Mutant Profilin1 Aggregation in Amyotrophic Lateral Sclerosis: An in Vivo Biochemical Analysis

INTRODUCTION: Profilin1 (PFN1) is a ubiquitously expressed protein known for its function as a regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to Amyotrophic Lateral Sclerosis (ALS), which is a fatal and progressive motor neuron disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and the formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested in investigating the aggregation of PFN1 further and searching for co-aggregated proteins in our mouse model overexpressing mutant PFN1. METHODS: We investigated protein aggregation in several tissues of transgenic and notransgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using an insoluble fraction of spinal cord lysates from hPFN1(G118V) transgenic mice. For this assay, we expressed His(6)-tagged PFN1(WT) and PFN1(G118V) in E. coli and purified these proteins using the Ni-NTA column. RESULTS: In this study, we demonstrated that mutant PFN1 forms aggregate in the brain and spinal cord of hPFN1(G118V) mice, while WT-PFN1 remains soluble. Among these tissues, spinal cord lysates were found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His(6)-PFN1(G118V) showed that Myelin Binding Protein (MBP) was present in the insoluble fraction. CONCLUSION: Our analysis of PFN1 aggregation in vivo revealed further details of mutant PFN1 aggregation and its possible complex formation with other proteins, providing new insights into the ALS mechanism.