Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development

Abstract

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

Keywords: Axon elongation, Brain malformation; Cerebellar cortical dysplasia; Cerebellar hypoplasia; NAV2; Neuron migration.

Fig. 1

Clinical and genetic findings of the NAV2-related neurodevelopmental disorder in the family of the proband. (A) Pedigree of the family showing the affected individual (shaded). + represents the reference allele. (B) Craniofacial dysmorphism of the affected subject (at the age of 3 years left panel and 7 years right panel) including deep set eyes, upslanting palpebral fissures, bulbous nasal tip, thin upper lip, and dimple and broad chin. (C) Retinal fundus photograph of the right and left eyes illustrating aberrant retinal vasculature with a subclinical retinal detachment in the right eye and retinal neovascularization left eye. Ultra-widefield fluorescein angiography of the right and left eyes demonstrating retinal ischemia and retinal neovascularization (inset). (D) RNA-seq tissue data generated by the Genotype-Tissue Expression (GTEx) project and reported as mean pTPM (protein coding transcripts per million), corresponding to the values of the different individual samples for respective subregion. Cerebellum has the highest expression (pTPM 20.4). (E) Depictions of the pathogenic variants p.(L1728Wfs*2) and p.(Ile2253*) and protein domains (CH, calponin homology domain; cytoskeletal interacting domain or CSID; CC, coiled coil domain; AAA, AAA-ATPase domain; purple indicates poly-Proline, Serine, and Lysine regions). (F) NAV2 protein sequences of different species based on the constraint-based alignment tool COBALT. The red colour indicates highly conserved protein regions among species and blue indicates less conserved ones. Percent of identity (indicating the percentage of the orthologous sequence matching the Human sequence according to the Ensembl database) and the percent of similarlity have been calculated using the EMBOSS Needle tool

Fig. 2

Neuroimaging features of the NAV2-related neurodevelopmental disorder in the proband. Brain MRI of the patient performed at 6 years of age (A–D) and of an age-matched control subject (E–F). A) Axial reformatted 3D T1-weighted images reveal abnormal cerebellar foliation fissuration, more pronounced at the level of the inferior cerebellar hemispheres (empty arrows). The pons is flattened and slightly asymmetric (dotted arrows). The superior cerebellar peduncles are thinned and splayed (arrows) with associated narrowing of the isthmic region (arrowhead) leading to a molar tooth-like appearance of the midbrain. Note the diffuse cortical dysgyria, with prevalent insular involvement (thick arrows), and the asymmetric enlargement of the lateral ventricles (asterisks). (B) Sagittal reformatted 3D T1-weighted image shows the hypoplasia and dysplasia with mild upward rotation of the cerebellar vermis, and prevalent involvement of the anterior and superior posterior lobes (empty arrows). There are also corpus callosum hypodysgenesis (arrow), agenesis of the anterior commissure (dotted arrow), narrow isthmus (arrowhead), and small pons (thick arrow). Note the small optic nerve chiasm. (C) Coronal T2-weighted image demonstrates the agenesis of the olfactory bulbs (arrows) and mild hypoplasia of the optic nerves (arrowheads). (D) Diffusion tensor imaging, axial color-coded fractional anisotropy (FA) maps reveal small asymmetric corticospinal tracts at the level of the pons (dotted arrows) and horizontal course of the superior cerebellar peduncles (arrows) with preservation of their decussation at the midbrain level (arrowhead). (E) Axial reformatted 3D T1-weighted images show the normal size and morphology of the cerebellar hemispheres. The cerebellar folia run parallel to the calvarium (onion-like configuration; thick arrows). (F) Axial color-coded FA maps, magnified view at 2 brainstem levels (middle pons and middle midbrain). Conventional color scheme: blue (inferior-superior), green (anteroposterior), and red (left–right). CST, corticospinal tract; MCP, middle cerebellar peduncles; SCP, superior cerebellar peduncles; SCPD, superior cerebellar peduncles’ decussation; SAF, somatosensory ascending fibers; TPF, transverse pontocerebellar fibers

Fig. 3

Comparison of the cerebellar volumes of the patient with an age-matched healthy subject. (A–C) Segmentation of the cerebellar volumes of the patient (green maps) and of an aged-matched control subject (red maps) overlayed on sagittal (A), coronal (B), and axial-reformatted (C) 3DT1-weighted images. (D–F) Volumetric reconstructions of cerebellar segmentations of the patient (green cerebellum, D), of the control subject (red cerebellum, E), and of the fusion of both cerebellar volumes (F) overlayed on 3D T1-weighted images. Note that the volume of both the cerebellar hemispheres and vermis of the patient is smaller compared to the age-matched control

Fig. 4

Brain and cerebellar abnormalities in the Nav2 hypomorphic mutant mice. Medial sagittal sections (panels a and b; panel b closest to midline) from a wild-type (WT) and homozygous hypomorph (HOM) show an overall reduction in cerebellar size, and a reduction in overall development of VIa,VIb/VII with absence of the intercrural fissue (arrowhead) in the HOM. In other brain regions, abnormalities noted in the HOM include thinning of the corpus callosum and a reduction in the size of the regions encompassing the thalamus/hypothalamus (Th/Hyp). The pons and medulla appear normal in size in both genotypes, with the pontine nucleus (PN) and inferior olive (IO) shown for reference. The cortex and tectum, anterior commissure (AC), and olfactory bulb showed no obvious dysmorphogenesis. Scale bar: 1 mm

Fig. 5

Cellular phenotype associated with the genetic loss of Neuron Navigator 2 in the NAV2 compound mutant patient. (A–C) Analysis of NAV2 protein and mRNA expression in fibroblasts obtained from 4 healthy individuals and the “NAV2 patient.” (A) Representative Western blot experiment showing immunodetection of NAV2 (top) and GAPDH (bottom) in the indicated fibroblasts lysates. (B) Bar graph showing the densitometric analysis of the upper band, corresponding to the full-length protein (2830 KDa); data are normalized for GAPDH expression. N = 3, *** p < 0.001 (ANOVA with Tukey’s post hoc test). (C) Bar graph showing NAV2 mRNA quantification by real time PCR. N = 3, ** p < 0.01 (ANOVA with Tukey’s post hoc test). (D and E) Analysis of cell migration by wound healing assay. Representative images (D) and analysis (E) of wounded areas of confluent fibroblasts at different time points. Wound edges, detected by image segmentation analysis, are outlined in green. N = 3; * p < 0.05, ** p < 0.01. (F) Representative image and summary graph showing results from cell morphology analysis. Fibroblasts were fixed, stained with phalloidin (red) and Hoechst 33,342 (blue) and analyzed by confocal microscopy. Scale bar = 10 µm. 200–250 cells/donor were analyzed. * p < 0.05

Fig. 6

The NAV2 ortholog in Drosophila, sick, is expressed in brain and mutants are semi-lethal with motor defects and heat-sensitive seizure-like behavior. (A) Schematics of sick^T2A−GAL4 acting as a gene trap: the insertion of SA-T2A-GAL4-polyA cassette leads to generation of truncated Sick and expression of GAL4 under the control of the regulatory sequences of the sick. (B) Complementation tests of sick^T2A−GAL4 with a corresponding deficiency (Df(2L)ED1303). (C–E) Gene expression of sick based on sick^T2A−GAL4; UAS-mCD8::RFP flies. Whole third instar larva (C), third instar larval brain (D), and adult brain (E) are shown. Note expression in the mushroom bodies (upper panel, dashed lines), olfactory glomeruli (upper panel, dashed ellipse), and antennal mechanosensory and motor center (upper panel, dashed ellipse). Scale bars = 100 µm. (F) Climbing assessment of sick^T2A−GAL4/Df flies in a negative geotaxis assay reveals motor deficits in sick mutants that are 12 days old. N are shown within the bars. Unpaired t tests, **** p < 0.0001. (G) sick mutants display heat-induced seizures in a 42 °C water bath (30 s). (H) Time to recover. N are shown within the bars. Unpaired t tests, *** p < 0.001, **** p < 0.0001