Investigating neurodegenerative diseases with small molecule modulators

Abstract

Investigating neurodegenerative diseases with small molecule modulators Reka Rebecca Letso Elucidation of the mechanisms underlying cell death in neurodegenerative diseases has proven difficult, due to the complex and interconnected architecture of the nervous system as well as the often pleiotropic nature of these diseases. Cell culture models of neurodegenerative diseases, although seldom recapitulating all aspects of the disease phenotype, enable investigation of specific aspects of these disease states. Small molecule screening in these cell culture models is a powerful method for identifying novel small molecule modulators of these disease phenotypes. Mechanistic studies of these modulators can reveal vital insights into the cellular pathways altered in these disease states, identifying new mechanisms leading to cellular dysfunction, as well as novel therapeutic targets to combat these destructive diseases. Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are quite rare. Small molecule protein upregulators with targeted activities would be of great value in the study of the mechanisms underlying many loss of function diseases. We developed a highthroughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease Spinal Muscular Atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analog designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via translation, an effect which may be associated with the translational regulator mammalian target of rapamycin (mTOR). These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA. Small molecule modulators of a disease phenotype, such as cell death, have the potential to reveal novel mechanisms regulating disease processes. This was exemplified by a screen for small molecule inhibitors of cell death caused by a pathogenic, misofolded mutant huntingtin protein in a cell culture model of Huntington’s Disease (HD). These cell death inhibitors were found to target protein disulfide isomerase (PDI), an oxidoreductase known to be important in endoplasmic reticulum quality control of protein folding. However, our studies utilizing the small molecule PDI inhibitors determined that the cell death observed in this system was due to a pro-apoptotic function of PDI involving proteins of the mitochondrial outer membrane. We have begun studies aimed at identifying the binding mode of these novel small molecule inhibitors of PDI, in efforts to develop more potent and efficacious analogs for testing in animal models of HD. These studies have helped defined a novel mechanism linking protein misfolding to cell death, and may prove to be relevant to a broader range of protein misfolding diseases. Table of contents List of Figures and Tables v Acknowledgements vii Chapter 1: Spinal Muscular Atrophy 1 I. Treating inherited neurodegenerative diseases 1 a. Gain of function neurodegenerative diseases 2 b. Loss of function neurodegenerative diseases 5 II. Spinal Muscular Atrophy Disease 7 a. SMA disease pathogenesis 8 b. SMA disease phenotypes 9 c. Genetics of SMA 10 d. Animal models of SMA 15 III. Survival of Motor Neuron protein: Structure and Function 23 a. SMN expression and localization 23 b. SMN domain structure and functions 25 c. SMN interacting partners 28 i. The SMN Complex 29 ii. SMN interacts with hnRNP Q/R and β-actin mRNA 30 iii. SMN and profilin 32 iv. The possible role of actin dynamics in Spinal Muscular Atrophy 33 d. Neuronal function(s) of SMN 34 IV. Current state of small molecule therapeutics for SMA 46