RTTN mutations link primary cilia function to organization of the human cerebral cortex

Abstract

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Figure 1

Brain Imaging of Individuals with PMG from Two Consanguineous Families MRI of individuals 1 (A–D, at age 1 year) from family 1 shows a diffuse and asymmetric abnormality of the cortex with the thickened and irregular surface typical of PMG, most pronounced in the perisylvian (A and C), parietal (A, C, and D), and occipital (C and D) areas and moderate dilatation of lateral ventricles (C and D). Bilateral temporal arachnoidal cysts and normal cerebellum are visible (B). The MRI of individual 2 from family 1 (E–G, at age 14 years) shows small cerebellar vermis (E) and hemispheres (F) and diffuse bilateral, slightly asymmetric PMG (G). The MRI of the individual from family 2 (H–J, at age 7 years) shows a bilaterally polymicrogyric cortex in the temporal areas around the sylvian fissure (H), in the parietal (J) and occipital (I) areas, with reduced occipital white matter and thin splenium of corpus callosum (I, arrow). Age-matched control of 13 years (K).

Figure 2

Identification of the RTTN Mutations in Families with PMG (A) Left, pedigree of family 1. Affected subjects have black symbols, and below, the RTTN genotype is indicated, which illustrates cosegregation of the c.2796A>T change with the phenotype. Middle, electropherogram showing the homozygous mutation (subject V-4), the heterozygous father (IV-4), and the normal sequence (healthy sibling V-1). PCR products were purified with ExoSAP-IT (USB), and direct sequencing of both strands was performed with BigDye Terminator chemistry v.3.1 on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Sequences were aligned and compared with consensus sequences obtained from the human genome databases (SeqScape v.2.5 software, Applied Biosystems). For annotation of DNA and protein changes, the mutation nomenclature guidelines from the Human Genome Variation Society were followed. Right, conservation of leucine 932 among species. (B) Visualization with the Genotyping Console browser of Affymetrix SNP 6.0 Array data, showing the overlapping areas of homozygosity among individuals from family 1 (upper three) and family 2 (lowest lane). (C) Left, pedigree of family 2. The affected subject has a black symbol, and below, the RTTN genotype at position c.80 is indicated. Middle, electropherogram showing the homozygous mutation (subject IV-2), the heterozygous father (III-2), and the normal sequence (control). Right, conservation of cysteine 27 among species. (D) Schematic representation of Rotatin, including the position of the amino acid change, Armadillo-like domains, and posttranslational modification sites. The c.2796A>T change was not present in 100 ethnically matched, healthy Turkish individuals and 165 individuals of European descent. The c.80G>A change was absent in 98 Cape Verdean ethnically matched individuals and 166 healthy individuals of European descent. Both the c.2796A>T and the c.80G>A changes were not annotated as polymorphic in dbSNP130, nor were they present in 679 control individuals of the 1000 Genomes database and the Exome Variant Sever (University of Washington), indicating an extremely low allele frequency in the healthy population. Algorithms PolyPhen-2, SNAP, and Mutation Taster predicted the changes as probably being deleterious (see Web Resources).

Figure 3

Immunofluorescent Staining of Rotatin and Cilia in Growth-Arrested Control Fibroblast, Affected-Individual Fibroblasts, and siRNA-Treated Controls A total of 1 × 10⁵ cells were seeded on glass coverslips in six-well cell culture plates and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (FCS) for 24 hr and for an additional 24 hr with 0.5% FCS (serum starvation), in order to arrest cell growth and allow ciliogenesis. Cells were fixed in cytoskelfix-20 (Cytoskeleton) for 5–10 min at −20°C. Afterward, cells were treated for 10 min at room temperature in blocking buffer (0.05 M Tris; 0.9 M NaCl; 0.25% gelatine; 0.5% Triton X-100; pH 7.4), then were incubated with primary antibodies for 2 hr (mouse monoclonal anti- acetylated tubulin [Sigma-Aldrich T7451;1:8,000], rabbit anti-gamma-tubulin [Sigma-Aldrich T7451;1:1,000], and rabbit anti-Rotatin [Santa Cruz Biotechnology, Sc-85129;1:200]). Cells were washed and incubated with Hoechst 33342 (Invitrogen;1:2,000), conjugated donkey anti-mouse-cy3 (Jackson ImmunoResearch; 1:200) and conjugated donkey anti-rabbit-cy2 (Jackson ImmunoResearch;1:200) for 1 hr. Fluorescence was visualized on an AXip-Axioplan2 imaging microscope (Zeiss) with a COOLSNAP-Pro camera (Zeiss). A total of 200 cells per well were examined and scored. During siRNA treatment, the cells were incubated for a longer time after trypsinization, which accounts for the higher percentage of control cells with normal cilia. (A) Control fibroblasts show a normal cilium with basal bodies (green) and axoneme (red, arrow). (B) Control fibroblasts stained with Rotatin antibodies (green) show localization of the protein to the basal bodies of the cilia; axoneme in red. (C) Fibroblasts from one individual with the c.2796A>T change (subject 1 in the pedigree) show normal Rotatin localization but abnormally short and bulbous axoneme. (D) Cell from the same individual shows normal basal bodies and abnormally short axoneme. (E) Graphical representation of cilia abnormalities in c.2796A>T cells. The total percentage of ciliated cells is comparable with the controls (n = 10), but the percentage of structural abnormalities (in red) is significantly higher in c.2796A>T cells (35%) compared to controls (10%) (Student’s t test; p < 0.01). All the experiments were blinded and performed in parallel cultures of affected individual and controls; length and shape of the axoneme was visually scored. (F and G) Control cells treated with RTTN-specific siRNA show downregulation of mRNA relative to housekeeping gene ACTB (F) and decreased Rotatin levels on western blot (G). Predesigned siRNA pools targeting transcripts of the human RTTN (L-031139-01) (Si-RTTN) and a nontarget control siRNA pool (D-001810-10-20, Dharmacon) (Si-Control) were used for knocking down RTTN in two separate control fibroblast cell lines and hNSCs (see Figure S4). siRNA (15 nM) was delivered into fibroblasts and hNSCs (3 × 10⁵ cells) with either the siLentFect Lipid Reagent (BioRad) or nucleofectin reagent (Amaxa Nucleofector Kit V). Cells were harvested after 96 hr, seeded, and transfected again. Protein levels were examined via western blot analysis 72 hr after a second transfection. Fifty μg of cell extracts was prepared in RIPA buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% NaDOC, and 0.1% SDS) and was resolved with SDS-PAGE. Primary antibodies used for blotting were Rotatin (Santa Cruz Biotechnology; Sc-85129) and Nucleophosmin (B23) (Abcam; ab37659) as a control. Blots were developed with the enhanced chemiluminescence detection kit (Pierce). The following abbreviations are used: anti-RTTN, human Rotatin antibodies; and anti-B23, Nucleophosmin antibodies. (H) Control cell after downregulation of RTTN expression by siRNA, showing an abnormal number of basal bodies and a short axoneme. Scale bars represent 5 μm; error bars represent SEM.