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.

Dietary restriction (DR) is a universal anti-aging intervention, which reduces age-related nervous system pathologies and neurological decline. The degree to which the neuroprotective effect of DR operates by attenuating cell intrinsic degradative processes rather than influencing non-cell autonomous factors such as glial and vascular health or systemic inflammatory status is incompletely understood. Following up on our finding that DR has a remarkably large beneficial effect on nervous system pathology in whole-body DNA repair-deficient progeroid mice, we show here that DR also exerts strong neuroprotection in mouse models in which a single neuronal cell type, i.e., cerebellar Purkinje cells, experience genotoxic stress and consequent premature aging-like dysfunction. Purkinje cell specific hypomorphic and knock-out ERCC1 mice on DR retained 40 and 25% more neurons, respectively, with equal protection against P53 activation, and alike results from whole-body ERCC1-deficient mice. Our findings show that DR strongly reduces Purkinje cell death in our Purkinje cell-specific accelerated aging mouse model, indicating that DR protects Purkinje cells from intrinsic DNA-damage-driven neurodegeneration.

BackgroundDietary restriction (DR) is a well-established universal anti-aging intervention, and is neuroprotective in multiple models of nervous system disease, including models with cerebellar pathology. The beneficial effects of DR are associated with a rearrangement of gene expression that modulate metabolic and cytoprotective pathways. However, the effect of DR on the cerebellar transcriptome remained to be fully defined.ResultsHere we analyzed the effect of a classical 30% DR protocol on the transcriptome of cerebellar cortex of young-adult male mice using RNAseq. We found that about 5% of expressed genes were differentially expressed in DR cerebellum, the far majority of whom showing subtle expression changes. A large proportion of down-regulated genes are implicated in signaling pathways, in particular pathways associated with neuronal signaling. DR up regulated pathways in large part were associated with cytoprotection and DNA repair. Analysis of the expression of cell-specific gene sets, indicated a strong enrichment of DR down genes in Purkinje cells, while genes specifically associated with granule cells did not show such a preferential down-regulation.ConclusionOur data show that DR may have a clear effect on the cerebellar transcriptome inducing a mild shift from physiology towards maintenance and repair, and having cell-type specific effects.