Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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.

SUBMITTER: Van't Sant LJ

PROVIDER: S-EPMC8139001 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Apoptosis inducing factor deficiency causes reduced mitofusion 1 expression and patterned Purkinje cell degeneration.

Project description:Alteration in mitochondrial dynamics has been implicated in many neurodegenerative diseases. Mitochondrial apoptosis inducing factor (AIF) plays a key role in multiple cellular and disease processes. Using immunoblotting and flow cytometry analysis with Harlequin mutant mice that have a proviral insertion in the AIF gene, we first revealed that mitofusion 1 (Mfn1), a key mitochondrial fusion protein, is significantly diminished in Purkinje cells of the Harlequin cerebellum. Next, we investigated the cerebellar pathology of Harlequin mice in an age-dependent fashion, and identified a striking process of progressive and patterned Purkinje cell degeneration. Using immunohistochemistry with zebrin II, the most studied compartmentalization marker in the cerebellum, we found that zebrin II-negative Purkinje cells first started to degenerate at 7 months of age. By 11 months of age, almost half of the Purkinje cells were degenerated. Subsequently, most of the Purkinje cells disappeared in the Harlequin cerebellum. The surviving Purkinje cells were concentrated in cerebellar lobules IX and X, where these cells were positive for heat shock protein 25 and resistant to degeneration. We further showed that the patterned Purkinje cell degeneration was dependent on caspase but not poly(ADP-ribose) polymerase-1 (PARP-1) activation, and confirmed the marked decrease of Mfn1 in the Harlequin cerebellum. Our results identified a previously unrecognized role of AIF in Purkinje cell degeneration, and revealed that AIF deficiency leads to altered mitochondrial fusion and caspase-dependent cerebellar Purkinje cell loss in Harlequin mice. This study is the first to link AIF and mitochondrial fusion, both of which might play important roles in neurodegeneration.

| S-EPMC3014456 | biostudies-literature

Quantitative peptidomics of Purkinje cell degeneration mice.

Project description:Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The Purkinje cell degeneration (pcd) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult pcd mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and pcd mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult pcd mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in pcd mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult pcd mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.

| S-EPMC3620535 | biostudies-literature

Conditional Niemann-Pick C mice demonstrate cell autonomous Purkinje cell neurodegeneration.

Project description:Pathways regulating neuronal vulnerability are poorly understood, yet are central to identifying therapeutic targets for degenerative neurological diseases. Here, we characterize mechanisms underlying neurodegeneration in Niemann-Pick type C (NPC) disease, a lysosomal storage disorder characterized by impaired cholesterol trafficking. To date, the relative contributions of neuronal and glial defects to neuron loss are poorly defined. Using gene targeting, we generate Npc1 conditional null mutant mice. Deletion of Npc1 in mature cerebellar Purkinje cells leads to an age-dependent impairment in motor tasks, including rotarod and balance beam performance. Surprisingly, these mice did not show the early death or weight loss that are characteristic of global Npc1 null mice, suggesting that Purkinje cell degeneration does not underlie these phenotypes. Histological examination revealed the progressive loss of Purkinje cells in an anterior-to-posterior gradient. This cell autonomous neurodegeneration occurs in a spatiotemporal pattern similar to that of global knockout mice. A subpopulation of Purkinje cells in the posterior cerebellum exhibits marked resistance to cell death despite Npc1 deletion. To explore this selective response, we investigated the electrophysiological properties of vulnerable and susceptible Purkinje cell subpopulations. Unexpectedly, Purkinje cells in both subpopulations displayed no electrophysiological abnormalities prior to degeneration. Our data establish that Npc1 deficiency leads to cell autonomous, selective neurodegeneration and suggest that the ataxic symptoms of NPC disease arise from Purkinje cell death rather than cellular dysfunction.

| S-EPMC2816612 | biostudies-literature

Transgene-driven expression of the Doppel protein in Purkinje cells causes Purkinje cell degeneration and motor impairment.

Project description:The Doppel (Dpl) and Prion (PrP) proteins show 25% sequence identity and share several structural features with only minor differences. Dpl shows a PrP-like fold of its C-terminal globular domain and lacks the flexible N-terminal tail. The physiological functions of both proteins are unknown. However, ubiquitous Dpl overexpression in the brain of PrP knockout mice correlated with ataxia and Purkinje cell degeneration in the cerebellum. Interestingly, a similar phenotype was reported in transgenic mice expressing an N-terminally truncated PrP (DeltaPrP) in Purkinje cells by the L7 promoter (TgL7-DeltaPrP). Coexpression of full-length PrP rescued both the neurological syndromes caused by either Dpl or DeltaPrP. To evaluate whether the two proteins caused cerebellar neurodegeneration by the same mechanism, we generated transgenic mice selectively expressing Dpl in Purkinje cells by the same L7 promoter. Such mice showed ataxia and Purkinje cell loss that depended on the level of Dpl expression. Interestingly, the effects of high levels of Dpl were not counterbalanced by the presence of two Prnp alleles. By contrast, PrP coexpression was sufficient to abrogate motor impairment and to delay the neurodegenerative process caused by moderate level of Dpl. A similar situation was reported for the corresponding TgL7-DeltaPrP mice supporting the concept that Dpl and DeltaPrP cause cell death, possibly by interfering with a common signaling cascade essential for cell survival.

| S-EPMC373516 | biostudies-literature

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1.

Project description:Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spinocerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory circuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compromising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcitable MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibition of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level deficits upstream of PNs are one of the main disease triggers in SCA1.

| S-EPMC10431915 | biostudies-literature

Sphingolipid metabolism governs Purkinje cell patterned degeneration in Atxn1[82Q]/+ mice.

Project description:Patterned degeneration of Purkinje cells (PCs) can be observed in a wide range of neuropathologies, but mechanisms behind nonrandom cerebellar neurodegeneration remain unclear. Sphingolipid metabolism dyshomeostasis typically leads to PC neurodegeneration; hence, we questioned whether local sphingolipid balance underlies regional sensitivity to pathological insults. Here, we investigated the regional compartmentalization of sphingolipids and their related enzymes in the cerebellar cortex in healthy and pathological conditions. Analysis in wild-type animals revealed higher sphingosine kinase 1 (Sphk1) levels in the flocculonodular cerebellum, while sphingosine-1-phosphate (S1P) levels were higher in the anterior cerebellum. Next, we investigated a model for spinocerebellar ataxia type 1 (SCA1) driven by the transgenic expression of the expanded Ataxin 1 protein with 82 glutamine (82Q), exhibiting severe PC degeneration in the anterior cerebellum while the flocculonodular region is preserved. In Atxn1[82Q]/+ mice, we found that levels of Sphk1 and Sphk2 were region-specific decreased and S1P levels increased, particularly in the anterior cerebellum. To determine if there is a causal link between sphingolipid levels and neurodegeneration, we deleted the Sphk1 gene in Atxn1[82Q]/+ mice. Analysis of Atxn1[82Q]/+; Sphk1-/- mice confirmed a neuroprotective effect, rescuing a subset of PCs in the anterior cerebellum, in domains reminiscent of the modules defined by AldolaseC expression. Finally, we showed that Sphk1 deletion acts on the ATXN1[82Q] protein expression and prevents PC degeneration. Taken together, our results demonstrate that there are regional differences in sphingolipid metabolism and that this metabolism is directly involved in PC degeneration in Atxn1[82Q]/+ mice.

| S-EPMC8433568 | biostudies-literature

A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation.

Project description:Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases.

| S-EPMC3098191 | biostudies-literature

Loss of the tuberous sclerosis complex protein tuberin causes Purkinje cell degeneration.

Project description:Tuberous sclerosis complex (TSC) is a neurogenetic disorder that often causes brain abnormalities leading to epilepsy, developmental delay, and autism. TSC is caused by inactivating mutations in either of the genes encoding the proteins hamartin (TSC1) and tuberin (TSC2). These proteins form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) pathway, controlling translation and cell growth. Loss of either protein results in dysregulated mTORC1 activation, an important aspect of TSC pathogenesis. About thirty percent of TSC patients have cerebellar pathology that is poorly understood. To investigate the effects of TSC on the cerebellum, we created a mouse model in which the Tsc2 gene was selectively deleted from Purkinje cells starting at postnatal day 6 (P6). The loss of Tsc2 caused a progressive increase in Purkinje cell size and subsequent death from apoptosis. Purkinje cell loss was predominantly cell type specific and associated with motor deficits. Immunohistochemical analysis showed that both endoplasmic reticulum (ER) and oxidative stress were increased in Tsc2-null Purkinje cells. The cell death and ER stress phenotypes were rescued by treatment with the mTORC1 inhibitor rapamycin. To assess whether the murine Purkinje cell loss has a correlate to the human TSC, we analyzed postmortem cerebellum samples from TSC patients and detected Purkinje cell loss in half of the samples. Our results establish a critical role for the TSC complex in Purkinje cell survival by regulating ER and oxidative stress and reveal a novel aspect of TSC neuropathology.

| S-EPMC3096682 | biostudies-literature

Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells.

Project description:Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a ?-subunit transmembrane protein 30?A (TMEM30A). Disruption of PS flippases led to several human diseases. However, the roles of TMEM30A in the central nervous system remain elusive. To investigate the role of Tmem30a in the cerebellum, we developed a Tmem30a Purkinje cell (PC)-specific knockout (KO) mouse model. The Tmem30a KO mice displayed early-onset ataxia and progressive PC death. Deficiency in Tmem30a led to an increased expression of Glial fibrillary acidic protein and astrogliosis in regions with PC loss. Elevated C/EBP homologous protein and BiP expression levels indicated the presence of endoplasmic reticulum stress in the PCs prior to visible cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis suggested that apoptotic cell death occurred in the cerebellum. Our data demonstrate that loss of Tmem30a in PCs results in protein folding and transport defects, a substantial decrease in dendritic spine density, increased astrogliosis and PC death. Taken together, our data demonstrate an essential role of Tmem30a in the cerebellum PCs.

| S-EPMC6125289 | biostudies-literature

Neurodegeneration and locomotor dysfunction in <i>Drosophila scarlet</i> mutants.

Project description:Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons, resulting in progressive locomotor dysfunction. Identification of genes required for the maintenance of these neurons should help to identify potential therapeutic targets. However, little is known regarding the factors that render dopaminergic neurons selectively vulnerable to PD. Here, we show that Drosophila melanogaster scarlet mutants exhibit an age-dependent progressive loss of dopaminergic neurons, along with subsequent locomotor defects and a shortened lifespan. Knockdown of Scarlet specifically within dopaminergic neurons is sufficient to produce this neurodegeneration, demonstrating a unique role for Scarlet beyond its well-characterized role in eye pigmentation. Both genetic and pharmacological manipulation of the kynurenine pathway rescued loss of dopaminergic neurons by promoting synthesis of the free radical scavenger kynurenic acid (KYNA) and limiting the production of the free radical generator 3-hydroxykynurenine (3-HK). Finally, we show that expression of wild-type Scarlet is neuroprotective in a model of PD, suggesting that manipulating kynurenine metabolism may be a potential therapeutic option in treating PD.This article has an associated First Person interview with the first author of the paper.

| S-EPMC6176922 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data