60 Impact of Reducing the Nuclear Mutant ATXN1 on Spinocerebellar Ataxia-Like Phenotype
Authors: Mather Kathleen B , Duvick Lisa A , Handler Hillary P , Orr Harry T 
Published: December 2023

Objective:Spinocerebellar ataxia type one (SCA1) is an autosomal dominant neurodegenerative disease caused by an expanded CAG repeat that encodes glutamine (polyQ) in the affected ATXN1 gene. SCA1 pathology is commonly characterized by the degeneration of the cerebellar Purkinje cells (PC) and brainstem. Symptoms include motor dysfunction, cognitive impairments, bulbar dysfunction, and premature death. Atxn1175Q/2Q knock-in mice were previously developed to model SCA1 by inserting 175 expanded CAG repeats into one allele of the Atxn1 gene, producing mice expressing ATXN1 throughout the brain and displaying SCA1 symptoms. Previous research has indicated the role of localization of the ATXN1 protein to the nucleus in pathology. Therefore, the Atxn1175QK772T/2Q mouse model was created by disrupting the NLS in the expanded Atxn1175Q/2Q mice by replacing lysine with threonine at position 772 in the nuclear localization sequence (NLS). Since this amino acid change previously blocked PC disease in another mouse model, the Atxn1175QK772T/2Q mice were created to examine how the NLS mutation affects neuronal cells. RNA sequencing analysis was previously performed and found differentially expressed genes (DEG) with Atxn1175Q/2Q downregulated compared to Atxn1175QK772T/2Q and Atxn12Q/2Q in the cerebellum, medulla, cortex, hippocampus, and striatum. The aim was to analyze these brain regions to validate the RNAseq differential gene expression at a protein level.Participants and Methods:Therefore, western blots were performed on the following mouse models (n=12): wild type mice (Atxn12Q/2Q), mice with the nuclear localization sequence mutation (Atxn12QK772T/2Q), and mice with 175 expanded CAG repeats (Atxn1175/2Q). Based off the RNAseq data, the cerebellum was tested with ion channel genes (Cav3.1, Kcnma1, and Trpc3) and the striatum was tested with a gene found in medium-spiny neurons (DARPP-32).Results:In the cerebellum, Atxn1175/2Q was significantly downregulated compared to Atxn1175QK772T/2Q in Cav3.1, Trpc3, and Kcnma1. Atxn1175Q/2Q was significantly downregulated compared to Atxn12Q/2Q in Trpc3 and Kcnma1. Atxn1175QK772T/2Q was significantly downregulated compared to Atxn12Q/2Q in Trpc3. In the striatum, there was significantly reduced DARPP-32 expression found between Atxn12Q/2Q and Atxn1175QK772T/2Q, Atxn12Q/2Q and Atxn1175Q/2Q, and Atxn1175Q/2Q and Atxn1175QK772T/2Q.Conclusions:Therefore, the significantly reduced gene expression at the protein level in the cerebellum and striatum validate RNAseq differentially expressed genes. Additionally, the downregulation of both the Atxn1175Q/2Q and Atxn1175QK772TQ/2Q compared to Atxn12Q/2Q in the striatum supports the lack of learning of those mouse models on the rotarod, suggesting that the nuclear localization mutation does not rescue learning. Interestingly, the downregulation of Atxn1175Q/2Q compared to Atxn1175QK772TQ/2Q likely supports the age-related motor decline rescue in the rotarod seen in Atxn1175QK772T/2Q and not Atxn1175Q/2Q.