MKS1 regulates ciliary INPP5E levels in Joubert syndrome

Abstract

Background: Joubert syndrome (JS) is a recessive ciliopathy characterised by a distinctive brain malformation 'the molar tooth sign'. Mutations in >27 genes cause JS, and mutations in 12 of these genes also cause Meckel-Gruber syndrome (MKS). The goals of this work are to describe the clinical features of MKS1-related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS.

Methods: We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a three-dimensional (3D) spheroid rescue assay to test the effects of disease-related MKS1 mutations.

Results: We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1-related JS carry ≥ 1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids.

Conclusions: MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalisation, is a key mechanism underlying JS, downstream of MKS1 and ARL13B.

Keywords: Cell biology; Genetics.

Figure 1. MRI findings and mutations in individuals with MKS1-related Joubert syndrome

(A-L) All affected individuals had classic imaging findings of JS including cerebellar vermis hypoplasia, and thick, horizontally-oriented superior cerebellar peduncles. (A–B) is JBTS-10, (C–D) is UW031-3, (E–F) is UW091-3, (G–H) is UW092-3, (I–J) is UW093-3, (K–L) is JBTS-3504 (A, C, E, I, K) are T2-weighted axial views, (G) is a T1-weighted axial view, (B, D, F, J) are T1-weighted sagittal views, (H, L) are T2-weighted sagittal views; (M) MKS1 mutations in individuals with JS and MKS based on sequence NM_017777.3. p.G471Lfs*92 extends the protein by 4 amino acids; p.Thr485Argfs*107 extends the protein by 33 amino acids; p.R510Pfs*81 extends protein by 40 amino acids; MKS1 protein (559 aa). Black arrows, truncating mutations; Gray arrows, non-truncating mutations; Outline arrows, splice site mutations. CC, coiled-coil; B9, B9-domain.

Figure 2. Fibroblasts from individuals with MKS1-related Joubert syndrome display primary cilia defects

(A) Immunostaining of fibroblasts derived from skin biopsies of JBTS-10, JBTS-153, JBTS-3504, MKS-158, and controls. ARL13B (green), gamma tubulin (g-tub; white) and cilia (acetylated tubulin, red; scale bar 5 μm). Brightness and contrast were identically adjusted across photos for visualization purposes; original data is in Figure S2. (B) Quantification of cilia frequency (mean and SEM) in fibroblasts from controls (Ctrl-10, Ctrl-117, Fetal Ctrl-26153), three individuals with JS (JBTS-10, JBTS-153 and JBTS-3504), the carrier parents of JBTS-3504 (Parent-3229 and Parent-1753), one fetus with MKS1-related MKS (MKS-158), and ARL13B (ARL13B-277) and INPP5E (INPP5E-171) mutants. * indicates p<0.05, *** p<0.001 (One-way ANOVA). (C) Kernel density plots depicting distribution of cilia length (x-axis) in fibroblasts obtained from individuals with different MKS1 mutations and controls (y-axis). Points and error bars represent medians and 99% confidence intervals respectively. JBTS-10 and JBTS-3504 have longer (L) cilia than the controls Ctrl-10 and Ctrl-117. *** indicates p<0.001 (Kruskal-Wallis test). Variance (σ²) in ciliary length was different between Fetal Ctrl-26153 and MKS-158, and between Ctrl-10 and Ctrl-117 compared to JBTS-10, JBTS-153, JBTS-3504 and ARL13B-277. *** indicates p<0.001 (F-test). Number of cells scored 100–300 in 2 batches.

Figure 3. Mks1 knockdown impairs ciliogenesis in 3D spheroid culture of IMCD3 cells

(A) Immunostaining of spheroids for cilia (acetylated tubulin, green), tight junctions (ZO1, white), and adherens junctions (β-catenin, red) with DAPI counterstaining (blue) shows loss of cilia after Mks1 siRNA transfection, and rescue by MKS1-WT. (B) Quantification of ciliary frequency in spheroids shows significant differences between control spheroids and spheroids depleted for MKS1 (indicates p<0.0003), and a potential dominant negative effect of transfection with MKS1-p.D19Y (p<0.001). Complete rescue of ciliary frequency was obtained upon transfection with MKS1-WT or MKS1-p.S403L (p<0.01), and a partial rescue upon transfection with MKS1-p.P218S (p<0.06). 50 spheroids were scored per condition. Data was normalized to IMCD3 cells transfected with siControl and empty vector, which was set to 1. Error bars represent SEM (n = 3 experiments), (C) Immunoblot for MKS1 in IMCD3 lysates (siControl) transfected with different MKS1 alleles. Upper band indicates equal endogenous levels of MKS1 in IMCD3 cells. Lower band indicates different MKS1 alleles (not full-length human MKS1 construct). β-actin is used as loading control.

Figure 4. Reduced ciliary ARL13B and INPP5E in fibroblasts from individuals with MKS1-related Joubert syndrome

(A) Immunostaining of fibroblasts derived from skin biopsies of JBTS-10, JBTS-153, JBTS-3504, MKS-158, and controls. INPP5E (green), gamma tubulin (g-tub; white) and cilia (acetylated tubulin, red; scale bar 5 μm). Brightness and contrast were identically adjusted across photos for visualization purposes; original data is in Figure S5. (B) MKS1-mutant fibroblasts have less ARL13B in the cilium than control cells (Tukey whiskers). *** indicates p<0.001 (Kruskal-Wallis test). (C) MKS1-mutant fibroblasts have less INPP5E in the cilium than control cells (Tukey whiskers). *** indicates p<0.001 (Kruskal-Wallis test). Cilium fluorescence intensity was calculated by subtracting cytoplasmic background from cilium signal and normalizing to Ctrl-117 intensity to be able to combine batches (n = 50–150 cilia in 2 batches, see Methods and Fig S1 for details).

Figure 5. MKS1 mutations associated with Joubert syndrome do not alter MKS1 protein localization to the ciliary TZ

(A) Immunostaining of fibroblasts derived from skin biopsies of JBTS-10, JBTS-153, JBTS-3504, MKS-158, INPP5E-171, and controls for MKS1 (red) and cilia (acetylated tubulin, green). Only the fetus with MKS1-related Meckel-Grüber syndrome (MKS-158) shows decreased MKS1 at the transition zone of the cilium. Experiment was performed twice independently. Scale bar 5 μm (B) Schematic overview of the roles of MKS1, ARL13B and INPP5E in Joubert syndrome. Loss of function mutations in MKS1 cause transition zone (TZ) dysfunction and disturb ciliary localization of ARL13B and INPP5E. Our data support the hypothesis that loss of ARL13B-dependent localization of INPP5E is a central mechanism underlying JS. The downstream events are hypothetical based on data in the literature.