Objective: To set up a robust (see ref 1 for definition)  96-well High Content Screening assay of synucleinopathy in primary cultures of cortical neurons.

Background: The initiation and spread of synucleinopathy in primary cultures of cortical neurons can be achieved by treating the latter with preformed  fibrillar assemblies of recombinant synuclein (PFFs) and appreciated by immunofluorescence (IF)  imaging. However, several  barriers have slowed the adoption of this type of assay for screening purposes: (i)  the ease and reliability of the isolation and culture protocols capable to insure long term survival (>21 DIV) of primary cortical neurons in a 96 well culture format, (ii) the batch-to-batch variability of artificial recombinant PFFs in terms of bioactivity, (iii) the relative lack of characterization of the anti-synuclein antibodies for a specific use in non-denaturing conditions such as IF imaging, and (iv) the absence of dedicated image analysis routines best serving the characterization of synucleinopathy in 2D neuronal cultures.

Method: We worked at raising specifically these 4 barriers by designing novel standard operating procedures and came up with a standardized assay system exploiting automation.

Results: The base High Content Screening assay was developped for WT mouse cortical neurons cultured in 96 well plates and challenged with specific human recombinant synuclein PFFs batches. In a first phase, the extent of  synucleinopathy was quantified by imaging  phosphorylated synuclein at S129. This assay was characteriez by a Z’ factor >0.5, and was thus  succesfully generalized to the other  key basic synuclein pools (unstructured and alpha-hecial monomeric,  alpha-helical multimeric, and beta-strand oligomeric & fibrillar), as well as the the study of the uptake and fate kinetics of the added PFFs. The assay was also successfully transposed to primary cortical cultures of transgenic mice and to primary cortical cultures transduced  using AAV infections.

Conclusion: These different assays can be routinely used to characterize the bioactivity of test-compounds.

References: 1- A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays (1999) Zhang JH, Chung TD, Oldenburg KR. J Biomol Screen. 4(2):67-73.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/high-content-screening-of-synucleinopathy-in-primary-cortical-neurons-dissecting-out-the-interplay-between-the-basic-synuclein-pools/