Drug Screening Identifies Sigma-1-Receptor as a Target for the Therapy of VWM Leukodystrophy

Vanishing white matter (VWM) disease is an autosomal genetic leukodystrophy caused by mutations in subunits of eukaryotic translation initiation factor 2B (eIF2B). The clinical symptoms exhibit progressive loss of white matter in both hemispheres of the brain, accompanied by motor functions deterioration, neurological deficits, and early death. To date there is no treatment for VWM disease. The aim of this work was to expedite rational development of a therapeutic opportunity. Our approach was to design a computer-aided strategy for an efficient and reliable screening of drug-like molecules; and to use primary cultures of fibroblasts isolated from the Eif2b5R132H/R132H VWM mouse model for screening. The abnormal mitochondria content phenotype of the mutant cells was chosen as a read-out for a simple cell-based fluorescent assay to assess the effect of the tested compounds. We obtained a hit rate of 0.04% (20 hits out of 50,000 compounds from the selected library). All primary hits decreased mitochondria content and brought it closer to WT levels. Structural similarities between our primary hits and other compounds with known targets allowed the identification of three putative cellular pathways/targets: 11β-hydroxysteroid dehydrogenase type 1, Sonic hedgehog (Shh), and Sigma-1-Receptor (S1R). In addition to initial experimental indication of Shh pathway impairment in VWM mouse brains, the current study provides evidence that S1R is a relevant target for pharmaceutical intervention for potential treatment of the disease. Specifically, we found lower expression level of S1R protein in fibroblasts, astrocytes, and whole brains isolated from Eif2b5R132H/R132H compared to WT mice, and confirmed that one of the hits is a direct binder of S1R, acting as agonist. Furthermore, we provide evidence that treatment of mutant mouse fibroblasts and astrocytes with various S1R agonists corrects the functional impairments of their mitochondria and prevents their need to increase their mitochondria content for compensation purposes. Moreover, S1R activation enhances the survival rate of mutant cells under ER stress conditions, bringing it to WT levels. This study marks S1R as a target for drug development toward treatment of VWM disease. Moreover, it further establishes the important connection between white matter well-being and S1R-mediated proper mitochondria/ER function

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel α-subunit expressed in mammalian cells

The rat brain IIA Na⁺ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na⁺ channel α-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (~10 channels/ µm² in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC₃H1 cells and failed to express in Ltk⁻ cells. However, voltage-dependent Drosophila Shaker H4 K⁺ channels and Escherichia coli β-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na⁺ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na⁺ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na⁺ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell- specific posttranslational modifications. IIA channels were blocked by ~90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX- resistant Na⁺ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection

Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

<p>Abstract</p> <p>Background</p> <p>Eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.</p> <p>Methods</p> <p>We used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of <it>ATF4</it>, and transcriptional induction of stress-specific mRNAs (<it>ATF4, CHOP, ASNS, GRP78</it>) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).</p> <p>Results</p> <p>Despite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.</p> <p>Conclusions</p> <p>Taken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.</p

Naturally occurring antisense: Transcriptional leakage or real overlap?

Naturally occurring antisense transcription is associated with the regulation of gene expression through a variety of biological mechanisms. Several recent genome-wide studies reported the identification of potential antisense transcripts for thousands of mammalian genes, many of them resulting from alternatively polyadenylated transcripts or heterogeneous transcription start sites. However, it is not clear whether this transcriptional plasticity is intentional, leading to regulated overlap between the transcripts, or, alternatively, represents a “leakage” of the RNA transcription machinery. To address this question through an evolutionary approach, we compared the genomic organization of genes, with or without antisense, between human, mouse, and the pufferfish Fugu rubripes. Our hypothesis was that if two neighboring genes overlap and have a sense-antisense relationship, we would expect negative selection acting on the evolutionary separation between them. We found that antisense gene pairs are twice as likely to preserve their genomic organization throughout vertebrates' evolution compared to nonantisense pairs, implying an overlap existence in the ancestral genome. In addition, we show that increasing the genomic distance between pairs of genes having a sense-antisense relationship is selected against. These findings indicate that, at least in part, the abundance of antisense transcripts observed in expressed data represents real overlap rather than transcriptional leakage. Moreover, our results imply that natural antisense transcription has considerably affected vertebrate genome evolution

Recurrent functional misinterpretation of RNA-seq data caused by sample-specific gene length bias.

Data normalization is a critical step in RNA sequencing (RNA-seq) analysis, aiming to remove systematic effects from the data to ensure that technical biases have minimal impact on the results. Analyzing numerous RNA-seq datasets, we detected a prevalent sample-specific length effect that leads to a strong association between gene length and fold-change estimates between samples. This stochastic sample-specific effect is not corrected by common normalization methods, including reads per kilobase of transcript length per million reads (RPKM), Trimmed Mean of M values (TMM), relative log expression (RLE), and quantile and upper-quartile normalization. Importantly, we demonstrate that this bias causes recurrent false positive calls by gene-set enrichment analysis (GSEA) methods, thereby leading to frequent functional misinterpretation of the data. Gene sets characterized by markedly short genes (e.g., ribosomal protein genes) or long genes (e.g., extracellular matrix genes) are particularly prone to such false calls. This sample-specific length bias is effectively removed by the conditional quantile normalization (cqn) and EDASeq methods, which allow the integration of gene length as a sample-specific covariate. Consequently, using these normalization methods led to substantial reduction in GSEA false results while retaining true ones. In addition, we found that application of gene-set tests that take into account gene-gene correlations attenuates false positive rates caused by the length bias, but statistical power is reduced as well. Our results advocate the inspection and correction of sample-specific length biases as default steps in RNA-seq analysis pipelines and reiterate the need to account for intergene correlations when performing gene-set enrichment tests to lessen false interpretation of transcriptomic data