Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response
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Abstract
Vanishing white matter disease (VWM) is a severe leukodystrophy of the central nervous system caused by mutations in subunits of the eukaryotic initiation factor 2B complex (eIF2B). Current models only partially recapitulate key disease features, and pathophysiology is poorly understood. Through development and validation of zebrafish ( Danio rerio ) models of VWM, we demonstrate that zebrafish eif2b mutants phenocopy VWM, including impaired somatic growth, early lethality, effects on myelination, loss of oligodendrocyte precursor cells, increased apoptosis in the CNS, and impaired motor swimming behavior. Expression of human EIF2B2 in the zebrafish eif2b2 mutant rescues lethality and CNS apoptosis, demonstrating conservation of function between zebrafish and human. In the mutants, intron 12 retention leads to expression of a truncated eif2b5 transcript. Expression of the truncated eif2b5 in wild-type larva impairs motor behavior and activates the ISR, suggesting that a feed-forward mechanism in VWM is a significant component of disease pathophysiology.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 22, 2020, follows.
Summary
Mutations in the five subunits of the eukaryotic initiation factor 2B (eIF2B1-5) complex cause a severe leukodystrophy called Vanishing White Matter (VWM) disease. In this study from the Bonkowsky lab, new mutants were either generated or imported and analyzed for CNS defects relevant to VWM. By studying growth, lethality, myelination, CNS cell development, and swimming behavior, the authors conclude that the new eif2b mutants phenocopy VWM patients. The authors also show that in mutants, a retained intron leads to expression of a truncated transcript, which they conclude acts in a dominant-negative fashion and suggest that this explains some pathology in human VWM patients.
Although modeling human …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 22, 2020, follows.
Summary
Mutations in the five subunits of the eukaryotic initiation factor 2B (eIF2B1-5) complex cause a severe leukodystrophy called Vanishing White Matter (VWM) disease. In this study from the Bonkowsky lab, new mutants were either generated or imported and analyzed for CNS defects relevant to VWM. By studying growth, lethality, myelination, CNS cell development, and swimming behavior, the authors conclude that the new eif2b mutants phenocopy VWM patients. The authors also show that in mutants, a retained intron leads to expression of a truncated transcript, which they conclude acts in a dominant-negative fashion and suggest that this explains some pathology in human VWM patients.
Although modeling human disease in model organisms like zebrafish is important, there are several major issues with the study that dampen enthusiasm as outlined below.
Essential Revisions
Significance of model
- For most of five the mutations most of them are heterozygous. Here the authors showed that only 2 out of 4 subunit mutants (eif2b5, eif2b2) exhibited phenotypes . Is it possible that other mutant alleles have mild phenotypes, such as increased ISR? More characterization of phenotypes in the other mutants (eif2b1, eif2b4) and the heterozygous siblings is needed to address relevance of this as a model of VWM.
Motor defects
It is unconvincing that the movement measurements in eif2b5zc103 mutants represent motor behavior deficits. Please address possible non-specific developmental delays cause the observed phenotypes. Can the authors perform the behavioral experiments at later stages or provide stage matched (rather than age matched) characterizations the mutants?
In Fig. 6, for the truncated EIF2B5 mis-expression experiments, it is unclear from the text and methods how the experiments were performed. If the truncated protein acts as a dominant-negative in the e1f2b mutants, why would expression of the wild-type gene rescue the mutant phenotype? The dominant-negative product would still be present. Also, Figure 6D: Compare motor behavior in actin:eif2b vs eif2b5 expression and clarify and address alternative interpretations of the data (See reviewer 3 comments).
Characterization of oligodendrocytes and myelin
Olig2 also labels motor neurons in the spinal cord and neural precursors in the hindbrain. Based on this marker alone it cannot be concluded they are only quantifying oligodendrocyte lineage cells in Figure 3. Please address.
Fig 3P-S: the TUNEL staining overlaps with Olig2 in the hindbrain but not in the rest of the brain. Please investigate which other cell types are undergoing apoptosis the optic nerve and optic tectum where the myelination and axon defects are evident in the eif2b5 zc103/103 mutants/
Fig. 3F-I:the eif2b2 and eif2b5 mutants show a striking change in proliferation pattern at 5dpf, including a loss of proliferation in the eyes and cerebellum and increased proliferation in the ventricles. To investigate which cell types are undergoing altered proliferation co-staining for cell-type-specific markers (eg: microglia, astrocytes, neurons) Is needed. See reviewer 1 comments.
The electron micrographs in Figure 4 are low quality and cannot be analyzed for G-ratio based on what is shown. Please address.
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