Antibody-assisted selective isolation of Purkinje cell nuclei from mouse cerebellar tissue

Abstract

Summary: We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability. Motivation: Cerebellar Purkinje cells (PCs) play a central role in controlling voluntary movements, coordination, and motor learning. PC degeneration and death are hallmarks of many human neurological disorders, including multiple forms of spinocerebellar ataxia. Unfortunately, PCs comprise a relatively small population of cells in the cerebellum, constituting less than 1% of the total cell count. This makes studying PC-related pathology using omics approaches exceptionally challenging. Here, we introduce a protocol that addresses the need for obtaining pure fractions of PC nuclei for subsequent cell-specific analysis.

Keywords

• CP: Neuroscience