Astrogliosis and Neuroinflammation Underlie Scoliosis Upon Cilia Dysfunction

Reviewer #1 (Public Review):

Summary:

In this study, Djebar et al. perform a comprehensive analysis of mutant phenotypes associated with the onset and progression of scoliosis in zebrafish ciliary transition zone mutants rpgrip1l and cep290. They determine that rpgrip1l is required in foxj1a-expressing cells for normal spine development, and that scoliosis is associated with brain ventricle dilations, loss of Reissner fiber polymerization, and the loss of 'tufts' of multi-cilia surrounding the subcommissural organ (the source of Reissner substance). Informed by transcriptomic and proteomic analyses, they identify a neuroinflammatory response in rpgrip1l and cep290 mutants that is associated with astrogliosis and CNS macrophage/microglia recruitment. Furthermore, anti-inflammatory drug treatment reduced scoliosis penetrance and severity in rpgrip1l mutants. Based on their data, the authors propose a feed-forward loop between astrogliosis, induced by perturbed ventricular homeostasis, and immune cell recruitment as a novel pathogenic mechanism of scoliosis in zebrafish ciliary transition zone mutants.

Strengths:

(1) Comprehensive characterization of the causes of scoliosis in ciliary transition zone mutants rpgrip1l and cep290.

(2) Comparison of rpgrip1l mutants pre- and post-scoliosis onset allowed authors to identify specific phenotypes as being correlated with spine curvature, including brain ventricle dilations, loss of Reissner fiber, and loss of cilia in proximity to the sub-commissural organ.

(3) Elegant genetic demonstration that increased urotensin peptide levels do not account for spinal curvature in rpgrip1l mutants.

(4) The identification of astrogliosis and Annexin over-expression in glial cells surrounding diencephalic and rhombencephalic ventricles as being correlated with scoliosis onset and severe curve progression is a very interesting finding, which may ultimately inform pathogenic mechanisms driving spine curvature

Weaknesses:

(1) The fact that cilia loss/dysfunction and Reissner fiber defects cause scoliosis in zebrafish is already well established in the literature, as is the requirement for cilia in foxj1a-expressing cells.

(2) Neuroinflammation has already been identified as the underlying pathogenic mechanism in at least 2 previously published scoliosis models (zebrafish ptk7a and sspo mutants).

(3) Anti-inflammatory drugs like aspirin, NAC, and NACET have also previously been demonstrated to suppress scoliosis onset and severe curve progression in these models.

Therefore, although similar observations in rpgrip1l and cep290 mutants (as reported here) add to a growing body of literature that supports a common biological mechanism underlying spine curvature in zebrafish, the novelty of reported findings is diminished.

(4) Although authors demonstrate that astrogliosis and/or macrophage or microglia cell recruitment are correlated with scoliosis, they do not formally demonstrate that these events are sufficient to drive spine curvature. Thus, the functional consequences of astrogliosis and microglia infiltration remain uncertain.

(5) The authors do not investigate the effect of anti-inflammatory treatments on other phenotypes they have correlated with spinal curve onset (like ventricle dilation, Reissner fiber loss, and multi-cilia loss around the subcommissural organ). This would help to identify causal events in scoliosis.

Reviewer #2 (Public Review):

Summary:

The manuscript by Djebar et al investigated the role and the underlying mechanism of the ciliary transition zone protein Rpgrip1l in zebrafish spinal alignment. They showed that rpgrip1l mutant zebrafish develop a nearly full penetrance of body curvature at juvenile stages. The mutant fish have cilia defects associated with ventricular dilations and loss of the Reissner fibers. Scoliosis onset and progression are also strongly associated with astrogliosis and neuroinflammation, and anti-inflammatory drug treatment prevents scoliosis in mutant zebrafish, suggesting a novel pathogenic mechanism for human idiopathic scoliosis. This study is quite comprehensive with high-quality data, and the manuscript is well written, providing important information on how the ciliary transition zone protein functions in maintaining the zebrafish body axis straightness.

Strengths:

Very clear and comprehensive analysis of the mutant zebrafish.

Weaknesses:

(1) In Figures 1D-G, magnified high-resolution pictures are required to show there are indeed no vertebral malformations.

(2) Are the transcriptome data and proteomic data consistent? Consistent targets in both analyses should be highlighted.

(3) What is the role of Anxa2 in neuroinflammation? Is increased Anxa2 expression in rpgrip1l mutant zebrafish reduced after anti-inflammatory drug treatment? What is the expression level of anxa2 in cep290 mutant zebrafish?

(4) More background about Rpgrip1l should be provided in the introduction, particularly the past studies of the mammalian homolog of Rpgrip11, if there are any.

(5) Is there any human disease associated with Rpgrip1l? Do these patients have scoliosis phenotype?

(6) A summary diagram at the end would be helpful for understanding the main findings.