{"id":{"repo_id":"edinburgh","oai_identifier":"oai:era.ed.ac.uk:1842/3887"},"canonical_url":"https://search.dev.ndltd.org/etd/edinburgh/oai:era.ed.ac.uk:1842/3887","repository":{"repo_id":"edinburgh","name":"University of Edinburgh","base_url":"https://era.ed.ac.uk/server/oai/request"},"display":{"title":"Non-toxic concentrations of α-synuclein exacerbate Parkinson's disease-like cell death by inducing mitochondrial dysfunction","abstract":"α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death.","abstract_html":"α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death.","abstract_has_math":false,"creators":["Williamson, Sally Joanne Mary"],"institution":"The University of Edinburgh","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Jones, Paul","Sharkey, John"],"committee_chairs":[],"committee_members":[],"year":2008,"date_issued":"2008","date_published":"2008","updated_at":"2026-07-24T02:14:14Z","subjects":["α-synuclein","Parkinson's disease"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1842/3887","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Jones, Paul","Sharkey, John"]},{"key":"dc:creator","label":"Author","values":["Williamson, Sally Joanne Mary"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2010-10-06T15:25:38Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2010-10-06T15:25:38Z"]},{"key":"dc:date.issued","label":"Date","values":["2008"]},{"key":"dc:publisher","label":"Institution","values":["The University of Edinburgh"]},{"key":"dc:type","label":"Dc Type","values":["Thesis or Dissertation"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["PhD Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["α-synuclein","Parkinson's disease"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1842/3887"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death."]},{"key":"dc:title","label":"Title","values":["Non-toxic concentrations of α-synuclein exacerbate Parkinson's disease-like cell death by inducing mitochondrial dysfunction"]}]}],"canonical_facts":{"dc:contributor.advisor":["Jones, Paul","Sharkey, John"],"dc:creator":["Williamson, Sally Joanne Mary"],"dc:date.accessioned":["2010-10-06T15:25:38Z"],"dc:date.available":["2010-10-06T15:25:38Z"],"dc:date.issued":["2008"],"dc:description.abstract":["α-Synuclein (α-syn), is a self-aggregating protein that has been identified as a pathologically important component in a number of diseases, such as Parkinson’s disease (PD). PD, a progressive neurological disorder affecting 1 in 500 people, results in motor dysfunction following the loss of dopaminergic neurones of the nigrastriatal pathway. A pathological hallmark of PD is the presence of α-syn containing Lewy bodies and Lewy neurites. Although α-syn has been linked to PD by both histology and genetic studies on familial PD, neither the physiological function nor the pathophysiological role of α-syn in PD has been fully elucidated. This thesis examines the cellular responses to exogenously applied recombinant α-syn under normal and disease-like conditions. Within this thesis large-scale expression and purification of α-syn was successfully established, reproducibly producing large quantities of pure recombinant α-syn that was utilised within in vitro experiments. In SHSY-5Y neuroblastoma cells, α-syn (10 and 30 μM) significantly decreased NAD(P)H levels after 48 h incubation, indicative of either cell death or disruption to energy metabolism of the cells. However, α-syn (0.1 - 30 μM) did not induce cell death, as determined by the LDH assay, even when the cells were exposed for 48 h. Therefore our studies show that under normal, physiological conditions, α-syn is not inherently toxic, but does result in a decrease of total cellular energy levels. The mitochondrial toxin, 1-methyl-4-phenylpyridinium ion (MPP+), induced cell death in SHSY-5Y cells that was both concentration- and time-dependent. α-Syn (30 μM) significantly exacerbated MPP+-induced cell death in this model of PD. This suggests that while α-syn is normally non-toxic, under PD-like conditions it can exacerbate the cell death process. We identified that α-syn (30 μM) significantly increased cytosolic Ca2+ levels in a time-dependent manner as well as increasing the levels of the apoptotic mediator, cytochrome c (cyt c). The release of cyt c from the mitochondria into the cytosol is indicative of mitochondrial dysfunction and pore formation within mitochondrial membranes. However, α-syn-induced increase in cytosolic Ca2+ was not blocked by the mitochondrial pore inhibitor, cyclosporine A. This suggests that α-syn effects were not mediated through the mitochondrial pore usually associated with dysfunction and cyt c release. α-Syn therefore releases cyt c and Ca2+ by a separate mechanism, such as the formation of α-syn protofibril pores. This was further compounded by data that showed that α-syn (30 μM) significantly decreased mitochondrial membrane potential after 48 h incubation. The loss of the mitochondrial membrane potential coincided with a decrease in NAD(P)H. These data would therefore suggest that physiologically α-syn induces a low, non-toxic effect on the mitochondrial membrane. Under pathological conditions similar to PD however, this mitochondrial stress mediated by α-syn acts to exacerbate cell death."],"dc:identifier.uri":["http://hdl.handle.net/1842/3887"],"dc:language.iso":["en"],"dc:publisher":["The University of Edinburgh"],"dc:subject":["α-synuclein","Parkinson's disease"],"dc:title":["Non-toxic concentrations of α-synuclein exacerbate Parkinson's disease-like cell death by inducing mitochondrial dysfunction"],"dc:type":["Thesis or Dissertation"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["PhD Doctor of Philosophy"]},"updated_at":"2026-07-24T02:14:14Z"}