Xenium-based spatial transcriptomic screening identifies candidate mRNAs localized in neuronal and glial processes of the adult mouse cerebellum

Abstract

Subcellular mRNA localization contributes to local protein synthesis and functional compartmentalization in polarized cells, including neurons and glial cells. Although process-localized mRNAs have been identified by in situ hybridization, reporter-based analyses, and compartment-based transcriptomic approaches, systematic screening of such mRNAs within intact brain tissue while preserving tissue architecture remains challenging. Here, we developed a Xenium-based spatial transcriptomic screening strategy to identify candidate mRNAs localized in neuronal and glial processes in the adult mouse cerebellum. We focused on the molecular layer, which is densely occupied by Purkinje cell dendrites, Bergmann glial radial processes, and granule cell parallel fibers, but contains relatively few cell bodies. Using a Xenium Prime 5K dataset from adult mouse cerebellar sections, we first identified 199 genes whose transcripts were enriched in the molecular layer. By further focusing on DAPI-negative, process-rich regions and reducing contributions from molecular layer cell bodies, we extracted 126 candidate process-localized genes. Comparison with Allen Brain Atlas in situ hybridization data supported molecular layer localization with process-like patterns for 28 of 32 evaluable candidates. Integration with a published adult cerebellar single-nucleus RNA-seq atlas provided information on the possible cellular origins of these candidates. Gene Ontology analysis revealed enrichment of terms related to intracellular transport, cell projection structures, and synaptic function. These findings support the usefulness of a Xenium-based spatial transcriptomic first-screening strategy for identifying candidate process-localized mRNAs in intact brain tissue and provide a resource for studying RNA localization in cerebellar neuronal and glial processes.