Regulators of Cytoplasmic TDP43 Condensates as Therapeutic Targets
Abstract
Our studies are a collaborative effort between two research institutes, Gladstone and Lawrence Livermore National Laboratory (LLNL), and a private biopharmaceutical company, Prosetta Biosciences. Our work focuses on understanding the molecular and cellular mechanisms that cause degeneration of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). ALS has been linked to the RNA binding protein (RBP) TDP43. TDP43 normally localizes to the nucleus, where it regulates transcription and RNA metabolism. But in 90% of ALS patients, TDP43 is found to mislocalize to the cytoplasm. Neurodegeneration in ALS is believed to result in part from this aberrant cytoplasmic accumulation of TDP43, which keeps it from performing its normal role in RNA processing. We propose to identify the mechanisms by which TDP43 gets trapped into the cytoplasm and, using that information, develop small molecule drugs that can return TDP43 to its normal functions and reduce neurodegeneration in ALS, thereby slowing disease progression. We found that TDP43-induced neurodegeneration is dependent on several other RBPs. When we reduce the expression of these RBPs in neurons, TDP43-induced neurodegeneration is diminished. These RBPs are found with TDP43 in cytoplasmic structures called stress granules (SGs). We will investigate if these RBPs cause TDP43 cytoplasmic mislocalization and how their association with TDP43 leads to neurodegeneration in ALS. Our initial studies will test if knocking down the RBPs in human MNs protects them from degeneration. To this end, we will use MNs derived from the cells of patients with familial ALS and from ALS patients with sporadic disease. An innovation of our work is the use of one of the first human neuronal models of sporadic ALS patients. This is important because sporadic ALS is more frequent than familial ALS. Results from our studies on these cells may be most relevant to the general population of ALS patients. The MNs are obtained by first deriving induced pluripotent stem cells (iPSC)s from the patients cells, and are referred to as iPSC-derived MNs (i-MNs). A second innovation of our work is our use of a time-lapse imaging technology we invented, robotic microscopy (RM), which allows us to study the slow cellular processes that underlie neurodegeneration in ALS i-MNs. Using RM and the novel high-resolution imaging technologies of LLNL, including lattice light sheet microscopy (LLSM), we will investigate if knocking down the three RBPs reduces the cytoplasmic mislocalization and aggregation of TDP43 and slows neurodegeneration. RM and LLSM will also help us identify three-dimensional features of TDP43 s association with the RBPs in cytoplasmic aggregates that may contribute to TDP43 mislocalization. A prime focus of our studies is to identify small molecule compounds that reduce TDP43 cytoplasmic mislocalization and neurodegeneration that could be rapidly developed into drugs to treat ALS patients. Prosetta is developing drugs that reduce TDP43 mislocalization and aggregation in SGs as a means to treat ALS. They have identified compounds that reduce TDP43 mislocalization to SGs in the fibroblasts of ALS patients. Some have also been shown to reduce TDP43-induced neurodegeneration in an animal model of ALS. Their prototypical drugs are stable and have limited toxicity in animal studies; they also have good pharmacokinetics and central nervous system availability. Prosetta has conducted an extensive medicinal chemistry program to optimize their ALS drugs. We will work with Prosetta to test their prototypical and optimized compounds for efficacy in slowing neurodegeneration in our human ALS neuronal models. Generally, developing therapeutics to treat a disease involves extensive testing in animal models for efficacy. While animal models of ALS exist, all are based on the expression of mutant proteins linked to the disease and therefore represent only a minor population of patient
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210721
Entities
People
- Steve Finkbeiner
Organizations
- The J. David Gladstone Institutes
- United States Army