SLAS 2019 Advanced 3D Human Models and High-Content Analysis Symposium

Several neurodegenerative diseases are characterized by axon degeneration. This is especially true for the peripheral neuropathies, a heterogeneous group of human diseases characterized by progressive degeneration of peripheral axons. Charcot-Marie-Tooth disease (CMT) is a group of peripheral neuropathies caused by a variety of genetic mutations leading to length-dependent axon loss. CMT is the most common neurogenetic disease, affecting 3 million people worldwide. It is a devastating, untreatable disorder. It limits patients’ ability to walk, balance themselves and use their hands and is associated with significant functional disability. It can also cause marked sensory impairment and neuropathic pain. Despite significant advances in our understanding of the pathophysiology of CMT, disease-modifying therapies are entirely lacking, partially due to lack of translational models suitable for drug discovery. One of the major challenges in creating such models is the need to specifically analyze axons in vitro. Two-dimensional neuronal cultures often exhibit overlap of cell bodies and neurites, making it difficult to analyze axonal morphology and potential screenable phenotypes in a high throughput manner. To overcome this limitation, we created a three-dimensional culture system based on induced pluripotent stem cell (iPSC)-derived motor neurons (spinal spheroids), one of the main cellular types affected by CMT. Spinal spheroids can be formed by culturing sorted iPSC-derived motor neurons in ultra-low-attachment 384-well plates. When transferred to laminin-coated plates, spinal spheroids attach to the bottom of the well, allowing for the robust growth of axons in a centrifugal fashion, optimal for high content imaging. Using this system, we identified a robust and reproducible axonal phenotype in spinal spheroids from patients with CMT2E, a particular type of CMT caused by missense mutations in NEFL (neurofilament light chain gene). This phenotype is characterized by the abnormal accumulation of neurofilaments in discrete areas of the motor axons and nicely recapitulates findings from a mouse model of this same disease. This phenotype is also easily quantifiable using automated image analysis. A focused, proof-of-concept experiment investigating the effect of kinase inhibitors on the CMT2E axonal phenotype identified two compounds capable of improving neurofilament distribution in motor axons, demonstrating the potential of this platform as a useful tool for drug discovery in CMT. High throughput drug screening of several compound libraries is expected to start soon.

Co-Authors:
Renata, Maciel, PhD, MBA
Banupriya, Sridharan, PhD
Louis, Scampavia, PhD
Timothy, Spicer, PhD