This protocol outlines a high-content screening (HCS) platform for investigating the impact of microRNA (miRNA) expression on primary motor neurons in culture. Such a system is applicable to automated screening of cell morphology in response to drug molecules, gene manipulation and other stimuli. When used with transfection reagents such as Neuro9™, this method can help with functional screening to identify the underlying pathways of disease and screen for therapeutic nucleic acids. The Hornstein Lab outlines, how this can be used to understand miRNA dysregulation in neurodegenerative diseases, and guide the design of therapeutic miRNAs.
The HCS system devised by the Hornstein lab consists of cell samples in a 384-well plate, an automated liquid handling system and automated fluorescence microscopy capable of addressing individual wells. They demonstrated automated fluorescence microscopy and morphometry measurements by inducing oxidative stress in primary neurons with sodium arsenite (NaAsO2), and imaging the cells 72h after treatment. NaAsO2 was found to reduce cell number, axon length and number of branches in a dose-dependent manner.
To modulate miRNA expression in this application, a transfection reagent needs to be efficacious, minimally toxic, and applicable by direct pipetting into cell culture without further intervention. Neuro9™ nanoparticles uniquely meet these criteria. To test Neuro9TM transfection in this system, the Hornstein Lab separated motor neurons from mouse embryonic spinal cord and plated them in 384-well plates. miRNA mimics encapsulated in Neuro9TM nanoparticles were applied by robotic multichannel pipette at two concentrations. The miRNA payload was measured in cell lysates at 24, 48 and 72h post-transfection to determine transfection levels. A dose-dependent response was observed, with over 100 fold increase in expression at a miRNA dose of 0.5 ng/µL. These results demonstrate how the ease of use of Neuro9TM transfection makes it well suited for screening applications. Furthermore, the potency of Neuro9TM transfection was also demonstrated.
Taken together, these experiments demonstrate how Neuro9™ can be used to transfect primary neurons and observe the phenotypic response in an automated, highly parallel functional screens for target validation and optimization of therapeutic nucleic acids.
In this chapter we provide a protocol for the design and usage of automated, high-content microscopy screening that enables the investigation of microRNA (miRNA) impact on primary motor neuron. High-content screening (HCS) platforms facilitate superior precision in research and are scalable to study in parallel multiple genetic, molecular, or cellular conditions. miRNAs are critical for neuronal function and for brain integrity and are considered attractive candidate targets for therapy in many neuropathologies. Therefore, HCS platforms provide a novel paradigm for exploring the impact of miRNA expression, applicable for functional pathways discovery in an academic setting, or towards development of therapeutics in the pharma industry.