Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease that affects motor neurons in the brain and spinal cord. Since motor neurons control the body's voluntary muscle activity, the loss of motors neurons leads to the paralysis of voluntary muscles such as those involved in speaking, walking, breathing, and swallowing. Approximately 5-10% of ALS cases are inherited, but most cases (90-95%) are of unknown cause. An established hallmark of ALS is the abnormal presence of protein clumps, called inclusions, within affected brain and spinal cord neurons. Recently, a protein named TAR DNA-binding protein-43 (TDP-43) was identified as the major component of these inclusions. In fact, TDP-43 inclusions are observed in ~95% of all ALS patients and mutations in the gene encoding TDP-43 have been identified in some sporadic and familial ALS patients, providing a direct link between abnormal TDP-43 and ALS. In healthy individuals, the TDP-43 present in neurons is mostly found within a specialized compartment: the nucleus. In affected motor neurons of ALS patients, however, TDP-43 is no longer present in the nucleus but instead is found in the cytoplasm, the fluid-filled region of neurons. As TDP-43 proteins accumulate in the cytoplasm, they begin to self-assemble and form the pathological inclusions. Additionally, some TDP-43 proteins are cleaved to generate shorter TDP-43 fragments. It is believed that these shorter fragments increase the speed at which inclusions are made. At present, there exists no effective treatment for ALS.

Given that we and others have shown that TDP-43 inclusions are toxic in various research models, we believe that drugs that prevent TDP-43 from forming inclusions will be an effective treatment for ALS. Therefore, we have developed a cell model that recapitulates the formation of TDP-43 inclusions observed in ALS. Specifically, we engineered human neuroblastoma cells to express a TDP-43 fragment that has a high tendency to self-assemble and form inclusions. In order to easily monitor the aggregation of this fragment into inclusions, we have attached it to another protein, the green fluorescent protein, which fluoresces green when exposed to blue light. Our goal is to use these cells to screen a small-molecule library for the identification of agents that block TDP-43 inclusion formation and the toxicity caused by the inclusions. Essentially, we will screen approximately 58,000 small-molecules with a high predictive value to cross the blood brain barrier. We have specifically chosen molecules that are likely to access the brain and spinal cord, as these are the regions affected in ALS. Of these ~58,000 compounds, our aim is to identify which ones decrease the amount of green fluorescence, and thus the amount of TDP-43 inclusions, present in cells. Additionally, we will analyze which of these small molecules also blocks the toxic effects caused by inclusions and validate all promising hits in at least two other cell models relevant to ALS. By the end of this three-year project, we expect to have discovered key small-molecule agents with strong evidence of therapeutic potential for ALS. Ultimately, in preparation for filing an investigational new drug (IND) application, the agents thus identified will subsequently be subjected to pre-clinical studies using animal models of ALS that are currently available in our laboratory. Because most ALS patients have TDP-43 inclusions within affected motor neurons, we believe that a treatment that targets TDP-43 inclusions would benefit the majority of ALS patients, including those patients that may have developed ALS due to their service with the military.