Abnormal Cerebrospinal Fluid Flow

As its name suggests, idiopathic scoliosis (IS) is a diagnosis made with unknown pathological factors that could give rise to observed scoliotic changes. It has been reported at rates of 3% to 5.2% in pediatric and adolescent populations and occurs more frequently in females than males (3:1).1 Disease progression and sequelae are largely a function of the location and severity of scoliotic curves, as well as the rate and manner in which these curves change over time. For example, curves in the thoracic spine have been reported as most vulnerable to progression and can cause cardiovascular or pulmonary pathology.2 Early detection has proven beneficial in patients treated conservatively (eg, bracing or casting) and surgically.3,4 However, as long as the cause of IS remains unknown, therapy can be initiated only after the scoliotic changes have begun, eliminating the opportunity for physicians to prevent these deformities from developing at all. With a series of experiments, a joint team including researchers at Princeton University and the University of Toronto has recently identified a possible pathogenic mechanism for IS in zebrafish as a model for human spinal development.5 Their data show that mutations in protein tyrosine kinase-7 (ptk7, a signaling pathway regulator) impair the growth and function of ependymal cell (EC) cilia, preventing the proper flow of cerebrospinal fluid (CSF). Importantly, these mutations and CSF flow irregularities are directly associated with deformities in the developing spine that parallel the human manifestations of IS. CSF flow during development is normally driven by the polarized beating of EC cilia. Therefore, the team first examined EC surface morphology under scanning electron microscopy, comparing cells from zebrafish sibling pairs: 1 fish was a scoliotic ptk7 mutant (ptk7), the other a ptk7-normal nonscoliotic control (ptk7/+). Although the control group had a normal distribution and arrangement of EC cilia, ptk7 mutants generally lacked EC cilia, and the few present cilia were disorganized and lacked polarization. The mutants also showed signs of hydrocephalus, which is typically associated with impaired EC cilia function and CSF flow abnormalities (Figure). Furthermore, by placing fluorescent microspheres across the EC surface, the team observed robust anterior-posterior flow in the ptk7-normal controls. In contrast, what little motion was observed in the ptk7 mutants was both erratic and significantly slower. In an attempt to show that IS develops directly from ptk7-related EC ciliary dysfunction, the team next used a transcription factor (foxJ1a) to restore ptk7 specifically in the midline structures of the brain and spinal cord in mutant lines. The mutants that were reintroduced ptk7 (ptk7 + Tg [foxj1a::ptk7]) developed normal EC ciliary function, organized CSF flow, and no hydrocephalus. Furthermore, microcomputed tomography in these lines showed normal spine development with no scoliotic curves.Figure: Scanning electron microscopy examination of ptk7 mutant (B) brain ventricles showed hydrocephalus (yellow line) compared with ptk7-normal control (A) and the mutants that were reintroduced ptk7 (C). A′ through C′ show the magnification of ciliary morphology from the green squares in A through C. The scoliotic spine curve seen in ptk7 mutant (D and D′) and the normal curve seen in the mutants that were reintroduced ptk7 (E and E′). From Grimes DT, Boswell CW, Morante NF, Henkelman RM, Burdine RD, Ciruna B. Zebrafish models of idiopathic scoliosis link cerebrospinal fluid flow defects to spine curvature. Science. 2016;352(6291):1341-1344. Reprinted with permission from AAAS.Having shown that IS was caused by ptk7 mutation, the consequent loss of EC motile cilia, and ultimately CSF flow defect, the team investigated other mutations that impair cilia development or function and so should in theory cause IS. However, these mutations generally cause death in the first 1 to 2 weeks of embryonic development, making their downstream effects on spinal development impossible to assess. To avoid early embryonic death, the team took advantage of a specific temperature-sensitive mutation (c21orf59TS) that impairs cilia motility and causes embryonic death at 30°C but has no impact on ciliary function or embryonic survival at 25°C. These mutants were raised at 25°C for 5 days to pass the threshold for embryonic death and then switched to 30°C. During the 25°C period, the mutant fish resembled wild-type controls and underwent normal vertebral formation. After the shift to 30°C, ventricular CSF flow was severely impaired, and the mutant fish developed spinal curves and signs of IS. In separate experiments, the team used wild-type mRNA injections during early stages of mutant development to prevent embryonic death. These fish went on to develop severe spine curves and malformations consistent with IS. In all, the team demonstrated that mutations in 4 distinct genes that affect cilia structure and motility (ccdc40, ccdc151, dyx1c1, and c21orf59) can lead to IS development. Interestingly, transient defects in these genes during early embryonic development do not lead to IS after birth, suggesting that the critical window for cilia-mediated spine development occurs after the embryonic period. Again using the temperature-sensitive mutation described above, the team attempted to define this critical period by switching mutant fish from 25°C (exhibiting normal ciliary function) to 30°C (exhibiting impaired ciliary function) at various periods of development. Although all mutant fish switched to 30°C at 19 days after fertilization developed IS by 5 weeks, mutants switched at 34 days after fertilization showed no signs of IS through adulthood. The sensitive period between 19 and 34 days after fertilization in this experiment demonstrates a critical time interval for the development of IS. Finally, the team showed that the scoliotic curve development in zebrafish could be inhibited or reversed if the cilia dysfunction is corrected. Again using the temperature-sensitive mutation, the researchers raised the mutant fish at 25°C for 1 week (preventing embryonic death) and then switched to 30°C until early signs of IS development were observed, at which point they were restored to 25°C. The return to 25°C (exhibiting normal ciliary function) halted the progression of spinal curves and IS signs, whereas the fish that remained at 30°C developed apparently worse scoliotic curves. This suggests that IS can potentially be corrected by restoring EC ciliary function. Together, this series of experiments demonstrates a pathogenic relationship among ciliary function impairment, CSF flow defect, and the development of scoliotic spine curves in a zebrafish model. Current literature contains more evidence (in human and animal studies) that scoliosis is associated with CSF flow problems.6,7 This study could lead scientists to investigate the development of human IS and its relationship with CSF flow. Beyond improving our understanding of IS development, this new model has great potential to expand our diagnostic capability in screening susceptible patients and initiating therapy before the gross onset of deformity. With the ever-growing field of genetic medicine, the pathogenesis implicated in this study suggests potential therapies to prevent IS rather than attempt to correct it.

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