Neurodegenerative diseases that affect motor neurons such as PD, ALS and HD represent an enormous unmet medical need that is growing with the aging population. Existing treatments have little or no effect on the course of disease, and patients have to cope with the loss of brain and body function for the rest of their lives. Defects in the transport of organelles and biomolecules through axons is a hallmark of early stage disease and there is strong evidence that it contributes to the dying back pathology seen in most motor neuron diseases, which is characterized by early degeneration of the synapse and the axon followed by damage to the soma and eventual neuronal loss. Therefore, discovery of drugs that prevent or rescue axonal transport defects is a compelling strategy for early intervention in neurodegeneration. However, the difficulty in tracking cargo movement through axons has prevented the scaling of assays for high throughput screening (HTS) and discouraged the use of physiologically relevant models incorporating glial cells and three dimensional (3D) tissue architecture. We will overcome these technical hurdles by using BellBrook's proprietary iuvo(R) microchannel plate technology to enable automated high content analysis (HCA) of axonal transport in 3D cocultures of neurons and astrocytes derived from induced pluripotent stem cells (iPSC). Flow-based collagen patterning in microchannels will be used to align both cell types with the longitudinal axis of the channel. Alignment of neurons combined with the height restrictions imposed by the microchannel will make it vastly simpler to track the movement of axonal cargoes, allowing the use of streamlined image acquisition that is scalable for high throughput screening (HTS). Moreover, a uniform cell polarity along collagen tracks will resemble in vivo tissue architecture much more closely than monolayers of randomly oriented neurons. This is a collaborative effort leveraging the microfluidics and expertise of Dr. David Beebe, John D. MacArthur Professor in the Department of Biomedical Engineering at UW- Madison and Dr. Robert Lowery's long track record in developing enabling HTS assay products at BellBrook Labs. We will optimize 3D coculture and neural cell alignment iuvo(R) Microchannel 5250 plate at BellBrook (Aim 1), while Dr. Beebe's methods in the existing group develops a new prototype microchannel slide (8 channels) with modifications to optimize optics and axon patterning for image based Aligned 3D neural coculture for HTS imaging of axonal transport. Microchannel plates analysis of axonal transport (Aim 2). Lastly, we will have arrays of precisely patterned channels in a standard SLAS footprint (shown is iuvo(R) use the new device to streamline methods for Microchannel 5250, with 192 channels). Longitudinal alignment of astrocytes (gray) and neurons along collagen fibrils combined with optimization of channel design will enable image based tracking of axonal cargo transport, rapid, scalable imaging of axonal transport. The microchannel plates are compatible establishing feasibility for scaling the device and with standard liquid dispensing and automated microscopy equipment, enabling robust high throughput assays for axonal transport in a 3D tissue-like microenvironment. methods to HTS in Phase II. To our knowledge, the proposed microchannel platform for aligned 3D cocultures will be the first in vitro model for probing axonal transport ina 3D, tissue-like microenvironment and the first HTS-scalable assay. An HTS-compatible phenotypic assay platform for axonal transport could enable the discovery of drugs that prevent or slow neuronal loss early in the disease process, and thereby accelerate the development of more effective treatments for devastating neurodegenerative diseases that affect a growing fraction of our aging population.
Transport of organelles and biomolecules between cell bodies and nerve endings, known as axonal transport, is critical to the function or neurons, and this process often becomes defective early in the course of neurodegenerative diseases like Parkinson's disease and Multiple Sclerosis. This proposal seeks to develop in vitro assays to identify new drugs that will prevent or rescue axonal transport defects, so that neurodegeneration can be slowed or prevented before irreversible damage occurs.
