Master's Theses

Date of Award

2014

Document Type

Thesis

Department

Biology

First Advisor

Itzhak Mano

Keywords

Excitotoxicity, genetic screens, neurodegeneration

Abstract

Excitotoxicity is an important and frequently observed neurodegenerative process. Excitotoxicity mediates brain damage in a range of diseases and conditions including stroke, and is triggered by excessive stimulation of glutamatergic synapses. In spite of extensive studies, the molecular mechanisms involved in excitotoxicity following the over-activation of postsynaptic glutamate receptors are not well understood, and clinical trials based on our partial understanding of the process ended with disappointment. Genetic screens in simple animal models offer a powerful alternative approach, since screens are unbiased, analysis is facilitated by strong research tools, and cellular mechanisms are highly conserved through evolution. We produced a reliable model for excitotoxicity in the nematode C. elegans (the Δglt-3;nuIs5 strain) and we now use this model to screen for genes whose mutation can alter the extent of neurodegeneration. We are using two approaches to systematically knock-down/modify C. elegans genes and test their involvement in exicitotoxic neurodegeneration: a) We are developing sensitive strains that will allow us to use an RNAi library to knock down each gene in the nematode’s genome. b) We are using EMS to introduce random mutations throughout the genome. In both cases we are monitoring the effect of genetic modification on the level of neurodegeneration. We have screened ~2,000 mutagenized genomes and identified 2 mutant strains that show enhanced excitotoxicity and 6 mutant strains that show suppression/decrease in the level of neurodegeneration. To identify the most interesting mutants, we are carrying out behavioral assays that can indicate if the mutation affects overall normal synaptic activity by changing presynaptic release or perisynaptic receptor activity. We are mostly interested in mutants where these functions are normal, suggesting that the modified levels of neurodegeneration come from a specific effect on postsynaptic cell-death pathways. Following the characterization of cell death pathway mutants, we will be using whole genome sequencing to identify enhancer or suppressor mutations and study their mechanism of action.

 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.