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- Vaikath, Nishant Narayanan
(författare)
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Development of specific monoclonal antibodies against different forms of alpha-synuclein as diagnostic tools for synucleinopathies
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 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 specificityand 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. Wefound 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 inDLB. 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.
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| 2. |
- Vaikath, Nishant Narayanan, et al.
(författare)
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Generation and characterization of novel conformation-specific monoclonal antibodies for α-synuclein pathology.
- 2015
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Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 79, s. 81-99
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Tidskriftsartikel (refereegranskat)abstract
- α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including β-syn, γ-syn, β-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer's disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.
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