Proteostasis regulation in ALS linked protein mediated toxicity

Abstract

The thesis entitled “Proteostasis regulation in ALS linked protein mediated toxicity” encompasses the proteostasis network in maintaining the cellular quality control system and its implication in the aspect of neurodegeneration. Neurodegenerative disease is often characterised as the accumulation of misfolded proteins which are highly aggregate prone and render toxicity to the neuronal cells. Autophagy, an important proteostasis mechanism, governs a key role in maintaining the balance between the protein aggregate formation and clearance; failing so leads to neurodegenerative diseases like ALS. The following chapters summarize the role of an ALS linked protein in neurodegeneration and its connection to autophagy thereafter. Chapter 1 is the literature summary about proteostasis machinery and its imbalance in context of neurodegeneration. This chapter narrates about the compromised proteostasis balance in neurodegenerative diseases like ALS and the implication of autophagy thereof. Chapter 2 contains the details of materials and reagents required in the experiments. The chapter content also includes the methodologies of the experiment performed. Here we have employed yeast model for Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease, to study the toxic effect rendered by the protein. For this, construct harbouring TDP43 (responsible factor for ALS) was transformed into wild type and Δatg1 yeast strains and subsequent experiments were performed. In chapter 3 we carried out various experiments like microscopy, spot dilution assay, growth assay to understand the toxic phenotype of the protein in yeast and its aggregate prone nature. Our result showed that there is defect in autophagy for the experimental strain. We sorted the cells by flow cytometry analysis to get the highest population of cells expressing the protein. Growth analysis revealed significant growth lag as compared to that of Wild type strain. We employed this evident growth lag as a tool to screen a small molecule library (ChemDiv library) for rescue of the growth defect. Chapter 4 summarizes the results we have found so far and concludes that indeed TDP43GFP shows toxic phenotype in yeast and this can be an appropriate model system to screen for the drugs and perform the experiments.