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

Landeck, Natalie LU ; Hall, Hélène LU ; Ardah, Mustafa T.; Majbour, Nour K.; El-Agnaf, Omar M A; Halliday, Glenda and Kirik, Deniz LU (2016) In Molecular Neurodegeneration 11(1).
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... (More)

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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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Alpha-synuclein, AlphaLISA, Multiplex assay, Phosphorylated alpha-synuclein, Synucleinopathy
in
Molecular Neurodegeneration
volume
11
issue
1
publisher
BioMed Central
external identifiers
  • Scopus:84983233341
  • WOS:000382729700001
ISSN
1750-1326
DOI
10.1186/s13024-016-0125-0
language
English
LU publication?
yes
id
7b0a4e67-f1f8-4c3c-9f2e-4bc0cd614f33
date added to LUP
2016-09-21 13:37:03
date last changed
2017-01-02 14:54:49
@article{7b0a4e67-f1f8-4c3c-9f2e-4bc0cd614f33,
  abstract     = {<p>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.</p>},
  articleno    = {125},
  author       = {Landeck, Natalie and Hall, Hélène and Ardah, Mustafa T. and Majbour, Nour K. and El-Agnaf, Omar M A and Halliday, Glenda and Kirik, Deniz},
  issn         = {1750-1326},
  keyword      = {Alpha-synuclein,AlphaLISA,Multiplex assay,Phosphorylated alpha-synuclein,Synucleinopathy},
  language     = {eng},
  month        = {08},
  number       = {1},
  publisher    = {BioMed Central},
  series       = {Molecular Neurodegeneration},
  title        = {A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein},
  url          = {http://dx.doi.org/10.1186/s13024-016-0125-0},
  volume       = {11},
  year         = {2016},
}