A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Natalie Landeck, Hélène Hall, Mustafa T. Ardah, Nour Majbour, Omar Ali El-Agnaf, Glenda Halliday, Deniz Kirik

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. Results: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. Conclusions: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.

Original languageEnglish
Article number125
JournalMolecular Neurodegeneration
Volume11
Issue number1
DOIs
Publication statusPublished - 22 Aug 2016

Fingerprint

alpha-Synuclein
Parkinson Disease
Post Translational Protein Processing
Phosphotransferases
Phosphorylation
Lewy Bodies
Neurons
Corpus Striatum
HEK293 Cells
Brain
Brain Diseases
Mesencephalon
human SNCA protein
Serine
Cerebrospinal Fluid
Dementia
Disease Progression
Pathology

Keywords

  • Alpha-synuclein
  • AlphaLISA
  • Multiplex assay
  • Phosphorylated alpha-synuclein
  • Synucleinopathy

ASJC Scopus subject areas

  • Molecular Biology
  • Clinical Neurology
  • Cellular and Molecular Neuroscience

Cite this

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein. / Landeck, Natalie; Hall, Hélène; Ardah, Mustafa T.; Majbour, Nour; Ali El-Agnaf, Omar; Halliday, Glenda; Kirik, Deniz.

In: Molecular Neurodegeneration, Vol. 11, No. 1, 125, 22.08.2016.

Research output: Contribution to journalArticle

Landeck, Natalie ; Hall, Hélène ; Ardah, Mustafa T. ; Majbour, Nour ; Ali El-Agnaf, Omar ; Halliday, Glenda ; Kirik, Deniz. / A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein. In: Molecular Neurodegeneration. 2016 ; Vol. 11, No. 1.
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abstract = "Background: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. Results: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 {\%} in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 {\%} in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. Conclusions: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.",
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AU - Halliday, Glenda

AU - Kirik, Deniz

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AB - Background: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. Results: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. Conclusions: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.

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