ASD Risk Gene KATNAL2 Affects Ependymal Ciliary Motion And Cerebrospinal Fluid Flow

Autism spectrum disorder（ASD）is a common neurodevelopmental disorder that significantly impacts patients’physical and mental health.KATNAL2（Katanin-like 2）is a high-risk gene for ASD,but the mechanism by which mutations in this gene affect brain development and function is poorly understood.Based on previous studies and preliminary findings of the laboratory,we hypothesize that molecular functions of Katnal2 is closely related to motility cilia in the brian.In this paper,by means of gene knockout and conditional knockout,different strains of mice were used as model animals to investigate the effect of Katnal2 gene deficits on brain development and the possible underlying mechanismWe first obtained the Katnal2 knockout（KO）mice and verified the gene ablation using RT-PCR.We found severe defects in the morphology and motility of sperms from homozygous KO male mice,consistent to previous reports.After cutting serial coronal brain sections and measuring the sizes of brain slices and the size of brain ventricles in each slice,we found an abnormal ventricular dilatation in KO mice compared to wild type and heterozygous littermates.Ventricular volume analysis at different time points showed that ventricular dilatation caused by Katnal2 gene deficit first occurred during the first postnatal week.Through the scanning electron microscopy analysis of the lateral ventricle wall,the analysis of Evans blue injection and its diffusion in the lateral ventricle,the analysis of choroid plexus tissue morphology,and the analysis of cerebrospinal fluid osmotic pressure,we found that the Katnal2 gene deficit may lead to the expansion of the ventricle mainly by impairing the motion of ependymal cilia,causing the slowing of the cerebrospinal fluid circulation However,the thickness and lamellar structure of the cerebral cortex were not significantly affected in KO rats.To further test this hypothesis,we utilized ependymal and choroid plexus-specific Foxj1^{Cre ERT2::GFP} tool mice crossbred with Katnal2-Flox mice to create ependymal and choroid plexus-specific Katnal2 conditional knockout mice(Foxj1^{Cre ERT2::GFP+/-};Katnal2-flox^+/+).We injected Tamoxifen（TAM）into mother mice to deliver the drug to the pups through breast milk and induce the specific ablation of Katnal2 in ependymal cells of newborn mice.To evaluate the gene recombination efficiency of this method,we crossed the Foxj1^{Cre ERT2::GFP} mice with Ai14 fluorescence reporter mice.After the maternal application of TAM,we analyzed the proportion of TAM-induced td Tomato⁺cells among total Foxj1⁺ependymal cells in a whole-mount preparation of the ventricle wall tissue from the pups using immmnofluorescent microscopy and confirmed that the recombination rate of the TAM-induction scheme was feasible.We found that the specific ablation of Katnal2 in ependymal cells in newborn mice was sufficient to cause ventricular dilation and produce a hydrocephalic phenotype.However,specific knockout of Katnal2 gene in ependymal cells induced by TAM injection did not cause ventricular dilatation after animal maturation.These results indicate that this gene plays an important role in maintaining ventricular morphology mainly through its expression the ependymal cell during the early postnatal period.In summary,our results highlight the relationship between motile cilia of ependymal cells and ASD and the intrinsic link between hydrocephalus and ASD.