The effects of skull malformations and intracranial hypertension on the glymphatic and meningeal lymphatic systems in craniosynostosis

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Abstract: Our work has shown that humans with craniosynostosis, the second most prevalent craniofacial disorder, have skull and cerebrovascular malformations that cause intracranial hypertension (i.e. raised pressure). The effects of raised intracranial pressure on cerebrospinal fluid (CSF) circulation and fluid balance in the central nervous system are unknown in craniosynostosis, a notable gap in the field. Our new findings in craniosynostosis animal models suggest that skull dysplasia and raised pressure impair CSF flow, brain perivascular waste clearance pathways (the glymphatic system), and the development of meningeal lymphatic vessels. Infusing fluorescent tracers into CSF reveals significant changes to circulation pathways, and less influx into brain tissue and outflow to surrounding meningeal lymphatic networks. Meningeal lymphatics, which grow in dura attached to the skull, are hypoplastic and less complex. Interestingly, highly branched segments specialized for CSF uptake and waste removal, known as “hotspots”, are poorly developed and/or missing. These novel findings are significant because the brain's glymphatic system and supporting meningeal lymphatic vessels are necessary to prevent β-amyloid plaque buildup and cognitive impairment. Collectively, these data form a central hypothesis; craniosynostosis causes raised intracranial pressure and altered CSF hydrodynamics that impair the development and functions of both the glymphatic and meningeal lymphatic systems. This, in turn, affects the clearance of waste and β- amyloid from the brain. We test this hypothesis using our innovative systems biology approach that integrates skull development with intracranial pressure, CSF flow, and the brain's glymphatic and lymphatic systems. In Aim 1, we use two genetic mouse models for craniosynostosis to test if impaired glymphatic (i.e. CSF) influx and efflux impede the clearance of β-amyloid oligomers injected into the brain. Next, we test if the clearance of β- amyloid plaques is reduced by generating mouse models for craniosynostosis and amyloid deposition that express five mutations found in familial Alzheimer's disease. We also test a mechanistic basis for glymphatic dysfunction in craniosynostosis, by examining if the polarization of Aquaporin-4 water channels to glial endfeet is perturbed. These glial (i.e. glymphatic) water channels facilitate CSF flow through perivascular spaces in the brain, and therefore waste exchange between CSF and brain interstitial fluid. In Aim 2, we test whether meningeal lymphatic deficits affect waste drainage to the cervical lymph nodes. We also explore a molecular mechanism for meningeal lymphatic deficits by testing if VEGF-C expression and the activation of VEGFR3 receptor signaling is affected. Collectively, our novel system and approaches can dissect interlocking mechanisms through which normal skull development controls CSF flow, brain waste clearance pathways, and meningeal lymphatic drainage. This work has profound clinical significance as deficits in glymphatic waste exchange and meningeal lymphatics in craniosynostosis may imply increased risks for β-amyloid plaque buildup and neurocognitive impairment.