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Adult mouse eIF2B? Arg191His astrocytes display a normal integrated stress response in vitro.

ABSTRACT: Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2B? (2b5 ^ho ), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- and long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.

SUBMITTER: Wisse LE

PROVIDER: S-EPMC5830650 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Adult mouse eIF2Bε Arg191His astrocytes display a normal integrated stress response in vitro.

Wisse Lisanne E LE Ter Braak Timo J TJ van de Beek Malu-Clair MC van Berkel Carola G M CGM Wortel Joke J Heine Vivi M VM Proud Chris G CG van der Knaap Marjo S MS Abbink Truus E M TEM

Scientific reports 20180228 1

Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated str ...[more]

PMID: 29491431

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Project description:Purpose: The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We performed RNA-seq analysis of cerebellum collected from mice harboring a homozygous point mutation in eIF2B5 (HO R191H), a mutation that causes a severe form of VWM in humans and compared gene expression to wildtype age-matched littermates to identify transcriptomic differences at early, mid and late time points during disease progression. We also performed RNA-seq on cerebellum collected from animals treated with the eIF2B activator 2BAct for 1 month to determine to what extent small molecule treatment could normalize the VWM transcriptome. Methods: Samples were collected at 2, 5, and 7 months of age. In addition, HO R191H and WT mice treated with and without with the eIF2B activator 2BAct for 1 month were profiled. RNA-seq libraries were prepared using purified RNA isolated from frozen tissue using the RNeasy Mini kit. RNA quality and concentration were assayed using a Fragment Analyzer instrument. RNA-seq libraries were prepared using the TruSeq Stranded Total RNA kit paired with the Ribo-Zero rRNA removal kit. Libraries were sequenced on an Illumina HiSeq 4000 instrument. For the experiment comparing different ages, N = 3 males/genotype/time point. For the 4-week 2BAct treatment experiment, N = 3 females/condition. Conclusions: HO R191H mutant brains show an elevated gene expression signature of the Integrated Stress Reponse in mice as early as 2 months, preceding the appearance of overt pathology in these animals. The expression of the ISR-related genes does not significantly increases at 5 months and 7 months of age. One month of treatment with 2BAct completely prevented the expression of ISR-related in genes in 2 month old animals.

Project description:Presenilin 1 (PS1) and Presenilin 2 (PS2) are predominantly known as the catalytic subunits of the γ-secretase complex that generates the amyloid-β (Aβ) peptide, the major constituent of the senile plaques found in the brain of Alzheimer's disease (AD) patients. Apart from their role in γ-secretase activity, a growing number of cellular functions have been recently attributed to PSs. Notably, PSs were found to be enriched in mitochondria-associated membranes (MAMs) where mitochondria and endoplasmic reticulum (ER) interact. PS2 was more specifically reported to regulate calcium shuttling between these two organelles by controlling the formation of functional MAMs. We have previously demonstrated in mouse embryonic fibroblasts (MEF) an altered mitochondrial morphology along with reduced mitochondrial respiration and increased glycolysis in PS2-deficient cells (PS2KO). This phenotype was restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized cells, and one bottom-line question is to know whether these observations hold true in central nervous system (CNS) cells. To that end, we carried out primary cultures of PS1 knockdown (KD), PS2KO, and PS1KD/PS2KO (PSdKO) neurons and astrocytes. They were obtained from the same litter by crossing PS2 heterozygous; PS1 floxed (PS2+/-; PS1flox/flox) animals. Genetic downregulation of PS1 was achieved by lentiviral expression of the Cre recombinase in primary cultures. Strikingly, we did not observe any mitochondrial phenotype in PS1KD, PS2KO, or PSdKO primary cultures in basal conditions. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. Likewise, mitochondrial morphology and content was unaltered by PS expression. We further investigated the differences between results we obtained here in primary nerve cells and those previously reported in MEF cell lines by analyzing PS2KO primary fibroblasts. We found no mitochondrial dysfunction in this model, in line with observations in PS2KO primary neurons and astrocytes. Together, our results indicate that the mitochondrial phenotype observed in immortalized PS2-deficient cell lines cannot be extrapolated to primary neurons, astrocytes, and even to primary fibroblasts. The PS-dependent mitochondrial phenotype reported so far might therefore be the consequence of a cell immortalization process and should be critically reconsidered regarding its relevance to AD.

Dataset Information

Adult mouse eIF2B? Arg191His astrocytes display a normal integrated stress response in vitro.

Publications

Adult mouse eIF2Bε Arg191His astrocytes display a normal integrated stress response in vitro.

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