Developing Cell-Based and Mechanism-Focused Preclinical Platforms with Diseased Upper Motor Neurons

Background: Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive degeneration of the motor neuron circuitry, which have components both in the brain and in the spinal cord. The cortical component of the motor neuron circuitry is especially important for the initiation and modulation of voluntary movement because the movement starts in the brain. However, the motor neurons in the brain (i.e., the upper motor neurons (UMNs), or Betz cells in humans and corticospinal motor neurons in mice) have been neglected in ALS drug discovery efforts. None of the compounds currently in clinical trials have been examined in relationship to the health and disease of UMNs. This is also true for more than 35 clinical trials that have failed in the past. Inclusion of the cortical component of ALS in drug discovery and drug development will improve the success rates of ALS clinical trials. Objective: The objective of this proposal is to develop the first semi high-throughput assay, which utilizes the improved health and stability of diseased UMNs as a measure of potential clinical effectiveness. We propose a cell-based and mechanism-focused approach to ALS drug discovery, based on the hypothesis that targeting diseased motor neurons for drug development will translate into improvement in the integrity of motor neuron circuitry and motor behavior in patients. This will be combined with a mechanism-focused approach to targeted drug discovery that will tailor drug discovery to address differing etiologies of motor neuron degeneration, such as TDP-43 pathology, lipid homology defects, problems with actin/tubulin dynamics, cytoarchitectural defects, misfolded SOD1 toxicity, and others. This approach is built on an understanding of the heterogeneity of ALS etiologies and its application to identifying compounds that will be effective for each patient population. Specific Aims: The specific aims of the project are to develop and establish a semi high-throughput assay, that will: 1) utilize genetically labeled primary cortical cultures, in which healthy and diseased UMNs allowing their precise identification and cellular assessment based on eGFP expression; 2) enable live tracking of cellular responses to treatment; 3) help visualize and assess the mechanism of action in response to treatment; and 4) deploy an array of UMNs representing various disease etiologies to enable alignment of disease mechanisms with responses to specific compounds of therapeutic interest. Study Design: The proposal leverages the UMN reporter lines generated in the Ozdinler lab and initial success with cell-based and mechanism-focused drug discovery effort. In partnership with the High Throughput Analysis Lab, we will establish preclinical assays using UMNs that are diseased due to mSOD1 toxicity, TDP-43 pathology, lack of Alsin function, mutations in the profilin and spastin genes. Because diseased UMNs can be distinguished among other cells and neurons, their responses will be recorded live and assessments will be based on their cellular responses. We will perform imaging studies to quantitatively assess dynamic changes in organelles in response to treatment using trackers for different organelles, such as mitotracker for mitochondria and lysotracker for lysosomes. We will perform unbiased assessment and statistical analyses with proper power to determine whether compound(s) of interest improve/s the health of diseased UMNs, and if so, which disease model shows the most significant response and at which dose. These data will enable us to determine the mode of act