to cleave the peptidoglycan component of gram-positive cell walls, allowing them to rupture. PMSF is a serine protease inhibitor that is commonly used to prevent protease-mediated proteolytic degradation of overexpressed proteins. After adding lysozyme and PMSF, the mixture was incubated and slowly inverted for 30 minutes at 20°𝐶. Thereafter, the lysate was incubated with 0.01 mg/ml DNase 1 (Merk, Germany) at 20°𝐶 for 30 minutes. DNase 1 was added to the bacterial DNA fragments to prevent DNA contamination in downstream applications and to reduce solution viscosity. Centrifugation (18 000 xg for 30 minutes at 4°𝐶) was used to separate the soluble and insoluble fractions of the lysates. The supernatant was then filtered through a 0.45 µm filter to remove insoluble matter before being used in the subsequent purification steps. 4.1.4.1. Immobilized metal-ion affinity chromatography (IMAC) IMAC is a purification technique commonly used to purify proteins which are fused to a short affinity peptide tag. IMAC relies on the interaction between multiple electron donors found on the tag with a transition metal cation such as Co2+, Ni2+, Cu2+, Zn2+ bound to the solid phase support (Sepharose) (Falke et al., 2013). A chelating agent such as iminodiacetic acid (IDA) is covalently bound to the solid phase support and is used to entrap the metal ions. 35 The affinity tag is a polyhistidine (6-12 residues) fused to either the N or C terminus of the protein, the most common form is the 6-His where the electron donor is the histidine imidazole ring. Since the LRP protein was fused with a His-tag, it was purified from the soluble crude cell lysate by IMAC. The protein was eluted using a high concentration of imidazole which competes for the metal ions with the histidine residues therefore displacing the protein from the IDA ligand (Falke et al., 2013). The LRP protein was purified on a 5 ml IMAC column (1.6 x 2.5 cm His Trap column (GE Healthcare, USA) attached to the ÄKTA Prime Liquid Purification System (GE Healthcare, USA) (Flow rate of 5 ml/minutes and pressure limit of 0.3 MPa). The column was packed with Sepharose, used the IDA ligand and was charged with either Ni2+ or Co2+ ions. Initially, 10 column volumes of equilibration buffer were used to equilibrate the IMAC column which ensured that the protein molecules interacted with the ligand. Subsequently, the column was loaded with 50 ml of filtered (0.45 µm) supernatant the flow through was collected. Thereafter, a salt wash (1.5 M NaCl, 30 mM imidazole and 20 mM tris-HCl, pH 7.5) was carried out by passing 5 column volumes through the column (and collected). A high concentration of salt is used to remove any DNA contamination while removing non- specific proteins bound to the column. Thereafter, the column was re-equilibrated with equilibration buffer (10 column volumes). The His-Tagged LRP was then eluted from the column using elution buffer (5 column volumes) (500 mM NaCl, 300 mM imidazole and, 20 mM tris-HCl, pH 7.5) and collected. 4.1.5. Protein concentration and purity 4.1.5.1. SDS-PAGE Smith et al. (1984) explained that Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is employed to segregate proteins based on their molecular size. In this technique, SDS is utilized as a negatively charged denaturing detergent that binds to the protein’s backbone, prompting the protein to unravel into primary chains and producing a net negative charge that is proportional to the protein’s molecular weight. The protein mixture is then placed on a porous acrylamide gel that is prepared at various concentrations. The concentration of the gel is inversely proportional to the protein size. The proteins move through the gel in response to an electric field, migrating from the cathode 36 (negative terminal) to the anode (positive terminal) of the electric field (Smith et al., 1984). Proteins do not have a constant charge to mass ratio, hence, they need to be linearised and coated with a uniform charge. The protein samples are reduced with β-mercaptoethanol and denatured by heat. The discontinuous buffer system has provided great resolution and consists of a stacking gel on top of a separating gel. In the stacking gel (pH 6.8) the large, negative chloride ions migrate at a fast rate due to their high ionic charge, followed by the negatively charged protein and the positive glycinate ions. Whereas, in the separating gel (pH 8.8), the glycinate ions migrate ahead of the protein due to their large negative charged creating a uniform electric field for the proteins to migrate in based on their molecular size. SDS-PAGE was used to identify contaminates and to determine the size of the LRP protein. The size of the protein was predicted to be ≈ 37 kDa based on the sequence. Initially, samples were prepared by diluting the protein into reducing sample buffer (4% (w/v) SDS, 125 mM tris-HCl (pH 6.8), 20% (v/v) glycerol, 3.5 μg/ml bromophenol blue and, 10% (v/v) β-mercaptoethanol) and heating the sample at 95°𝐶 for 5 minutes. This ensured that the protein sample was denatured, linearized and in a uniform negative charge. SDS-PAGE was performed using the Bio-Rad Mini Protean™ Tetra Cell electrophoresis system (Bio Rad Laboratories, USA). Once the gel plates were setup, 12.5% acrylamide separating gel (12.5% (w/v) acrylamide, 1.08% (w/v) bis-acrylamide, 250 mM tris-HCl (pH 8.8), 0.1% (w/v) SDS, 0.2% (v/v) TEMED and, 0.05% (w/v) ammonium persulfate) was prepared and poured. Once it had polymerised, a 4% acrylamide stacking gel (4% (w/v) acrylamide, 0.36% (w/v) bis-acrylamide, 50 mM tris-HCl (pH 6.8), 0.1% (w/v) SDS, 0.2% (v/v) TEMED and, 0.005% ammonium persulfate) was prepared and poured. A 10-well comb was inserted into the stacking gel after it was poured. Once polymerization had occurred, the gels were placed into the electrophoresis tank containing the SDS-PAGE electrophoresis buffer (250 mM tris-HCl pH 8.3, 192 mM glycine and, 0.1% (w/v) SDS). The samples were then loaded into each well (10 µl) and electrophoresis was carried out at 120 V until the dye front reached the 1 cm from the bottom of the gel. Gels were carefully removed and stained with Coomassie stain solution (0.1% (w/v) Coomassie Brilliant Blue R- 250 in 1:5:4 (v/v/v) acetic acid:methanol:water) for 3 hours. Subsequently, destain solution (1:5:4 (v/v/v) acetic acid:methanol:water) was added to the gels for 24 hours. The unknown 37 molecular weight of the samples were calculated relative to the migration of the proteins in the BLUeye Prestain protein Ladder (Merck, Germany). Protein purity was assessed through densitometric analysis and protein samples that were at least 85% pure were used for the downstream experiments. 4.1.6. Protein characterisation 4.1.6.1. Absorbance spectroscopy Due to tyrosine, tryptophan, and phenylalanine (aromatic) amino acid residues, proteins absorb light at 280 nm (𝐴"*$). Therefore, 𝐴"*$ is quantitively used to determine protein concentration using the Beer-Lambert law. Additionally, nucleic acids are known to absorb light at 260 nm (𝐴")$) due to the resonance structure in purine and pyrimidine bases, therefore the 260 nm absorbance is used to detect nucleic acid contamination. Thus, 𝐴"*$/")$ is used as an indicator for the amount of nucleic acid contamination in a purified protein (≥1.8). Briefly, absorbance spectroscopy was used to determine the protein concentration, as well as to quantify contamination. Initially, the protein was centrifuged (13 200 rpm for 15 minutes, at 4°𝐶) to remove any aggregation. Using a Jasco V630 spectrophotometer (Jasco, USA), the absorbance spectrum was obtained from 240 to 340 nm, in triplicate with 3 biological repeats. The resultant 𝐴"*$ was used to calculate the molar concentration of the protein according to the Beer lambert law 𝑐 = 𝐴"*$ 𝜀 ∙ ℓ 𝑐 𝑖𝑠 𝑡ℎ𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀), 𝐴"*$ 𝑖𝑠 𝑡ℎ𝑒 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 280 𝑛𝑚, 𝜀 𝑖𝑠 𝑡ℎ𝑒 𝑚𝑜𝑙𝑎𝑟 𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑎𝑡 280 𝑛𝑚 𝑎𝑛𝑑 ℓ 𝑖𝑠 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ (𝑐𝑚) The molar coefficient is the sum of the aromatic contributions of all the aromatic amino acids present in the protein and was predicted to be 67045 M,-. cm,- for LRP protein. 38 4.1.6.2. Circular Dichroism (CD) Spectroscopy CD is a method used in absorption spectroscopy based on the unequal absorption of left and right circularly polarized light. Optically active chiral molecules absorb one of the directions of circularly polarized light, and it is possible to quantify the disparity in absorption between the left and right circularly polarized light. UV CD is commonly used to determine aspects of the secondary structure in a protein (Li et al., 2011). CD is typically reported as a difference between the absorbance of E. and E/(∆E) by an asymmetrical molecule and can be expressed as degrees of ellipticity. For proteins, the ∆E is representative of the secondary structure composition when measured in the far UV range, producing a distinct CD-spectra for different secondary structural features found in proteins. The far-UV CD spectrum of α- helical protein has troughs at 208 nm and 222 nm, and a peak at around 190 nm. Whereas, in β-sheeted protein there is a trough at 218 nm and a peak at 195 nm (Greenfield, 2006). Far UV CD spectropolarimetry was used to characterize the secondary structure of the LRP protein. This was done to confirm that the protein was predominantly α-helical as expected, which would indicate that the protein is correctly folded. The far-UV CD spectra were obtained from 200-250 nm, in triplicates, on the Jasco J-1500 spectropolarimeter (Jasco, USA). Each replicate was averaged from 10 accumulations. The spectra were obtained using a bandwidth of 5 nm, a scanning spread of 200 nm/minute and a path length of 2 mm (20°𝐶). The protein sample consisted of 10 μM of protein, diluted five times with Milli-Q® water such that the final NaCl concentration was only 20 mM (to minimize background noise caused by the presence of chloride ions). The average spectrum of the samples was corrected by subtracting the spectrum of the buffer. The signal of the far UV was obtained in millidegrees (mdeg) and normalised to mean residue ellipticity using; 𝜃012 = 𝜃 (𝑚𝑑𝑒𝑔) × 10) ℓ ∙ 𝐶 ∙ 𝑛 𝜃012 𝑖𝑠 𝑡ℎ𝑒 𝑚𝑒𝑎𝑛 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 𝑒𝑙𝑙𝑖𝑝𝑖𝑡𝑖𝑐𝑖𝑡𝑦 (𝑑𝑒𝑔. 𝑐𝑚". 𝑑𝑚𝑜𝑙,-), 𝜃 (𝑚𝑑𝑒𝑔) 𝑖𝑠 𝑡ℎ𝑒 𝑟𝑒𝑐𝑜𝑟𝑑𝑒𝑑 𝑠𝑖𝑔𝑛𝑎𝑙, ℓ 𝑖𝑠 𝑡ℎ𝑒 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ (𝑚𝑚), 𝐶 𝑖𝑠 𝑡ℎ𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (µM) and n is the number of peptide bonds in the protein backbone 39 4.1.6.3. Intrinsic tryptophan fluorescence (ITF) spectroscopy Tryptophan, tyrosine, phenylalanine, and cysteine amino acids act as fluorophores and exhibit an intrinsic fluorescence in proteins. Specifically, tryptophan contributes significantly to fluorescence as it has a high quantum yield as compared to tyrosine, and can be selectively excited at 295 nm (Ghisaidoobe et al., 2005). ITF is commonly used as a probe to assess the tertiary structure of a protein due to the indole group of tryptophan which is solvatochromic, meaning that the emission wavelength is dependent on the polarity of its environment. When the tryptophan residues are buried within the protein core, a blue shift in the fluorescence emission occurs (hypochromic) due to the decreased tryptophan exposure to the polar solvent. Whereas, when the tryptophan residues are exposed to the polar solvent (unfolded protein), a red shift occurs in the fluorescence emission (bathochromic) (Royer, 1995). ITF spectroscopy was utilised to determine if the LRP protein is folded correctly. The spectra was obtained both in the presence and absence of denaturants (8 M Urea), which allowed for comparisons between the native and unfolded protein. Initially, samples were incubated with the allocated denaturant for 2 hours, at 20°𝐶. On the Jasco FP-6300 spectrofluorometer (Jasco, USA), using the excitation wavelength of 280 and 295 nm, the emission spectra were obtained from 280 – 350 nm, in triplicates. The spectra were obtained at 20°𝐶 using excitation and emission bandwidths of 5 and 2.5 nm, a scanning speed of 200 nm/minute and a path length of 1 cm. In the absence of a denaturant, the sample consisted of 2 μM of protein, while in the presence of the denaturant, the sample contained 2 μM protein and 8 M Urea. Both samples were normalised by subtracting the spectrum of the buffer. Thereafter, the data was corrected by dividing the corrected fluorescence intensity value by the maximum fluorescence intensity value of the protein. 4.2. LRP encapsulated nanoparticle production and in vitro studies 4.2.1. LRP encapsulated PLGA Nanoparticle production The purified 37 kDa LRP protein was encapsulated using PLGA nanoparticle technology. PLGA is the most popular and well-known polymer for controlled release drug delivery systems because of its biocompatibility, biodegradability, and mechanical strength (Kumari et al., 2010; Makadia et al., 2011). There are multiple methods used to prepare nanoparticles, the drug can either be trapped within the core or entrapped on the surface of the matrix. The 40 double-emulsion-evaporation method is utilised for the encapsulation of hydrophilic drugs such as nucleic acids and proteins whereas the single emulsion technique is suitable for hydrophobic drugs such as steroids. In this study, the single emulsion technique was not performed with the LRP protein since it is hydrophilic and will result in the LRP diffuse into the aqueous solution before encapsulation (McCall et al., 2013). In preparation, glass test tubes were marked at the 1 ml level, a polymer solution was prepared with 100 mg of PLGA dissolved in 1 ml of ethyl acetate. The test tubes were tightly sealed and incubated overnight at room temperature. Due to the high evaporation rate of ethyl acetate, the 1 ml level was monitored, and the solvent was replaced if lost. Emulsification of LRP with the polymer solution was prepared by adding 700 ug/µl of LRP protein into the polymer solution. The drug was emulsified with the polymer until a homogenous, opaque solution was achieved. Subsequently, 50 ml of 0.3% (w/v) Vitamin E-TPGS was prepared, and 4 ml of the solution was added to a new 50 ml centrifuge tube. The tube was vortexed, and the polymer solution was added in a dropwise manner using a glass Pasteur pipette. Once added, the emulsion solution was vortexed for an additional 15 seconds and thereafter sonicated on ice (3x for 10 seconds at 40% amplitude) with a 700 W probe sonicator. Subsequently, 45 ml of the 0.3% Vitamin E-TPGS solution was added to a 100 ml beaker and placed on a magnetic stirrer. The emulsion solution was added into the stirring Vitamin E- TPGS solution and incubated 3-4 hours at room temperature. The emulsion solution was partially covered with foil to ensure evaporation of the ethyl acetate. After 3-4 hours, the emulsion solution was left overnight at 4°𝐶 allowing the nanoparticles to harden. Thereafter, the nanoparticles were purified to collect uniform and smaller particles of the PLGA particles. Initially, the larger nanoparticles were separated out by low centrifugation (8 000 xg for 10 minutes) and the supernatant (containing smaller nanoparticles) was transferred into 1.5 ml Eppendorf tubes. The smaller nanoparticles were purified out by high centrifugation (17 000 xg for 15 minutes) and the clear supernatant was discarded. The nanoparticles were washed by resuspending the pellet in 200 µl dH2O using a water bath sonicator. Thereafter, the solution was centrifuged (17 000 xg for 15 minutes) and the clear supernatant was discarded, this step was repeated for a total of three times, and subsequently resuspended in 500 µl of dH2O and placed at -80 ºC in preparation for lyophilisation. Once frozen, the solution was transferred to the lyophiliser where the lid of the tube was removed 41 and covered with parafilm (with small holes) to allow for water sublimation. The lyophilised nanoparticles were stored at -80°𝐶 until further use. 4.2.1.1. Treatments of cells with empty and LRP PLGA nanoparticles The HEK293 and SH-SY5Y cells were treated with 1-10% (v/v) of empty and LRP- encapsulated PLGA nanoparticles. This was done to assess their effect on cellular viability, LRP levels and telomerase activity. Briefly, the cell lines were seeded into a 24-well plate and incubated overnight (at 37°𝐶 and 5% CO2) until confluency reached 60-70% (see 4.2.2.). Thereafter, the cells were treated with 1, 5 and 10% (v/v) of empty and LRP-encapsulated PLGA nanoparticles in a final volume of 1 ml/well for 48 hours under standard conditions. The next day, media was changed to remove nanoparticles that were not taken up by the cells and to replenish with 1 ml/well. The treated cells were either used to extract protein for western blotting (see 4.2.4.-4.2.7.), cellular viability (see 4.2.8.) and telomerase activity (see 4.2.9.). 4.2.2. Cell culture This study utilized two different cell lines: the SH-SY5Y human neuroblastoma cell line and the HEK293 human embryonic kidney cell line. The HEK293 cell line was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) antibiotics, while the SH-SY5Y cell line was grown in a mixture of 1:1 DMEM and Ham’s F12 nutrient with the same supplements. These media were used to supplement cells with vital nutrients and prevent bacterial and/or fungal growth. The cells were cultured in an incubator at 37°𝐶 and 5% CO2 to simulate in vivo conditions, and were sub-cultured when they reached 70-90% confluency. Seeding and sub-culturing involved washing the cells with 1x phosphate buffered saline (PBS), detaching them with 1x Trypsin/EDTA, and suspending them in fresh media. 4.2.3. pCIneo-moLRP::FLAG stable transfection To overexpress LRP::FLAG, cell transfections were carried out using the pCIneo- moLRP::FLAG DNA construct (Vana & Weiss, 2006). The cells were seeded at a confluency of 60-70% in a 6-well plate, and 5 μg of the plasmid DNA was mixed with 100 µl XfectTM Reaction Buffer and 1.5 ml XfectTM Polymer which formed nanoparticle complexes after 10 42 minutes. The complexes were added dropwise to the cell culture, and incubated at 37°𝐶 and 5% CO2 for 4 hours. After incubation, the media was replaced with fresh growth media, and the cells were incubated for an additional 48 hours at 37°𝐶. To select for transfected cells, cells were treated with 800 ng/ml Geneticin followed by 400 ng/ml, which removed the population of non-transfected cells. This was done to investigate the effects of overexpressing LRP::FLAG in TBHP/ Aβ!" and MPP# mediated cytotoxicity (see 4.2.8.). 4.2.4. Extraction of protein The cells were detached from the culture flasks by adding 2 ml of Trypsin/EDTA and incubating for 5 minutes at 37°𝐶. To inactivate the Trypsin/EDTA, 8 ml of cell culture media was added, and the resulting cell suspension was added to a 15 ml falcon centrifuge tube and centrifuged for 10 minutes at 5000 rpm. The supernatant was removed, and the cells were lysed by adding 0.5-1 ml of radioimmunoprecipitation assay (RIPA) buffer (1% Triton X- 100, 10 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 50 mM tris-HCl (pH 8.0), and 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), followed by incubation at 4°𝐶 for 10 minutes. The total protein concentration was determined using a BCA assay for western blot analysis. 4.2.5. BCA™ Protein Assay The (Bicinchoninic acid assay) BCA assay is used to measure the quantity of protein and ensure that the samples are loaded evenly for western blot analysis. This assay works by detecting a colour change, which can be quantified using an absorbance spectrophotometer. This reaction is due to the protein’s peptide bonds and the temperature. Peptide bonds within the protein reduce copper ions (Cu"#) from copper sulphate (Cu#) to form a purple-coloured product with bicinchoninic acid. The more peptide bonds present, the greater the amount of copper ions that are reduced, allowing for a direct measurement of the protein concentration. Subsequently, bovine serum albumin (BSA) standards were prepared through a serial dilution, for protein concentration quantification a standard curve was constructed. Briefly, the following concentrations of BSA were prepared: 0, 0.025, 0.125, 0.25, 0.5, 0.75, 1.0, 1.5, and 2 mg/ml. In a 96 well plate, 25 µl of each standard was added. Subsequently, a 1:5 ratio of diluted cell lysates (25 µl) were added into each well. Accordingly, in a 1:50 and 49:50 ratio, the bicinchoninic assay (BCA) and copper (II) sulphate (CuSO!) reagents were 43 combined. In the plate containing the cell lysate sample and BSA standards, 200 µl of the BCA and CuSO! solution was added. Until colour change occurred, the 96 well plate was Incubated at 37°𝐶 for 40 minutes. Using the Multiskan® GO ELISA plate reader (Thermo Scientific, Massachusetts, USA) absorbance was measured at 562 nm. Thereafter, using the absorbance of the standards, a linear regression curve was constructed where the equation of the curve was used to calculate protein concentration of the samples. 4.2.6. SDS PAGE SDS-PAGE was performed as mentioned above, 4.1.5.1. The protein samples were heated at 95°𝐶 for 5 minutes in the Blue Loading buffer (New England Biolabs) containing 40 mM dithiothreitol. Initially, 2-3 µl of PageRuler Prestained Protein Ladder (Thermo Fisher) was loaded into the first well of each gel. For β-actin and LRP, 10 ug was loaded, while 20 ug of PINK1 was loaded into wells. The samples were separated on a 12% polyacrylamide gel in 1x running buffer for 60-90 minutes at 120 V using the Mini Protean gel system (Bio-rad, California, USA). 4.2.7. Western Blotting Once the proteins were separated by SDS PAGE, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane via electro-blotting. At first, the Whatman papers were trimmed to the required size and immersed in a 1x transfer buffer containing 20% methanol, 25 mM tris, and 19.2 mM glycine. At the same time, the PVDF membranes were trimmed to the desired size and soaked in methanol for a duration of 5 minutes. Next, the gels were trimmed to the appropriate size and three sheets of filter paper were arranged on the semi-dry electrophoretic transfer device. The membranes were then placed on top of the filter papers, and the gels were positioned above them, followed by an additional three sheets of filter paper. The apparatus used for the transfer was closed and placed into the Trans-blot® Turbo™ Transfer system (Bio-rad, California, USA) for 30 minutes at 25 V - 1.0 A. After transferring the proteins onto the membranes, the membranes were placed in a blocking buffer comprising of 5% low-fat milk powder in PBST (1x PBS and 0.1% Tween 20) for a period of 1 hour. This step aimed to prevent the primary and secondary antibodies from binding non-specifically. Subsequently, the membrane was incubated with the primary antibody (Table 1) overnight at a temperature of 4°𝐶 on a shaker. Subsequently, the 44 membranes were washed in 1x PBST 5 times, for 5 minutes each, to remove primary antibodies which were not bound. Thereafter, the membrane was incubated with the secondary antibody (Table 1) for 2/3 hours (in the dark at room temperature). Following the incubation, the membranes were subjected to the washing step. They were rinsed 5 times with 1x PBST for 5 minutes each time. The Clarity™ Western ECL Blotting Substrate (Bio- Rad) and the ChemiDoc™ Imaging System (Bio-Rad) were used to visualise and capture the membranes. Densitometric analysis was calculated using The Image Lab 5.1 software (Bio- Rad) where β-actin was used as the loading control to normalise all values. Table 1: Antibodies (primary and secondary) used for western blotting Target Protein Primary antibody Dilution Secondary antibody Dilution LRP::FLAG Murine anti- FLAG® M2, (Sigma F3165) 1:4000 Anti-murine IgG-HRP (Sigma A4416) 1:2500 LRP/LR Human anti- LRP/LR IgG- iS18, (Affimed) 1:6500 Anti-human IgG-HRP, (abcam 6858) 1:6500 PINK1 Rabbit anti- Pink-1 (Sigma P0076) 1:1500 Anti-rabbit IgG- HRP, (Cell Signaling Technology® 7074S) 1:2500 β-actin Murine anti-β- actin- peroxidase, (Sigma A3854) 1:5000 4.2.8. MTT Assay The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl diphenyltetrazolium bromide) assay was used for cellular viability assessment of LRP::FLAG overexpressed SH-SY5Y and HEK293 cells. Furthermore, The assay was used to determine cellular viability in SH-SY5Y cells treated with exogenous LRP encapsulated in PLGA nanoparticles. The MTT assay employs a colorimetric method that relies on the capacity of metabolically active cells to convert a yellow tetrazolium salt into purple formazan crystals. This reduction reaction is facilitated by specific oxidoreductase enzymes that are present in viable cells and depend on the coenzyme 45 NAD(P)H. The extent of formazan crystal formation is directly proportional to the mitochondrial activity of the cells and is indicative of their viability (Mosmann et al., 1983). Briefly, TBHP/Aβ and MPP# were used to treat transfected and non-transfected cell lines while LRP encapsulated PLGA nanoparticles were used to treat SH-SY5Y cells. Furthermore, PCA is a well-known agent that triggers apoptosis and was utilized as a positive control in this study. Briefly, non-transfected and transfected LRP::FLAG cells were seeded in a 48 well plate at 1 x 103 cells/well and incubated overnight at standard conditions to allow for attachment. Once confluency of 60-70% was obtained, cells were treated with either 250 nM, 500 nM and 750 nM Aβ!"/ml, 50 µM, 100 µM and 200 µM TBHP/ml or 500 µM and 750 µM MPP#/ml for 48 hours. Thereafter, to each well the MTT solution (100 µl of 1 mg/ml) was added, and incubated at 37°𝐶 for 3-4 hours. Post incubation, the media was carefully removed from each well and 300 µl of dimethyl sulfoxide (DMSO) was used to dissolve the purple formazan crystal. In a 96 well-plate, 100 µl of each sample was transferred into the wells in triplicates. Using the VICTOR® Nivo™ Multimode Microplate Reader the absorbance was measured at 570 nm. 4.2.9. Telomerase activity detection using quantitative polymerase chain reaction (qPCR) Telomerase activity in cells treated with empty and LRP-encapsulated nanoparticles was quantified using the TRAPeze® RT Telomerase Detection Kit. The method is sensitive and uses fluorescence to detect telomerase activity in real time, and it relies on the telomeric repeat amplification protocol (TRAP), which makes use of the fluorescence energy transfer primers (Amplifluor® primers) that directly measure the fluorescence emission to detect and quantify telomerase activity. Initially, in an extracted sample the telomerase adds telomeric repeats to the 3' end of the substrate over a period of time. Subsequently, the extended products are amplified by Taq polymerase, using the Amplifluor® primers. Once incorporated into the telomeric repeat amplification products, these primers generate a fluorescent signal. The quantity of fluorescence emission generated is directly proportional to the quantity of TRAP products produced. At first, the RNA and protein were extracted following the specified protocol. In this step, the samples were suspended in 200 µl of CHAPS lysis buffer and left on ice for 30 minutes. 46 Afterwards, the samples were subjected to centrifugation at 12 000 xg for 20 minutes at 4˚°𝐶. The supernatant was transferred to a 1.5 ml Eppendorf tube and snap frozen (-80°𝐶) in liquid nitrogen. Subsequently, the NanoDrop® ND-1000 (Thermo Fishe Scientific, Massachusetts, USA) was used to quantify the protein where it was and standardized to 500 ng/µl. In a 96- well plate, the TRAP reaction mixture and 1 μg/μl of protein samples was loaded into each well to 12.5 μl as the final volume. This reaction Mastermix consisted of Luna® Universal qPCR Mastermix (New England Biolabs, Massachusetts, USA), TS and ACX primers, EGTA (to inhibit RNases), and PCR-grade water. The CFX96™ Touch qPCR System from Bio-Rad, California, USA, was employed to perform qPCR analysis on all samples. The cycling parameters used in this study consisted of an initial cycle of 30 minutes at 37°𝐶, followed by 2 minutes at 95°𝐶, and subsequently 45 cycles of amplification with denaturation at 95°𝐶 for 15 seconds, annealing at 59°𝐶 for 60 seconds, and extension at 45°𝐶 for 10 seconds. To ensure that the buffer was not contaminated and had no telomerase activity, a minus telomerase negative control (consisting only of CHAPS lysis buffer) was included. A no template negative control (PCR-grade water) was included to adjust for any primer dimer formation in the absence of telomerase activity. Additionally, a heat-treated negative control was included, where the samples were incubated at 90°𝐶 for 10 minutes to inactivate any telomerase activity. A positive cell extract was provided as a positive control and made up as per protocol. The Bio-Rad CFX Maestro software was used to analyse the Cq values. The values were normalized as a percentage of the untreated controls, with the heat-treated samples serving as the background after subtraction. 4.2.10. Statistical Analysis The statistical analysis will be conducted using Microsoft Excel 2018 (Microsoft Corporation) and QuickCalcs Outlier Calculator ©2017 GraphPad Software. Each experiment will include at least three biological replicates with error bars representing the standard deviation. The ANOVA and Student’s t-test will be conducted at a 95% confidence interval, with p values less than 0.05 being considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). 47 5. Results 5.1. LRP::FLAG plasmid in vitro study The study was conducted in three parts. The first part focused on evaluating the impact of LRP::FLAG overexpression on HEK293 cells, with the aim of comparing its effects with LRP protein treatment to assess their potential role in impeding the progression and pathogenesis of AD and PD. This investigation aimed to determine whether targeting LRP could provide a new and effective strategy for treating these diseases. The second part of the study aimed to overexpress, purify and characterise the 37 kDa LRP protein in preparation for its encapsulation in PLGA nanoparticles. Finally, the third part of the study evaluated the in vitro therapeutic potential of PLGA encapsulated LRP nanoparticles in treating PD and AD. The study aimed to provide insights into the potential of LRP as a therapeutic target and the usefulness of PLGA encapsulated LRP nanoparticles as a novel treatment approach for these debilitating diseases. 5.2.1. Confirmation of transfection with LRP::FLAG The main method used to achieve stable overexpression of LRP::FLAG within the HEK293 cell line was by transfecting the cells with the pCIneo-moLRP::FLAG plasmid. This resulted in a new cell line called transfected HEK293. The HEK293 cells were selected for these experiments since they express neuronal markers and are easily transfectable. Briefly, 48 hours after the cells were transfected, they were treated with geneticin, whereafter geneticin resistant cells were pooled and analyzed. Subsequently, western blot analysis was used to determine the effect of LRP::FLAG overexpression on LRP/LR and PINK1 protein levels. PINK1 protein levels were determined, as it is a protein known to play a significant role in the implication of PD. Thus, PINK1 was assessed to determine if LRP::FLAG overexpression may alter the protein levels of PINK1. The results of the western blot analysis (shown in Figure 8, lanes 4-6) confirmed that the HEK293 cells that were transfected with pCIneoLRP::FLAG successfully expressed LRP::FLAG. This indicates that the transfection was successful and the subsequent experiments using LRP/LR to study PD and AD could be carried out. 48 A. Figure 8. Transfection of HEK293 with pCIneo-LRP::FLAG plasmid was successful (A) The presence of LRP::FLAG was determined via western blotting in transfected and non- transfected HEK293 cells. Accordingly, it was discovered that the transfected cells exclusively expressed a band at 38 kDa, which indicates the presence of LRP::FLAG, with an absence of bands in the non-transfected samples (A, lanes 4-6). LRP::FLAG was detected using the rabbit anti-FLAG HRP-conjugated antibody. 5.1.2. LRP::FLAG overexpression in HEK293 cells increase LRP/LR and PINK1 protein levels Western blotting was used to determine the effects of LRP::FLAG on LRP and PINK1 protein levels