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 death of motor neurons (MNs), cells in the spinal cord responsible for movement and breathing. Many attempts have been made to search for agents that can slow or even halt the ALS disease process. 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 and Harvard Universities involved in this project have made some groundbreaking observations. They showed that MNs can be taken from mouse spinal cord or produced from mouse embryonic stem cells (ES-MNs). Such ES-MNs normally survive for long periods in culture, but when they are grown with astrocytes -- a class of non-neuronal cells that surround MNs in the spinal cord -- that express high levels of ALS-mutant SOD1, the ES-MNs undergo a cell death that mimics that observed in ALS. The ability to replicate ALS-like MN death in a dish now makes it possible to screen for compounds that may protect MNs against ALS. Furthermore, because our ALS model can be expanded infinitely, large quantities of the necessary cells (i.e., ES-MNs bearing the disease-causing mutation) can be produced, thereby allowing us to test a large number of small molecules in a short period of time.

We have assembled a bi-institutional team of experts committed to this 4-year project that proposes sequential validation steps of small molecules for the treatment of ALS. The first goal of this project will be devoted to high-throughput screening of our large libraries of small chemical compounds. By testing each compound individually, we will identify those that are most potent in preventing the death of mutant ES-MNs grown with mutant astrocytes. All active compounds will be tested at both Columbia and Harvard and the 100 most promising compounds will be selected for further investigation. The second goal of this project will be to confirm the beneficial activities of these 100-hit compounds and to analyze their relevance to ALS. To determine whether they are active in the human context, we will examine the effects of the compounds on the survival of human ES-MNs derived from Presidential human ES cell lines cultured with mutant astrocytes. We will also confirm their activity on MNs isolated directly from ALS animal models and determine whether they act on MNs or astrocytes, or both. Finally, we will assay their capacity to protect not only the cell bodies but also the axonal processes of MNs, which degenerate in ALS, leading to a loss of contact with target muscles.

The third goal of this project will begin by selecting ~20 lead compounds from those tested in the first two parts of the project, based on their potency, efficacy, and predicted drugability. To select the optimal compounds to be tested for protection against MN death in the animal model of ALS, compounds will be studied to determine their solubility and stability in fluids that mimic those found in the body. We will also determine their optimal route of administration in mice to achieve maximal brain concentrations with minimal adverse effects. The fourth and final goal of this project will focus on the actual preclinical testing of two compounds with the most promising drug-like properties as determined in the first three aims. For each compound, we will also select two others from our chemical compound collections that belong to the same chemical families but which show some structural differences. These will also be tested to determine if such analogs may prove even more beneficial. Each of the six compounds will be administered chronically in parallel at Columbia and Harvard in the best current mouse model of ALS to determine whether they can slow the progression of 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, making it feasible to complete the project within the period of the Award. Strict criteria for compound selection have been set at the different key steps of the project so that from ca. 180,000 molecules at the start, we expect to end this 4-year project by having identified two to six novel compounds with activity in the most relevant experimental models of ALS currently available. Subsequent studies geared toward elucidating how these lead compounds work and modifying their chemical properties should lead to identification of new drug candidates for this currently incurable disease. This proposal thus contains a comprehensive set of experiments that may have far-reaching implications for the treatment and prevention of ALS.