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. 2014 Jun;7(6):711-22.
doi: 10.1242/dmm.015222. Epub 2014 Apr 24.

A Novel Mouse Model of Warburg Micro Syndrome Reveals Roles for RAB18 in Eye Development and Organisation of the Neuronal Cytoskeleton

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Free PMC article

A Novel Mouse Model of Warburg Micro Syndrome Reveals Roles for RAB18 in Eye Development and Organisation of the Neuronal Cytoskeleton

Sarah M Carpanini et al. Dis Model Mech. .
Free PMC article

Abstract

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.

Keywords: Cataract; Neurofilament; Warburg Micro syndrome.

Figures

Fig. 1.
Fig. 1.
Rab18−/− mice recapitulate the Warburg Micro syndrome phenotype. (A) Representation of the FlipRosaβGeo genetrap cassette inserted into intron 2 of Rab18. LTR, long terminal repeat; frt and F3, target for FLPe recombinase; loxP and lox5171, targets for Cre recombinase; SA, splice acceptor; βgeo, β-galactosidase and neomycin phosphotransferase fusion gene as a marker of protein expression; pA, polyadenylation sequence, adapted from Schnütgen et al., 2005. (B) Reverse-transcriptase PCR on cDNA taken from whole embryos of wild-type (WT), heterozygote (HET) and Rab18−/− mice. The products were amplified using oligonucleotides that targeted exons 1 and 5 (red arrows in A) and showed weak residual Rab18 transcript in Rab18−/− mice. Amplification using oligonucleotides that targeted exons 3 and 7 (blue arrows in A) showed slightly reduced transcript levels from the 3′-end of the cassette insertion site in Rab18−/− mice. β-actin was used as a reaction control. (C) Quantitative RT-PCR on heterozygous (HET) and Rab18−/− mouse embryonic fibroblasts that amplified exons 3–4 showed no Rab18 transcript in Rab18−/− mice compared with heterozygous littermate controls. The relative expression of Rab18 to the expression of TBP is shown. (D) Representative western blot analysis on 10 μg of sciatic nerve isolated from wild-type (WT), heterozygous (HET) and Rab18−/− mice using an antibody targeting the C-terminus of RAB18. The western blot shows a lack of RAB18 protein in Rab18−/− mice. β3 tubulin was used as a loading control. (E) Representative western blot analysis of heterozygous (HET) and Rab18−/− mouse embryonic fibroblasts. RAB18 protein expression was lacking in Rab18−/− mouse embryonic fibroblasts, α tubulin was used as a loading control. (F) Slit-lamp picture of the eye of a control mouse. (G) Slit-lamp pictures showed dense cataracts in adult Rab18−/− mice. (H) Representative images of hind limb clasping in Rab18−/− mice. When elevated by the tail, wild-type mice (WT, left) spread their hind limbs, whereas their Rab18−/− littermates (right) clasped their hind paws together. (I) Kaplan-Meirer plot showing the survival of wild-type (WT), heterozygote (HET) and Rab18−/− mice. Rab18−/− mice were culled following onset of hind limb weakness in accordance with Home Office guidelines.
Fig. 2.
Fig. 2.
Defective pre-natal development of the lens in Rab18−/− mice. (A,B) At E12.5, Rab18−/− mice showed a delay in the filling of the lens vesicle through the delayed migration of lens fibre cells from the posterior (B, arrow), whereas wild-type littermates had a closed lens vesicle (A). By E15.5, the Rab18−/− lens vesicle had closed, but the first signs of cataract development (small vacuoles at the lens periphery) were evident (D, arrow). Signs of cataract formation were absent in controls (C). By P1.5, large vacuoles and centralised pyknotic nuclei were seen in Rab18−/− lenses (F, arrow) but not in controls (E). All other ocular structures in control (G) and Rab18−/− lenses (H) appeared normal, and no retinal degeneration was observed. Note that the Rab18−/− eye was unpigmented, owing to the origin of the mutation in 129P2-derived embryonic stem cells; consequently, the retinal pigment epithelium (arrow in G) was not easily observed. Optical projection tomography undertaken on adult wild-type (I) and Rab18−/− unpigmented eyes (J) showed a dense nuclear cataract in the centre of the Rab18−/− lens (arrow in J). Scale bars: 100 μm (A–F); 50 μm (G,H); 400 μm (I,J).
Fig. 3.
Fig. 3.
Loss of RAB18 results in the enlargement of lipid droplets in MEFs. Control (A) and Rab18−/− (B) MEFs that had been treated with oleic acid for 24 hours were fixed and stained with the neutral lipid stain BODIPY 493/503 (green), cell nuclei were stained by using DAPI (blue). (C) Quantification of lipid droplet size showed enlarged lipid droplets in Rab18−/− MEFs. n=3 mice, mean±s.e.m. (mean lipid droplet size – 8.9 pixels in control MEFs and 12.8 pixels in Rab18−/− MEFs), *P<0.05 using an unpaired Student’s t-test. Scale bars: 10 μm.
Fig. 4.
Fig. 4.
Rab18−/− mice have large accumulations of neurofilament at the neuromuscular junction. (A–F) Confocal micrographs showing the neuromuscular junction in (A,D) flexor digitorum brevis (FDB), (B,E) lumbrical and (C,F) transverse abdominus muscle preparations from control (A–C) and Rab18−/− mice (D–F). TRITC-conjugated α-bungarotoxin staining is shown in red, staining of 165-kDa neurofilament and synaptic vesicle protein SV2 are both shown in green. (G–I) Bar charts (mean±s.e.m.) showing that, in all muscles examined – (G) FDB, (H) lumbrical and (I) TVA – the majority of endplates from both early- and mid-late-symptomatic Rab18−/− mice were fully occupied (where SV2 staining overlayed the endplate). Statistical significance was assessed using a Mann–Whitney U test. Early symptomatic: FDB P-value=0.5553, muscles from control mice (n=8) and muscles from Rab18−/− mice (n=7); lumbrical P-value=0.8120, muscles from control mice (n=10) and muscles from Rab18−/− mice (n=8); TVA muscle (n=1). Mid-late symptomatic: FDB P-value=0.3523, muscles from control mice (n=9) and muscles Rab18−/− mice (n=8); lumbrical P-value=1.0, muscles (n=10); TVA P-value=1.0, muscles (n=3). ns, not significant. (J–L) Bar charts (mean±s.e.m.) showing the percentage of endplates that exhibited large accumulations (stained green) in Rab18−/− mice at early- and mid-late-symptomatic timepoints. Statistical significance was assessed using a Mann–Whitney U test. Early symptomatic: FDB P-value=0.0026, muscles from control mice (n=8) and muscles from Rab18−/− mice (n=7); lumbrical P-value=0.0096, muscles from control mice (n=10) and muscles from Rab18−/− mice (n=8); TVA muscle (n=1). Mid-late symptomatic: FDB P-value=0.0005, muscles from control mice (n=9) and muscles from Rab18−/− mice (n=8); lumbrical P-value=0.0002, muscles (n=10); TVA P-value=0.0765, muscles (n=3). ns, not significant, **P<0.005, ***P<0.001. (M–R) Confocal micrographs showing neuromuscular junctions from Rab18−/− lumbrical muscles that had been immunostained with TRITC-conjugated α-bungarotoxin (red) and one of either neurofilament (NEFM) (M–O) or SV2 (P–R). Note the large neurofilament accumulations in Rab18−/− endplates (O) but normal levels of synaptic vesicle marker SV2 (R). Scale bars: 10 μm.
Fig. 5.
Fig. 5.
Rab18−/− mice have grossly disorganised cytoskeletons in peripheral nerves and accumulate microtubules at the neuromuscular junction. (A–D) Electron micrographs of the sciatic nerve in control (A,C) and mid-late-symptomatic Rab18−/− mice (B,D) showed normal myelination and normal Remak bundles in both genotypes. (C,D) Higher-magnification images showed disorganisation of the cytoskeleton in the sciatic nerve of Rab18−/− mice (D) compared with that of controls (C). The images are representative of that found in three animals for each genotype. (E–G) Quantification of myelination identified no abnormalities in the G-ratio (G-ratio is the diameter of an axon divided by the diameter of axon plus myelin). Statistical significance was tested by using an unpaired two-tailed Student’s t-test. G-ratio P-value=0.7610, mice (n=3) (E) or spread of axon diameter compared with the G-ratio in control (F) and Rab18−/− mice (G). (H,I) FDB muscles from wild-type (H) and mid-late-symptomatic Rab18−/− mice (I) were co-stained for β3 tubulin (green, immunostaining) and TRITC-conjugated α-bungarotoxin (red). Large accumulations of microtubules at the NMJ of Rab18−/− mice were observed. Scale bars: 2 μm (A–D); 10 μm (H,I).

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