Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the lives of at least 30,000 people in the United States, annually. It is often categorized by the progressive neurodegeneration of motor neurons. TAR DNA Binding Protein (TDP-43) is normally found within the nucleus having known roles in RNA splicing as well as DNA-binding. Mislocalization of TDP-43 to the cytoplasm, either due to mutations or to environmental stressors leads to accumulation of TDP-43 in cytoplasmic inclusions. Notably, in the cytoplasm, TDP-43 associates with RNA stress granules and also affects mRNA translation, both of which are thought to contribute to disease pathology. In order to study the effects that cytoplasmic TDP-43 mutations have on translation, a technique called polysome fractionation is utilized. This technique uses sucrose gradients to effectively separate different ribosomal populations. The mRNAs that are bound to multiple ribosomes, or polysomes, are separated from the mRNAs bound to single ribosomes. This allows the pinpointing of specific defects in protein production as well as potential explanations to how it may affect cellular metabolism. Furthermore, this technique is able to provide information about the halting of protein production in response to stress which is a potential factor that contributes to ALS. Western blotting and quantitative PCR (qPCR) allow us to observe the protein and transcript distribution within the polyribosome fractions, respectively. Using these approaches we found that TDP-43 associates with both translating polyribosomes as well and untranslated fractions (RNP and the ribosomal subunits). qPCR of futsch, an mRNA target of TDP-43 showed a shift in transcript levels from actively translated to untranslated fractions in the context of TDP-43, indicating that futsch mRNA is being repressed by TDP-43 in motor neurons. Taken together, these findings indicate that TDP-43 regulates the translation of specific mRNAs and defects in translation may contribute to ALS. Experiments will focus on additional candidate mRNA targets that will be tested for their distribution within polysome fractionations to determine their translational status in disease. Candidate mRNAs that are identified by Translating Ribosome Affinity Purification (TRAP) that also associate with TDP-43 in complex will be tested. Specifically, qPCR will used to distinguish shifts in translational targets in polysomes. Targets that are downtranslated should exhibit a shift into the RNP fractions while uptranslated targets will exhibit a shift into the polysomes.