Real Time Videomicroscopy and Semiautomated Analysis of Brain Cell Culture Models of Trinucleotide Repeat Expansion Diseases

Bráz, Sandra; Dincã, Diana Mihaela; Gourdon, Geneviève; Gomes‐Pereira, Mário

doi:10.1007/978-1-4939-9784-8_14

Cited by 3 publications

(6 citation statements)

References 30 publications

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“…We next asked if the lower cell index of DMSXL astrocytes could be a consequence of modified cell-substrate interactions. We plated primary DMSXL or WT astrocytes and monitored their adhesion and spreading by live cell videomicroscopy 28 . To exclude the confounding effect of different starting cell numbers, we first confirmed that the cell density of DMSXL and WT cultures was identical 45 min after plating (Fig.…”

Section: Resultsmentioning

confidence: 99%

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

et al. 2022

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Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

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Section: Resultsmentioning

confidence: 99%

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

et al. 2022

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“…The column flow-through, containing mainly astrocytes, was cultured for 14 DIV in astrocyte medium, to further enrich this cell type, eliminate contaminating cells and promote astroglia differentiation. This method allows the establishment of primary cell cultures with a degree of purity of at least ∼90% ( Braz et al, 2020 ), which we assessed by the immunostaining of proteins markers specifically enriched in neurons (TUBB3), oligodendroglia (OLIG2), oligodendrocytes (MBP), and astrocytes (GFAP).…”

Section: Resultsmentioning

confidence: 99%

“…Primary dissociated cell cultures of mouse cortical neurons, astrocytes, and oligodendrocytes were produced as previously described ( Braz et al, 2020 ). Astrocyte precursors were extracted by magnetic cell sorting with magnetic anti-ACSA-2 microbeads (Miltenyi, 130-097-678), following the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Astrocyte precursors were extracted by magnetic cell sorting with magnetic anti-ACSA-2 microbeads (Miltenyi, 130-097-678), following the manufacturer’s instructions. The purity of primary cell cultures was estimated by immunofluorescence of cell type-specific protein markers: neurons, TUBB3; OPC, OLIG2; OL, MBP; Astrocytes, GFAP ( Braz et al, 2020 ).…”

Section: Methodsmentioning

confidence: 99%

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Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1

González-Barriga

Lallemant

Dincã

et al. 2021

Front. Cell. Neurosci.

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Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

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“…We next asked if the lower cell index of DMSXL astrocytes could be a consequence of modi ed cellsubstrate interactions. We plated primary DMSXL or WT astrocytes and monitored their adhesion and spreading by live cell videomicroscopy 26 . To exclude the confounding effect of different starting cell numbers, we rst con rmed that the cell density of DMSXL and WT cultures was identical 45 minutes after plating (Fig.…”

Section: Dmsxl Astrocytes Show Abnormal Growth Dynamics Reduced Cell ...mentioning

confidence: 99%

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Dincã

González-Barriga

Sicot

et al. 2021

Preprint

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Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types, we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading and migration. RNA-dependent toxicity and phenotypes was also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses revealed extensive expression and accumulation of toxic RNA in astrocytes, which resulted in RNA spliceopathy that was remarkably more severe than in neurons. Astrocyte missplicing affected primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis, and it was confirmed in human brain tissue. We demonstrate for the first time that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

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Real Time Videomicroscopy and Semiautomated Analysis of Brain Cell Culture Models of Trinucleotide Repeat Expansion Diseases

Abstract: Real-time monitoring of brain cell models of trinucleotide expansion diseases.

Cited by 3 publications

References 30 publications

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

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