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

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.

Purkinje-cell-specific DNA repair-deficient mice reveal that dietary restriction protects neurons by cell-intrinsic preservation of genomic health

“…Previously, we have shown that ERCC1-deficient mice develop multiple age-dependent neurological deficits and progressive neuropathology characterized by a diversity of degenerative changes throughout the nervous affecting both neurons and glia ( de Waard et al, 2010 ; Borgesius et al, 2011 ; Vegh et al, 2012 ; Raj et al, 2014 ; Vermeij et al, 2016a ). The nervous system pathology is consistent with a model where stochastic accumulation of DNA damage causes transcription stress, i.e., a variety of yet incompletely defined transcription problems and down-stream effects that can affect cell function in multiple ways, for instance by blocked or deregulated expression of essential genes or problems resulting from persistent transcription stalling ( Vermeij et al, 2016b ; Niedernhofer et al, 2018 ; Lans et al, 2019 ; Mulderrig and Garaycoechea, 2020 ; Apelt et al, 2021 ; Jia et al, 2021 ; Van't Sant et al, 2021 ). Here, we focused on stereological analysis of residual Purkinje cell numbers as the principal readout, representing an unequivocal quantitative readout that reflects the final outcome of multiple neurodegenerative pathways.…”

“…We generated a Cre-lox-based Purkinje cell-specific ERCC1-deficient ( Pcp2-Ercc1 Δ/f ) mouse model that carried a floxed Ercc1 allele ( Ercc1 f ), a C-terminally truncated “delta” Ercc1 allele ( Ercc1 Δ ; Vermeij et al, 2016b ), and a Pcp2-Cre transgene (Mpin line) that drives Cre recombinase expression in postnatal Purkinje cells ( Van't Sant et al, 2021 ). After recombination by Cre, the floxed Ercc1 allele becomes a null allele, resulting in Purkinje cells that have one null and one truncated Ercc1 allele, and that reproduce the severe (but incomplete) reduction in ERCC1/XPF nuclease function of whole body Ercc1 Δ/− mice ( Figure 1A ), affecting nucleotide excision repair, inter-strand crosslink repair, and single-strand annealing of double-strand breaks ( Vermeij et al, 2016b ; Mulderrig and Garaycoechea, 2020 ; Apelt et al, 2021 ).…”

“…Ercc1 ∆/− , Pcp2-Ercc1 Δ/f , and Pcp2-Ercc1 f/− were generated as previously described ( de Waard et al, 2010 ; de Graaf et al, 2013 ; Vermeij et al, 2016a ; Van't Sant et al, 2021 ). To obtain Ercc1 ∆/− mice, we crossed Ercc1 ∆/+ with Ercc1 +/− mice (both in pure FVB or C57BL6J backgrounds respectively) to yield Ercc1 ∆/− offspring with a genetically uniform C57BL6J/FVB F1 hybrid background ( Weeda et al, 1997 ; Vermeij et al, 2016a ).…”

“…Subsequently, female Pcp2-Cre + /Ercc1 ∆ /+ were crossed with male Ercc1 f/f mice in FVB background to yield Pcp2-Cre + /Ercc1 ∆ /f in a uniform C57BL6J/FVB F1 hybrid background. Pcp2-Cre + /Ercc1 +/f and Pcp2-Cre − /Ercc1 ∆ /f transgenic littermates that do not develop a degenerative phenotype served as controls ( Van't Sant et al, 2021 ). Additional controls for this study were wild-type animals from the same C57BL6J/FVB F1 hybrid genetic background.…”

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

“…4-SUrd was shown to affect cell viability upon prolonged exposure and to inhibit the synthesis of rRNA, which significantly impacts the population of RNA observed upon analysis . Similarly, 5-EUrd was also shown to reduce cell viability with prolonged exposure and can cause neurodegeneration in mice . These undesired effects of modified nucleosides present a clear need for a novel nucleoside suitable for RNA metabolic labeling with minimum perturbation to endogenous systems.…”