High Content-based Quantification of α-Synuclein Aggregation in a Dopaminergic Model System

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by tremor, akinesia, and lack of balance. These symptoms are caused by dopaminergic motor neuron death or dysfunction in the substantia nigra of the mesencephalon. Aggregates of the pre-synaptic protein α-synuclein are considered the primary biomarkers of PD, and evidence suggests that α-synuclein aggregates directly mediate neuronal cell death. Consequently, strategies aimed at reducing α-synuclein aggregation and toxicity may possess therapeutic potential.

To date, cellular models available to probe α-synuclein biology either possess only limited predictive and translational value because of their poor resemblance to terminally differentiated dopaminergic neurons or experimental timelines and throughput are insufficient to drive drug discovery programs. In a fee-for-service contract between the Michael J. Fox Foundation for Parkinson’s Research, we have developed an in vitro neuroscience assay to measure α-synuclein aggregation in an immortalized human dopaminergic cell model using an aggregate-selective anti-α-synuclein antibody (MJFR14). Experimental timing and format (96-well) are amenable to testing a considerable number of candidate therapeutics using industry standard quality criteria. This α-aynuclein assay and a complimentary TOM20 loss assay are available to the scientific community to support translation in vivo.

α-Synuclein Assay Principle

Immortalized human mesencephalic progenitor cells (ReNcell VM) are differentiated by withdrawal of growth factors (bFGF, EGF) and addition of pro-differentiation factors (cAMP, GDNF), followed by delivery of full-length wildtype human α-synuclein using our proprietary adenoviral FleXSelect® technology. After 24 hours, cells are treated with test compounds and α-synuclein expression and aggregation are detected six days later by immunocytochemistry using antibodies detecting α/β-synuclein (Syn205; cell signaling technology) and aggregated α-synuclein (MJFR14; Abcam), followed by quantification using in-house developed high content analysis based (HCA) algorithms.

α-Synuclein Aggregation Assay Setup

  • Cells → ReNcell VM cells
  • Seeding density → 10,000 cells/well in laminin-coated 96-well plates
  • Differentiation → Differentiation medium (- bFGF/EGF, + cAMP/GDNF) on D0
  • Transduction → Adenovirus encoding WT human α-synuclein on D0
  • Assay controls → 10 μM KU 0063794 (positive control) and 0.1% DMSO (negative control) on D1 and D4
  • Immunocytochemistry → MJFR14 rabbit Mab and Syn205 mouse Mab on D7
  • Readout → HCA-based quantification of total- (Syn205) and aggregated (MJF-14) α-synuclein immunoreactivity

Flow chart showing the process and timeframe for the alpha synuclein assay.

α-Synuclein Assay Performance

Representative data is shown below for the α-synuclein in vitro neuroscience assay.

 

The Reduction of α-synuclein Expression

Data images illustrating the reduction in α-synuclein expression following treatment with the positive control compound.

Immunocytochemical images of cells treated with increasing concentrations of the autophagy-inducing compound KU 0063794 and stained with MJFR14, illustrating the reduction in α-synuclein expression following treatment with this compound, used as positive control.


High Content-Based Quantifications

Scatter plot line graph showing the high content-based quantification of aggregated α-synuclein.

High content-based quantification of total and aggregated α-synuclein species in combination with determination of nuclei counts in differentiated ReNcell VM cells treated with increasing concentrations of KU 0063794.


Alpha Synuclein Assay Screen Data: Assay Window MJFR14

Bar chart showing the immunoreactivity data of the alpha synuclein assay.

Plate-to-plate comparison of MJFR14-immunoreactivity for assay positive (KU 0063794) and negative (0.1% DMSO) control samples, illustrating data consistency over screening batches.


The Reduction of α-synuclein Immunoreactivity

Data image of the cells treated in the alpha synuclein assay.

Immunocytochemical images of cells treated with vehicle (0.1% DMSO) or two test compounds and immunostained with MJFR14 (top panels) and Syn205 (bottom panels), demonstrating the assay’s ability to identify compounds that reduce overall α-synuclein immunoreactivity (compound 1) and compounds that specifically reduce MJFR14 immunoreactivity (compound 2).


Summary

The therapeutic candidates are evaluated in 8-point concentration response curves at 0.5Log-fold dilution steps in biological triplicates. In addition, potential cytotoxicity of the tested therapeutic candidates is assessed by HCA-based quantification of nuclei loss derived from DAPI-staining. As a positive control for inhibition of α-synuclein expression and aggregation, the autophagy inducer KU 0063794 is tested on each plate as a benchmark for assay quality. Results are provided as percentage inhibition (PIN values) and percentage viable cells.

Complementary Neuroscience Assays

TOM20 Loss Assay

 

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