Modeling the mature CNS: A predictive screening platform for neurodegenerative disease drug discovery

Abstract

Background: Mature primary neuronal cultures are an important model of the nervous system, but limited scalability has been a major challenge in their use for drug discovery of neurodegenerative diseases. This work describes a method for improving scalability through the use of larger format microtiter plates while preserving culture quality. New Method: Here we describe a method and quality control procedures for growing embryonic day 18 rat hippocampal/cortical neuronal cultures in 384-well microtiter plates for three weeks in vitro . Results: We use these cultures in two assays measuring intracellular lipid vesicle trafficking and synapse density for routine screening of small molecule libraries. Together this culture system and screening platform have successfully identified therapeutics capable of improving cognitive function in transgenic models of Alzheimer’s disease that have advanced to clinical trials, validating their translational applicability. Comparison with Existing Methods: Our method enables the growth of healthy, mature neurons in larger format microtiter plates than in traditional primary neuronal culturing protocols, making it ideal for drug screening and mechanism of action studies. Conclusion: The predictive capacity of this culture system and screening platform provides a method for rapidly identifying novel disease-modifying neurodegenerative therapeutics.

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity

“…We used confocal microscopy to assess the morphology of DIV21 (21 days in vitro ) primary hippocampal neurons following 7 days of precursor trapping or Tau variant over-production by transient transfection. DIV21 neurons equate to ‘mature’ neurons with spines and fully formed synapses ( Grabrucker et al, 2009 ; LaBarbera et al, 2021 ), and therefore allow an analysis of synapses and neuronal complexity. Axons were identified by staining for ankyrin-G, a scaffolding protein important for the formation of the initial segment of the axon ( Grubb and Burrone, 2010 ).…”

Perturbation of the mitochondrial import machinery by aggregation prone Tau affects organelle morphology and reduces neuronal complexity

“…We used confocal microscopy to assess morphology of DIV21 (21 days in vitro ) primary hippocampal neurons following 7 days of precursor trapping or Tau variant over-production. DIV21 neurons equate to ‘mature’ neurons with spines and fully formed synapses (Grabrucker et al, 2009; LaBarbera et al, 2021) and therefore allow analysis of synapses and neuronal complexity. Axons were identified by staining for Ankyrin G, a scaffolding protein important in formation of the initial segment of the axon (Grubb & Burrone, 2010).…”

Long-Term Cultures of Spinal Cord Interneurons

“…Our goal was to create a culture of spinal cord neurons that models the maturing and mature CNS and which is suitable for future experiments designed to enhance the ability of the neurons to regenerate. The viability of embryonic neuronal cultures is a great advantage in culture preparation, but the immaturity of the neurons after seeding is a problem as they display entirely different properties compared to mature neurons that have lost their intrinsic ability to regenerate (LaBarbera et al, 2021). To evaluate the maturity of our cultures, we investigated the expression of DBC, NF70, synaptic development, and electrophysiological properties of neurons during cultivation.…”