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1- Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.
2- Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.
3- Department of Pharmacology and Toxicology, Department of Neurology, Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Abstract:  
Abstract
Introduction:
Profilin1 (PFN1) is a ubiquitously expressed protein that is well known for its function as regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to ALS as a fatal and progressive motor neurons disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested to further investigate the aggregation of PFN1 and search for co-aggregated proteins in our mouse model overexpressing mutant PFN1.
Methods:
We investigated protein aggregation in several tissues of transgenic and no-transgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using Ni-NTA column.
Results:
In this study, we demonstrated that mutant PFN1 forms aggregates in the brain and spinal cord of hPFN1G118V mice, while WT PFN1 remains soluble. Among these tissues, spinal cord lysates found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V 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 complexes formation with other proteins which may provide new insights in ALS mechanism.
Abstract
Introduction:
Profilin1 (PFN1) is a ubiquitously expressed protein that is well known for its function as regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to ALS as a fatal and progressive motor neurons disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested to further investigate the aggregation of PFN1 and search for co-aggregated proteins in our mouse model overexpressing mutant PFN1.
Methods:
We investigated protein aggregation in several tissues of transgenic and no-transgenic mice using western blotting. To gain further insight into the neurotoxicity of mutant PFN1, we conducted a pull-down assay using insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using Ni-NTA column.
Results:
In this study, we demonstrated that mutant PFN1 forms aggregates in the brain and spinal cord of hPFN1G118V mice, while WT PFN1 remains soluble. Among these tissues, spinal cord lysates found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V 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 complexes formation with other proteins which may provide new insights in ALS mechanism.
 
Type of Study: Original | Subject: Cellular and molecular Neuroscience
Received: 2018/12/15 | Accepted: 2019/04/6

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