A Cell-Based Assay To Identify Neuroprotective Molecules for the Treatment of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disease that affects adults. There is, at present, no effective treatment for this devastating disorder. Most cases of ALS occur spontaneously, although sometimes it can be inherited. Mutations in the antioxidant enzyme superoxide dismutase-1 (SOD1) are a cause of inherited ALS, and animals that express mutated SOD1 mimic the hallmarks of ALS, including the loss of the specific brain cells, namely motor neurons (MNs). The search for agents that can slow or even stop ALS has always been paramount. However, this quest has been hampered by a lack of screenable disease models with clear relevance to the human condition. Recently, research groups at Columbia University, who are involved in this proposal, have made the following groundbreaking observations: MNs can be taken from mouse brains or produced from mouse embryonic stem cell (ES-MNs); and, when these MNs are placed in a dish with non-neuronal supportive brain cells called astrocytes, which express mutant SOD1, the MNs die exactly as in ALS. Subsequently, this observation has been confirmed and extended by other groups to show that the toxicity of astrocytes on MNs also occurs in a dish if one uses human astrocytes from sporadic ALS patients (i.e., patient with ALS but no SOD1 mutations) and in living animals transplanted with ALS astrocytes.

Because of our replication of the ALS-like MN death in a dish, it may now be possible to screen for compounds that are protective against ALS. Furthermore, because our ALS model can be expanded infinitely, large quantities of the necessary cells (i.e., MNs) can readily be produced, thereby allowing, for the first time, the testing of a large number of small molecules in a short period of time. Since these recent discoveries have opened unprecedented avenues for drug development, we have, for the past 3 years, thanks to a previous Department of Defense (DoD) award, successfully adapted this ALS model in a dish as a powerful screening tool to search for small molecules for the treatment of ALS.

The first goal of this project will be devoted to high-throughput screening of our 80,000 small molecule library. For each compound, how potent it is in preventing the death of ES-MNs caused by mutant astrocytes will be calculated, and the best protective compound will be selected for further investigations. The second goal of this project will be dedicated to confirming the beneficial effects of ~100 hit compounds that we anticipate identifying in the preceding part of this project and to strengthening their ALS relevance. Indeed, ALS is a human condition, and MNs, which are selectively affected in this disorder, have long processes whose integrity is essential for proper motor control and neuromuscular connections. Thus, experiments in this second part of the project will ultimately examine the effects of the hit compounds on the survival of human MNs derived from the Presidential human ES cell lines cultured with human ALS astrocytes and on their capacity to protect not only the cell bodies but also the processes of MNs.

In agreement with the intent of this program announcement, our DoD project proposes a comprehensive set of investigations geared toward the screening of therapeutics. We anticipate that, by the end of this 3-year DoD project, our entire library of ~80,000 small molecules will have been tested in full and that the ~5 lead candidate compounds with the highest potency, efficacy, relevance to the human condition, and predicted drugability will advance into a therapy development project for ALS. The latter will consist of preclinical studies based on a set of toxicity and pharmacologic investigations in living animals, and testing of the lead candidates in the best available animal models of ALS for their ability to modify the expression and progression of the paralysis in these animals.

All of the techniques, reagents, and expertise necessary for the performance of this project are available in the laboratories of the research team. Thus, it is anticipated that no difficulties would be encountered in completing this research project within the period of the award. Ultimately, we expect that, at the end of the above investigations, several small molecules with neuroprotective properties for ALS will not only have been identified, but will also have been extensively characterized, making them ready for regulatory testing in preparation for review by the Federal Drug Administration, the mandatory step prior to the clinical use of any of the identified small molecules.