The Effect of Arf Knockdown on ALS Pathology Using Model ALS Motor Neurons from Mice




Emeritus Professor of Neurology

Project Summary

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s Disease, is a neurodegenerative disease in which progressive death of motor neurons occurs in the brain, brainstem, and spinal cord. Arf proteins are highly conserved GTPases that are involved in membrane trafficking, lipid metabolism/signaling, actin remodeling, and lipid droplet formation. Recent discoveries show that Arfrelated cellular processes may be mutated and disturbed in ALS motor neurons. For example, defective membrane trafficking caused by impaired dynein function can be linked to motor neuron diseases and neurodegeneration generally and lipid metabolism abnormalities can be found in mutant SOD mice and TDP-43 knockout mice. These discoveries make the Arf family of GTPases a very interesting subject to study for disease cell signaling in cell and animal-based moles of ALS. Vertebrates and C. elegens have six different Arf GTPases which share common general functions and high sequence homology but have different and specific targets in membrane trafficking. The purpose of this study is to design and test microRNAs that will silence the protein production of the six primary Arf proteins in mice specifically. These miRNA constructs will then be used in future studies directed to elucidate the different Arf signaling pathways within model ALS diseased neurons from mice. miRNA mARF4-508 was successful in specifically knocking down mARF4 and not the other ARFs and ARF5 knockdown was rescued by hARF4, showing that miRNA mARF4-508 was specific to mouse ARF4. miRNA mARF5-58 was successful in knockdown mARF5 and not the other ARFs, and ARF5 knockdown was rescued by hARF5, showing that miRNA mARF5-58 was specific to mouse ARF5. Given that the generated RNAi constructs of Arf 4 and 5 have successfully knocked down the target Arf specifically and Arf protein production could be rescued by the human protein homolog, a possible next step of this project would involve using biochemical assays to follow the how protein trafficking (ER-Golgi) in ALS disease neurons in mice may be mutated and Arf’s role in such protein trafficking. While the RNAi constructs generated thus far for Arf 1, 2, 3, and 6 has not worked in silencing protein production of these Arfs, continued work will be done to design better RNAi constructs and testing the efficacy of these constructs