Purkinje Cell Nuclear Purification and Enrichment: A Single-Nucleus Exploration of Pathomechanisms in Polyglutamine Ataxias

Abstract

Spinocerebellar ataxia type 7 (SCA7) is a genetic neurodegenerative disorder caused by a CAG-trinucleotide repeat expansion coding for an elongated tract of glutamine residues in the Ataxin-7 protein. SCA7 is one of nine polyglutamine (polyQ) expansion disorders, each of which causes selective degeneration of neurons in various regions of the central nervous system (CNS). While the genetic basis of these disorders is well characterized, the cellular mechanisms which lead to neurodegeneration are still poorly understood, and no effective therapies exist for patients with these diseases. SCA7, and the five related polyglutamine SCAs, show preferential vulnerabilities in the cerebellum with degeneration of Purkinje cells (PCs) that are primarily responsible for the symptoms of ataxia in these patients. While PCs are central to the pathology of ataxias, it has been difficult to deeply characterize the molecular mechanisms associated with polyQ SCAs since PCs only represent ~1% of the cells in the cerebellum. Here I detail my research into the molecular mechanisms of SCA7 and related polyQ SCAs through the development of new methods to purify and enrich PC nuclei from mouse cerebellar tissues. I apply this Purkinje nuclear enrichment method to the SCA7-266Q mouse model followed by single-nucleus RNA-sequencing to profile differentially expressed genes (DEGs) in all cerebellar cell types across the timespan of symptom onset in SCA7. This experiment revealed hundreds of DEGs in symptomatic SCA7-266Q animals, leading to my discovery of increased inhibitory synapses, reduced PC spiking frequency, and signals of cell identity loss in PCs and glial cell types. Zebrin-II subtype dysregulation emerged as the predominant signal in SCA7 PCs, leading to the loss of zebrin-II striping concurrent with motor symptom onset. I show that zebrin-II striping degradation is a shared characteristic across SCA1, SCA2 and SCA3 mouse models, and provide evidence for zebrin-subtype dysregulation through snRNA-seq in SCA7 human cerebellar tissues. These results suggest that a breakdown of zebrin-subtype regulation is a unifying pathological feature of polyQ ataxias, and reveal several promising biological pathways for future therapy development.