In vivo 5-ethynyluridine (EU) labelling detects reduced transcription in Purkinje cell degeneration mouse mutants, but can itself induce neurodegeneration

Lisanne J Van’T Sant,Jan H J Hoeijmakers,Dick Jaarsma,Joshua J White,Wilbert P Vermeij

doi:10.1186/s40478-021-01200-y

Abstract

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress. Here, we tested whether EU labelling can be used to examine transcription in vivo in mouse models of nervous system disorders. We show that injection of EU directly into the cerebellum results in reproducible labelling of newly transcribed RNA in cerebellar neurons and glia, with cell type-specific differences in relative labelling intensities, such as Purkinje cells exhibiting the highest levels. We also observed EU-labelling accumulating into cytoplasmic inclusions, indicating that EU, like other modified uridines, may introduce non-physiological properties in labelled RNAs. Additionally, we found that EU induces Purkinje cell degeneration nine days after EU injection, suggesting that EU incorporation not only results in abnormal RNA transcripts, but also eventually becomes neurotoxic in highly transcriptionally-active neurons. However, short post-injection intervals of EU labelling in both a Purkinje cell-specific DNA repair-deficient mouse model and a mouse model of spinocerebellar ataxia 1 revealed reduced transcription in Purkinje cells compared to controls. We combined EU labelling with immunohistology to correlate altered EU staining with pathological markers, such as genotoxic signalling factors. These data indicate that the EU-labelling method provided here can be used to identify changes in transcription in vivo in nervous system disease models.

Highlights

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress
We examined EU labelling in the cerebellum of two Purkinje cell-specific mutant mouse models for, respectively, a severe progeroid DNA repair deficiency syndrome (Ercc1d/fPcp2-Cre mice) [11] and spinocerebellar ataxia 1 (SCA1, ATXN1[82Q] mice) [7]
Cerebellar EU injections result in differential intensities and temporal changes of EU labelling between cell types In previous in vivo studies in mice, EU was injected intraperitoneally (330–750 μmol/g animal), resulting in labelling of nascent RNA in cells of multiple organs and tissues, but not in the central nervous system [29, 39]

Summary

Introduction

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress. We show that injection of EU directly into the cerebellum results in reproducible labelling of newly transcribed RNA in cerebellar neurons and glia, with cell type-specific differences in relative labelling intensities, such as Purkinje cells exhibiting the highest levels. We examined EU labelling in the cerebellum of two Purkinje cell-specific mutant mouse models for, respectively, a severe progeroid DNA repair deficiency syndrome (Ercc1d/fPcp2-Cre mice) [11] and spinocerebellar ataxia 1 (SCA1, ATXN1[82Q] mice) [7]. In both mouse models, we found reduced EU-labelling intensities consistent with transcriptional abnormalities. Our data show that EU may be neurotoxic for Purkinje cells in the long-term, the EU labelling method can be used to investigate changes in transcription in the central nervous system in vivo using short post-injection survival times

Methods

Results

Conclusion