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Development of specific monoclonal antibodies against different forms of alpha-synuclein as diagnostic tools for synucleinopathies

Vaikath, Nishant Narayanan LU (2019)
Abstract
Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies
(DLB) and multiple system atrophy (MSA), are a group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble alpha-synuclein (α-syn) aggregates in the brain. The detection of α syn pathology has relied vastly on the use of several antibodies, which were mostly generated against different forms of α-syn. Recent studies have shown that the intermediate prefibrillarlar, oligomeric α-syn species formed during the aggregation process are the neurotoxic species. Hence, generating antibodies against α-syn conformations that would specifically target the neurotoxic species would lead to improved understanding of the pathogenesis of... (More)
Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies
(DLB) and multiple system atrophy (MSA), are a group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble alpha-synuclein (α-syn) aggregates in the brain. The detection of α syn pathology has relied vastly on the use of several antibodies, which were mostly generated against different forms of α-syn. Recent studies have shown that the intermediate prefibrillarlar, oligomeric α-syn species formed during the aggregation process are the neurotoxic species. Hence, generating antibodies against α-syn conformations that would specifically target the neurotoxic species would lead to improved understanding of the pathogenesis of synucleinopathies. We generated mouse monoclonal antibodies specifically against various α-syn species, including oligomeric-, phosphorylated S129- and total-α-syn (o-, p-S129-and t-α-syn). These antibodies were characterized extensively for their specificity
and affinity by various biochemical and immunohistochemical analysis. Firstly, we utilized these antibodies to optimize and develop highly specific and sensitive ELISA assays to measure α-syn species in biological samples. The efficacy of the developed ELISA assays was validated and confirmed by measuring α-syn species in CSF samples obtained from PD patients comparing with age-matched healthy controls. Moreover, utilizing the logistic regression analysis, we also found that the combination of multiple CSF α-syn species (o-/t-α-syn, p-S129-α-syn) with the Alzheimer’s disease (AD) biomarker (p-tau) can provide the best fitting predictive model for distinguishing PD patients from controls. Thereafter, we utilized the assay to measure t-, o- or p-S129-α-syn in CSF samples from a longitudinal Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism (DATATOP) study cohort. We
found that, there was an increase in the t- and o-α-syn levels and a decrease in the pS129-α-syn levels during the 2-year study follow-up period. Moreover, we also observed an association between the changes in CSF α-syn species with PD phenotypes, which in turn supports their role as a PD progression biomarker. Finally, we investigated the occurrence of α-syn species in post-mortem brain tissues from PD, DLB, AD and compared with age matched control cases. Using ELISA and western blot analysis on the sequentially extracted soluble and insoluble/aggregated α-syn, we found there was an elevated level of non-phosphorylated o- α-syn in PD, DLB as well in AD. On the other hand increased level of pS129-α-syn was observed in detergent soluble fragment of both PD and AD. However, in the urea soluble tissue lysates, pS129-α-syn was elevated only in
DLB. Hence the presence of α-syn species in Lewy body disease and in AD suggests a heterogeneous nature of α-syn across the spectrum of neurodegenerative disorders. Taken together, we can conclude that the generation of antibodies portrayed here that target specifically the neurotoxic species of α-syn could serve as an invaluable tool for research, biomarkers development, diagnosis and even immunotherapy for synucleinopathies. (Less)
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author
supervisor
opponent
  • professor Jensen, Henning, Aarhus University, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
pages
93 pages
publisher
Lund University, Faculty of Medicine
defense location
Segerfalksalen, BMC A10, Sölvegatan 17 i Lund
defense date
2019-01-31 09:15
ISBN
978-91-7619-742-4
language
English
LU publication?
yes
id
7b12b22d-6d4a-45a6-af52-4a211c23321f
date added to LUP
2019-01-11 19:09:36
date last changed
2019-01-16 09:33:36
@phdthesis{7b12b22d-6d4a-45a6-af52-4a211c23321f,
  abstract     = {Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies<br/>(DLB) and multiple system atrophy (MSA), are a group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble alpha-synuclein (α-syn) aggregates in the brain. The detection of α syn pathology has relied vastly on the use of several antibodies, which were mostly generated against different forms of α-syn. Recent studies have shown that the intermediate prefibrillarlar, oligomeric α-syn species formed during the aggregation process are the neurotoxic species. Hence, generating antibodies against α-syn conformations that would specifically target the neurotoxic species would lead to improved understanding of the pathogenesis of synucleinopathies. We generated mouse monoclonal antibodies specifically against various α-syn species, including oligomeric-, phosphorylated S129- and total-α-syn (o-, p-S129-and t-α-syn). These antibodies were characterized extensively for their specificity<br/>and affinity by various biochemical and immunohistochemical analysis. Firstly, we utilized these antibodies to optimize and develop highly specific and sensitive ELISA assays to measure α-syn species in biological samples. The efficacy of the developed ELISA assays was validated and confirmed by measuring α-syn species in CSF samples obtained from PD patients comparing with age-matched healthy controls. Moreover, utilizing the logistic regression analysis, we also found that the combination of multiple CSF α-syn species (o-/t-α-syn, p-S129-α-syn) with the Alzheimer’s disease (AD) biomarker (p-tau) can provide the best fitting predictive model for distinguishing PD patients from controls. Thereafter, we utilized the assay to measure t-, o- or p-S129-α-syn in CSF samples from a longitudinal Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism (DATATOP) study cohort. We<br/>found that, there was an increase in the t- and o-α-syn levels and a decrease in the pS129-α-syn levels during the 2-year study follow-up period. Moreover, we also observed an association between the changes in CSF α-syn species with PD phenotypes, which in turn supports their role as a PD progression biomarker. Finally, we investigated the occurrence of α-syn species in post-mortem brain tissues from PD, DLB, AD and compared with age matched control cases. Using ELISA and western blot analysis on the sequentially extracted soluble and insoluble/aggregated α-syn, we found there was an elevated level of non-phosphorylated o- α-syn in PD, DLB as well in AD. On the other hand increased level of pS129-α-syn was observed in detergent soluble fragment of both PD and AD. However, in the urea soluble tissue lysates, pS129-α-syn was elevated only in<br/>DLB. Hence the presence of α-syn species in Lewy body disease and in AD suggests a heterogeneous nature of α-syn across the spectrum of neurodegenerative disorders. Taken together, we can conclude that the generation of antibodies portrayed here that target specifically the neurotoxic species of α-syn could serve as an invaluable tool for research, biomarkers development, diagnosis and even immunotherapy for synucleinopathies.},
  author       = {Vaikath, Nishant Narayanan},
  isbn         = {978-91-7619-742-4},
  language     = {eng},
  pages        = {93},
  publisher    = {Lund University, Faculty of Medicine},
  school       = {Lund University},
  title        = {Development of specific monoclonal antibodies against different forms of alpha-synuclein as diagnostic tools for synucleinopathies},
  year         = {2019},
}