Search

Background: Dietary 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. Results: Here 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. Conclusion: Our 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.

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.

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) and rapamycin extend healthspan and life span across multiple species. We have recently shown that DR in progeroid DNA repair-deficient mice dramatically extended healthspan and trippled life span. Here, we show that rapamycin, while significantly lowering mTOR signaling, failed to improve life span nor healthspan of DNA repair-deficient Ercc1( increment /-) mice, contrary to DR tested in parallel. Rapamycin interventions focusing on dosage, gender, and timing all were unable to alter life span. Even genetically modifying mTOR signaling failed to increase life span of DNA repair-deficient mice. The absence of effects by rapamycin on P53 in brain and transcription stress in liver is in sharp contrast with results obtained by DR, and appoints reducing DNA damage and transcription stress as an important mode of action of DR, lacking by rapamycin. Together, this indicates that mTOR inhibition does not mediate the beneficial effects of DR in progeroid mice, revealing that DR and rapamycin strongly differ in their modes of action.

Purkinje cells are the primary processing units of the cerebellar cortex and display molecular heterogeneity that aligns with differences in physiological properties, projection patterns, and susceptibility to disease. In particular, multiple mouse models that feature Purkinje cell degeneration are characterized by incomplete and patterned Purkinje cell degeneration, suggestive of relative sparing of Purkinje cell subpopulations, such as those expressing Aldolase C/zebrinII (AldoC) or residing in the vestibulo-cerebellum. Here, we investigated a well-characterized Purkinje cell-specific mouse model for spinocerebellar ataxia type 1 (SCA1) that expresses human ATXN1 with a polyQ expansion (82Q). Our pathological analysis confirms previous findings that Purkinje cells of the vestibulo-cerebellum, i.e., the flocculonodular lobes, and crus I are relatively spared from key pathological hallmarks: somatodendritic atrophy, and the appearance of p62/SQSTM1-positive inclusions. However, immunohistological analysis of transgene expression revealed that spared Purkinje cells do not express mutant ATXN1 protein, indicating the sparing of Purkinje cells can be explained by an absence of transgene expression. Additionally, we found that Purkinje cells in other cerebellar lobules that typically express AldoC, not only display severe pathology but also show loss of AldoC expression. The relatively preserved flocculonodular lobes and crus I showed a substantial fraction of Purkinje cells that expressed the mutant protein and displayed pathology as well as loss of AldoC expression. Despite considerable pathology in these lobules, behavioral analyses demonstrated a relative sparing of related functions, suggestive of sufficient functional cerebellar reserve. Together, the data indicate that mutant ATXN1 affects both AldoC-positive and AldoC-negative Purkinje cells and disrupts normal parasagittal AldoC expression in Purkinje cells. Our results show that, in a mouse model otherwise

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involv

Dietary restriction (DR) and rapamycin extend healthspan and life span across multiple species. We have recently shown that DR in progeroid DNA repair-deficient mice dramatically extended healthspan and trippled life span. Here, we show that rapamycin, while significantly lowering mTOR signaling, failed to improve life span nor healthspan of DNA repair-deficient Ercc1∆/− mice, contrary to DR tested in parallel. Rapamycin interventions focusing on dosage, gender, and timing all were unable to alter life span. Even genetically modifying mTOR signaling failed to increase life span of DNA repair-deficient mice. The absence of effects by rapamycin on P53 in brain and transcription stress in liver is in sharp contrast with results obtained by DR, and appoints reducing DNA damage and transcription stress as an important mode of action of DR, lacking by rapamycin. Together, this indicates that mTOR inhibition does not mediate the beneficial effects of DR in progeroid mice, revealing that DR and rapamycin strongly differ in their modes of action.