Establishment of a high-content neurodegenerative disease model screening platform
- Authors: Swanepoel, Bresler
- Date: 2023-12
- Subjects: Molecular neurobiology , Nervous system -- Diseases , Nervous system -- Degeneration
- Language: English
- Type: Doctorial theses , text
- Identifier: http://hdl.handle.net/10948/62644 , vital:72906
- Description: The identification of viable therapeutic targets and new treatments for central nervous system disorders, especially neurodegenerative diseases, remain major challenges in the field of drug discovery. Over the past few years there has been a steady decline in the turnaround time of current screening processes to yield viable drugs. Therefore, an increasing need exists for better technologies, protocols, and the screening of larger libraries. High-throughput screening provides the best solution to this problem and has become a key part in the drug discovery and development process. Likewise, high-content analysis has gained popularity over the past few years and is suitable for high-throughput screening. The aim of this study was to establish a comprehensive in vitro neuroprotective screening platform incorporating high throughput screening, using Parkinson’s disease as the neurodegenerative disease of interest. To evaluate the success of this platform, the neuroprotective potential of two mushrooms (Hericium erinaceus and Phlebopus sudanicus), two plants (Lippia javanica and Myrothamnus flabellifolia) and two seaweeds (Eucheuma denticulatum and Kappaphycus alvarezii) were investigated. Aqueous and ethanolic extracts of the selected natural products were evaluated across 21 parameters associated with four hallmarks of neurodegeneration: acquiring senescence, acquiring cell death, neuroinflammation and altered metabolism/cell survival. Based on the effects of these selected natural products on the 21 parameters, their potential mechanisms of action were elucidated. In addition to this, the natural products were scored under each of these therapeutic targets in an attempt to identify the most suitable animal models for future studies. The scoring system indicated that animal models investigating anti-senescence ability would be more suited for extracts of H. erinaceus, P. sudanicus and E. denticulatum whereas studies investigating the prevention of cell death would be more suited for extracts of E. denticulatum, L. javanica and K. alvarezii. Likewise, models based on neuroinflammation would be best suited for extracts of H. erinaceus, E. denticulatum and L. javanica while studies examining altered metabolism/cell survival would be best suited to extracts of H. erinaceus, E. denticulatum, K. alvarezii and M. flabellifolia. Considering the pleiotropic nature of the selected natural products, models that can combine these therapeutic targets could be of great interest. 6-OHDA proved to be capable of inducing favourable effects, in all the parameters investigated, with regard to a neurodegenerative state. However, it is known to have some disadvantages when it comes to pathological features such as the lack of the ability to induce Lewy body formation. Choosing the correct inducer remains a daunting task and no model, whether cell-based or animal-based, exists yet in which all the features of neurodegenerative diseases have been successfully replicated. The limitations of the current models, however, does not mean that they do not produce valuable insights. This is especially true if the mechanism of action for a specific compound or natural product is unknown. Animal models are still indispensable for the validation and interpretation of the results obtained from cell models with particular importance to toxicity. Therefore, this study assessed the best studied extract with the highest overall score for its toxicity using a zebrafish larvae-based model. Assessment of the toxicity of H. erinaceus revealed that both aqueous and ethanolic extracts resulted in death at the highest concentrations. This was supported by the results obtained in the in vitro cytotoxicity screening. In conclusion, this highlighted the importance of using physiologically relevant concentrations and supported the translational value of the current cell-based screening model to animal models and possibly humans. The findings of the present study suggest that a scoring system, which categorizes the different activities of selected natural products into distinct groups, can be a useful tool to improve the translatability of in vitro results to animal models. Furthermore, the current study arguably provides the most comprehensive neuroprotective screening platform in existence. Future research can look at expanding the platform through incorporation of additional parameters based on other hallmarks of neurodegeneration, not covered in this study, including protein folding and aggregation, altered epigenetics and the examination of other neuronal markers such as the involvement of astrocytes, oligodendrocytes, and microglia. In addition to this, future research can make use of more sophisticated cell models such as differentiated, human induced pluripotent stem cells and three-dimensional cultures. , Thesis (PhD) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Swanepoel, Bresler
- Date: 2023-12
- Subjects: Molecular neurobiology , Nervous system -- Diseases , Nervous system -- Degeneration
- Language: English
- Type: Doctorial theses , text
- Identifier: http://hdl.handle.net/10948/62644 , vital:72906
- Description: The identification of viable therapeutic targets and new treatments for central nervous system disorders, especially neurodegenerative diseases, remain major challenges in the field of drug discovery. Over the past few years there has been a steady decline in the turnaround time of current screening processes to yield viable drugs. Therefore, an increasing need exists for better technologies, protocols, and the screening of larger libraries. High-throughput screening provides the best solution to this problem and has become a key part in the drug discovery and development process. Likewise, high-content analysis has gained popularity over the past few years and is suitable for high-throughput screening. The aim of this study was to establish a comprehensive in vitro neuroprotective screening platform incorporating high throughput screening, using Parkinson’s disease as the neurodegenerative disease of interest. To evaluate the success of this platform, the neuroprotective potential of two mushrooms (Hericium erinaceus and Phlebopus sudanicus), two plants (Lippia javanica and Myrothamnus flabellifolia) and two seaweeds (Eucheuma denticulatum and Kappaphycus alvarezii) were investigated. Aqueous and ethanolic extracts of the selected natural products were evaluated across 21 parameters associated with four hallmarks of neurodegeneration: acquiring senescence, acquiring cell death, neuroinflammation and altered metabolism/cell survival. Based on the effects of these selected natural products on the 21 parameters, their potential mechanisms of action were elucidated. In addition to this, the natural products were scored under each of these therapeutic targets in an attempt to identify the most suitable animal models for future studies. The scoring system indicated that animal models investigating anti-senescence ability would be more suited for extracts of H. erinaceus, P. sudanicus and E. denticulatum whereas studies investigating the prevention of cell death would be more suited for extracts of E. denticulatum, L. javanica and K. alvarezii. Likewise, models based on neuroinflammation would be best suited for extracts of H. erinaceus, E. denticulatum and L. javanica while studies examining altered metabolism/cell survival would be best suited to extracts of H. erinaceus, E. denticulatum, K. alvarezii and M. flabellifolia. Considering the pleiotropic nature of the selected natural products, models that can combine these therapeutic targets could be of great interest. 6-OHDA proved to be capable of inducing favourable effects, in all the parameters investigated, with regard to a neurodegenerative state. However, it is known to have some disadvantages when it comes to pathological features such as the lack of the ability to induce Lewy body formation. Choosing the correct inducer remains a daunting task and no model, whether cell-based or animal-based, exists yet in which all the features of neurodegenerative diseases have been successfully replicated. The limitations of the current models, however, does not mean that they do not produce valuable insights. This is especially true if the mechanism of action for a specific compound or natural product is unknown. Animal models are still indispensable for the validation and interpretation of the results obtained from cell models with particular importance to toxicity. Therefore, this study assessed the best studied extract with the highest overall score for its toxicity using a zebrafish larvae-based model. Assessment of the toxicity of H. erinaceus revealed that both aqueous and ethanolic extracts resulted in death at the highest concentrations. This was supported by the results obtained in the in vitro cytotoxicity screening. In conclusion, this highlighted the importance of using physiologically relevant concentrations and supported the translational value of the current cell-based screening model to animal models and possibly humans. The findings of the present study suggest that a scoring system, which categorizes the different activities of selected natural products into distinct groups, can be a useful tool to improve the translatability of in vitro results to animal models. Furthermore, the current study arguably provides the most comprehensive neuroprotective screening platform in existence. Future research can look at expanding the platform through incorporation of additional parameters based on other hallmarks of neurodegeneration, not covered in this study, including protein folding and aggregation, altered epigenetics and the examination of other neuronal markers such as the involvement of astrocytes, oligodendrocytes, and microglia. In addition to this, future research can make use of more sophisticated cell models such as differentiated, human induced pluripotent stem cells and three-dimensional cultures. , Thesis (PhD) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
β-N-Methylamino-L-Alanine is a developmental neurotoxin
- Authors: Scott, Laura Louise
- Date: 2019
- Subjects: Neurotoxic agents , Nervous system -- Diseases
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/43633 , vital:36949
- Description: β-N-methylamino-L-alanine (BMAA) has been implicated in the development of the neurodegenerative diseases Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC), Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s Disease (AD), but to date no animal model has adequately substantiated this link at environmentally relevant or even exaggerated BMAA exposure levels. The resulting controversy over a possible role for BMAA in neurodegenerative diseases was further hampered by a lack of evidence for mechanistic explanation for the disease pathology associated with these diseases However, the different responses to BMAA that have been observed in neonatal compared to adult rats, together with the findings of epidemiological studies that exposure to environmental factors in utero or in the early stages of life may be important for the development of ALS several years later, suggested that age of exposure might be the determining factor of BMAA neurotoxicity. This study therefore specifically addresses the developmental nature of BMAA as a neurotoxin, and investigates the pathology and progressive nature of that pathology after exposure to the toxin at the most susceptible age. This study demonstrated the importance of BMAA exposure age over total BMAA dose by showing that the administration of a single neonatal dose of BMAA to rodents on postnatal day (PND) 3, 4 and 5, and not prenatally or on PND 6, 7 and 10, caused behavioural, locomotor, emotional and long-term cognitive deficits, clinical symptoms of neurodegeneration as well as pathological hallmarks of AD, PD and ALS in the central nervous system. Furthermore, the observed behavioural deficits and distribution of neuronal loss and proteinopathies in the rodent central nervous system following exposure to BMAA on PND 3, 4 and 5 (corresponding to the developing age of an infant during the third trimester of pregnancy) is consistent with that typically associated with the disruption of normal dopamine and/or serotonin signaling in the brain and the consequent alteration in normal hippocampal and striatal neurogenesis that is modulated, in part, by dopamine. The pattern of spread and rate of propagation of pathology in this neonatal rat BMAA model provided further evidence that BMAA potentially exerts its effect by acting on neurotransmitter signaling. The observed late onset of typical ALS symptoms and pathology suggest that in this BMAA model AD and/or PD related symptoms develop first, followed by the start of ALS symptoms only after the AD and/or PD neuropathological deficits have severely progressed. This study also demonstrated that BMAA exposure at different doses and at different developmental ages resulted in the development of different combinations of either AD and/or PD and/or ALS pathology and/or symptoms in rats, and it is therefore feasible that in humans the age and/or frequency of exposure as well as the BMAA dose might similarly be a major determinant of the variant of AD, PD and/or ALS that might develop in adulthood. Based on the low BMAA dose that was able to cause AD and/or PD-like neuropathological abnormalities in rats in this study, it is feasible that a pregnant human could over the course of her pregnancy, and specifically during the third trimester of pregnancy, consume sufficient BMAA to result in her unborn child developing AD and/or PD and/or ALS up to 30-50 years later. This neonatal BMAA model is the only non-transgenic rodent model that reproduces the behavioural deficits, neuropathology and clinical symptoms that are typically associated with AD, PD and ALS in humans and that, more importantly, mimics the delayed onset of disease symptoms and typical slow progression of these neurodegenerative diseases with age. It now seems very likely that BMAA is a developmental neurotoxin that, as a result of perinatal, but probably prenatal exposure, causes or contributes significantly to the development of neurodegenerative diseases in humans.
- Full Text:
- Date Issued: 2019
- Authors: Scott, Laura Louise
- Date: 2019
- Subjects: Neurotoxic agents , Nervous system -- Diseases
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/43633 , vital:36949
- Description: β-N-methylamino-L-alanine (BMAA) has been implicated in the development of the neurodegenerative diseases Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC), Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s Disease (AD), but to date no animal model has adequately substantiated this link at environmentally relevant or even exaggerated BMAA exposure levels. The resulting controversy over a possible role for BMAA in neurodegenerative diseases was further hampered by a lack of evidence for mechanistic explanation for the disease pathology associated with these diseases However, the different responses to BMAA that have been observed in neonatal compared to adult rats, together with the findings of epidemiological studies that exposure to environmental factors in utero or in the early stages of life may be important for the development of ALS several years later, suggested that age of exposure might be the determining factor of BMAA neurotoxicity. This study therefore specifically addresses the developmental nature of BMAA as a neurotoxin, and investigates the pathology and progressive nature of that pathology after exposure to the toxin at the most susceptible age. This study demonstrated the importance of BMAA exposure age over total BMAA dose by showing that the administration of a single neonatal dose of BMAA to rodents on postnatal day (PND) 3, 4 and 5, and not prenatally or on PND 6, 7 and 10, caused behavioural, locomotor, emotional and long-term cognitive deficits, clinical symptoms of neurodegeneration as well as pathological hallmarks of AD, PD and ALS in the central nervous system. Furthermore, the observed behavioural deficits and distribution of neuronal loss and proteinopathies in the rodent central nervous system following exposure to BMAA on PND 3, 4 and 5 (corresponding to the developing age of an infant during the third trimester of pregnancy) is consistent with that typically associated with the disruption of normal dopamine and/or serotonin signaling in the brain and the consequent alteration in normal hippocampal and striatal neurogenesis that is modulated, in part, by dopamine. The pattern of spread and rate of propagation of pathology in this neonatal rat BMAA model provided further evidence that BMAA potentially exerts its effect by acting on neurotransmitter signaling. The observed late onset of typical ALS symptoms and pathology suggest that in this BMAA model AD and/or PD related symptoms develop first, followed by the start of ALS symptoms only after the AD and/or PD neuropathological deficits have severely progressed. This study also demonstrated that BMAA exposure at different doses and at different developmental ages resulted in the development of different combinations of either AD and/or PD and/or ALS pathology and/or symptoms in rats, and it is therefore feasible that in humans the age and/or frequency of exposure as well as the BMAA dose might similarly be a major determinant of the variant of AD, PD and/or ALS that might develop in adulthood. Based on the low BMAA dose that was able to cause AD and/or PD-like neuropathological abnormalities in rats in this study, it is feasible that a pregnant human could over the course of her pregnancy, and specifically during the third trimester of pregnancy, consume sufficient BMAA to result in her unborn child developing AD and/or PD and/or ALS up to 30-50 years later. This neonatal BMAA model is the only non-transgenic rodent model that reproduces the behavioural deficits, neuropathology and clinical symptoms that are typically associated with AD, PD and ALS in humans and that, more importantly, mimics the delayed onset of disease symptoms and typical slow progression of these neurodegenerative diseases with age. It now seems very likely that BMAA is a developmental neurotoxin that, as a result of perinatal, but probably prenatal exposure, causes or contributes significantly to the development of neurodegenerative diseases in humans.
- Full Text:
- Date Issued: 2019
Evaluation of model systems for the study of protein association / incorporation of Beta-Methylamino-L-Alanine (BMAA)
- Authors: Visser, Claire
- Date: 2011
- Subjects: Neurotoxic agents , Nervous system -- Diseases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10314 , http://hdl.handle.net/10948/1451 , Neurotoxic agents , Nervous system -- Diseases
- Description: β-methylamino-L-alanine (BMAA) is thought to be a contributing factor of Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS/PDC). It has been shown that the levels of toxin ingestion by humans are too low to cause disease. However, it has recently been theorized that this toxin is bioaccumulated within cells. Via a process of slow release from this reservoir, the BMAA is able to bring about neurotoxicity. Mechanisms of uptake and bioaccumulation of BMAA have been proposed in several publications; however the mechanism of protein incorporation of BMAA has not yet been identified. Identifying suitable model systems would be a prerequisite in order for future studies on BMAA protein incorporation. Three specific models were therefore chosen for investigation; mammalian cell lines including C2C12 and HT29, a prokaryotic (E. coli) expression system and yeast cells. The cytotoxity of BMAA was established for the mammalian cell lines and further investigation of BMAA incorporation into cellular proteins was performed on all three above mentioned models. Samples were run on HPLC-MS in order to determine uptake of BMAA into cells or lack thereof. Results indicate negligible cytotoxicity as measured by MTT and CellTitre Blue assays, limited uptake and protein incorporation of BMAA within the prokaryotic model and insignificant uptake of BMAA by yeast cells. Although the uptake of BMAA in the prokaryotic model was not extensive, there was indeed uptake. BMAA was not only taken up into the cells but was also observed in inclusion body protein samples after hydrolysis. After further investigation and use, this model could very well provide researchers with information pertaining to the mechanism of association of BMAA with proteins. Although the other models provided negative results, this research was valuable in the sense that one can narrow down the number of possible model systems available. Also, in seeking models for studying protein association/incorporation, the use of the final target cell is not relevant or necessary as the purpose of the research was to identify a model system in which the mechanism of protein association/incorporation can, in future, be studied.
- Full Text:
- Date Issued: 2011
- Authors: Visser, Claire
- Date: 2011
- Subjects: Neurotoxic agents , Nervous system -- Diseases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10314 , http://hdl.handle.net/10948/1451 , Neurotoxic agents , Nervous system -- Diseases
- Description: β-methylamino-L-alanine (BMAA) is thought to be a contributing factor of Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS/PDC). It has been shown that the levels of toxin ingestion by humans are too low to cause disease. However, it has recently been theorized that this toxin is bioaccumulated within cells. Via a process of slow release from this reservoir, the BMAA is able to bring about neurotoxicity. Mechanisms of uptake and bioaccumulation of BMAA have been proposed in several publications; however the mechanism of protein incorporation of BMAA has not yet been identified. Identifying suitable model systems would be a prerequisite in order for future studies on BMAA protein incorporation. Three specific models were therefore chosen for investigation; mammalian cell lines including C2C12 and HT29, a prokaryotic (E. coli) expression system and yeast cells. The cytotoxity of BMAA was established for the mammalian cell lines and further investigation of BMAA incorporation into cellular proteins was performed on all three above mentioned models. Samples were run on HPLC-MS in order to determine uptake of BMAA into cells or lack thereof. Results indicate negligible cytotoxicity as measured by MTT and CellTitre Blue assays, limited uptake and protein incorporation of BMAA within the prokaryotic model and insignificant uptake of BMAA by yeast cells. Although the uptake of BMAA in the prokaryotic model was not extensive, there was indeed uptake. BMAA was not only taken up into the cells but was also observed in inclusion body protein samples after hydrolysis. After further investigation and use, this model could very well provide researchers with information pertaining to the mechanism of association of BMAA with proteins. Although the other models provided negative results, this research was valuable in the sense that one can narrow down the number of possible model systems available. Also, in seeking models for studying protein association/incorporation, the use of the final target cell is not relevant or necessary as the purpose of the research was to identify a model system in which the mechanism of protein association/incorporation can, in future, be studied.
- Full Text:
- Date Issued: 2011
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