Loss of Dynein Axonemal Heavy Chain 5 Causes Cortical Development Disorders and CSF Flow Stagnation

Background: Dynein axonemal heavy chain 5 (Dnah5) has been identified as a key gene associated with primary ciliary dyskinesia in humans. Studies have demonstrated that mice lacking Dnah5 (Dnah5-/-) develop acute hydrocephalus shortly after birth due to impaired ciliary motility, resulting in cerebrospinal fluid (CSF) stagnation and ultimately death within approximately a month. Notably, such hydrocephalus has not been reported in humansharboring this mutation. We aimed to elucidate the pathogenesis of hydrocephalus in Dnah5-deficient mice and by that improve our understanding of the role these pathogenetic mechanisms play in human hydrocephalus. Methods: Using CRISPR/Cas9, we targeted exon 2 of the Dnah5 gene on chromosome 15, introducing a 4-base pair deletion to generate Dnah5-/- mice. We analyzed gene expression in the cerebral cortex using microarrays. Subsequently, we performed immunostaining of the cerebral cortex and ventricular wall using specific antibodies against dynein, n-cadherin, and nestin, and determined the gene expression levels and protein quantities through real-time Polymerase Chain Reaction and Western blot analysis. Results: Hydrocephalus was observed in all Dnah5-/- mice. Electron microscopy images revealed an absence of the axonemal outer dynein arm of the peripheral doublet microtubules. Ventricular size of Dnah5-/- mice was enlarged immediately after birth and it progressed through life. The number of mature neurons in the cerebral motor cortex of Dnah5-/- mice was reduced by approximately 25% compared to wild-type mice. The level ofexpression of the Dynein Cytoplasmic 1 Heavy Chain 1 (Dync1h1) gene was decreased. Cytoplasmic dynein in the cerebral cortex of Dnah5-/- mice showed a 60% decrease compared to the wild-type mice. It was also observed a 32% reduction in nestin and 35% reduction in N-cadherin in the lateral ventricular wall of Dnah5-/- mice. Conclusion: The reduction of cytoplasmic dynein resulted in the suppression of axonal growth and disrupted neurogenesis, and, consequently, a decrease in cell density in the ventricular wall layers. Hydrocephalus in the Dnah5-/- mouse model may arise from the stagnation of CSF due to impaired motile cilia function, and cortical malformations caused by cytoplasmic dynein deficiency.