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, 7 (1), 10220

Wfs1- Deficient Rats Develop Primary Symptoms of Wolfram Syndrome: Insulin-Dependent Diabetes, Optic Nerve Atrophy and Medullary Degeneration

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Wfs1- Deficient Rats Develop Primary Symptoms of Wolfram Syndrome: Insulin-Dependent Diabetes, Optic Nerve Atrophy and Medullary Degeneration

Mario Plaas et al. Sci Rep.

Abstract

Wolfram syndrome (WS) is a rare autosomal-recessive disorder that is caused by mutations in the WFS1 gene and is characterized by juvenile-onset diabetes, optic atrophy, hearing loss and a number of other complications. Here, we describe the creation and phenotype of Wfs1 mutant rats, in which exon 5 of the Wfs1 gene is deleted, resulting in a loss of 27 amino acids from the WFS1 protein sequence. These Wfs1-ex5-KO232 rats show progressive glucose intolerance, which culminates in the development of diabetes mellitus, glycosuria, hyperglycaemia and severe body weight loss by 12 months of age. Beta cell mass is reduced in older mutant rats, which is accompanied by decreased glucose-stimulated insulin secretion from 3 months of age. Medullary volume is decreased in older Wfs1-ex5-KO232 rats, with the largest decreases at the level of the inferior olive. Finally, older Wfs1-ex5-KO232 rats show retinal gliosis and optic nerve atrophy at 15 months of age. Electron microscopy revealed axonal degeneration and disorganization of the myelin in the optic nerves of older Wfs1-ex5-KO232 rats. The phenotype of Wfs1-ex5-KO232 rats indicates that they have the core symptoms of WS. Therefore, we present a novel rat model of WS.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Creation and characterization of the Wfs1 mutation in rats. (a) Zink finger nuclease (ZFN) design and cutting site. Genotyping primers are in bold and underlined, ZFN binding site in red, ZFN cut site in lower case red, and blue indicates the start and end of exon 5 of the rat Wfs1 gene. (b) DNA Sequence of exon 5 of the Wfs1 deficient rat line 232. Rat line Wfs1-ex5-KO232 lost 184 bp (17,833–18,017) in the Wfs1 gene, including 55 bp in exon 5; blue indicates the start and end of exon 5. (c) Comparison of cDNA and protein sequences from wild-type (WT) and Wfs1-ex5-KO232 rat lines. Line Wfs1-ex5-KO232 has lost all nucleotides from coding exon 5 of the Wfs1 gene. According to cDNA sequencing and ORF analysis, the resulting strain had lost exon 5 in the Wfs1 gene. Protein sequence is predicted from cDNA analysis; deletion of 55 bp from exon 5 of the rat Wfs1 gene did not result in a frame shift mutation. Thus, there is a loss of 27 amino acids (from coding exon 5 of the Wfs1 gene) and a new GCC codon (coding A - alanine) at the junction of exon 4 and exon 6 in the Wfs1-ex5-KO232 rats (marked red).
Figure 2
Figure 2
Development of diabetes mellitus in Wfs1-ex5-KO232 rats. (a) Wfs1-ex5-KO232 animals are slightly lighter than wild-type (WT) rats of similar ages and begin to lose weight after 10 months of age. (b) Basal blood glucose levels were similar for both genotypes up to 11 months of age; thereafter, Wfs1-ex5-KO232 rats develop hyperglycaemia. (c) Wfs1-ex5-KO232 rats developed glycosuria after 10 months of age. (d) Insulin levels were lower in older Wfs1-ex5-KO232 animals compared to levels in WT animals of the same age. For the insulin tolerance tests (ITTs), human insulin was administered (1 U/kg, s.c.), and blood glucose levels were measured at the indicated time points. There were no genotype-associated changes in insulin sensitivity in either (e) young or (f) old animals. For intraperitoneal glucose tolerance tests (IPGTTs), blood glucose levels were measured after administration of glucose (2 g/kg i.p.). (g,h) Glucose tolerance was similar for both genotypes at 1 and 2 months. (i) At 3 months of age, Wfs1-ex5-KO232 rats showed a slight glucose intolerance compared with WT rats, which was exacerbated at (j) 6 months. (k) Area under the curve for IPGTT results at different ages. (l) Glucose-stimulated increases in blood insulin levels (relative to baseline) 30 minutes after glucose administration, Wfs1-ex5-KO232 animals showed a defect in insulin secretion after 3 months of age. Size distribution of islets of Langerhans in rats at (m) 3, (n) 7 and (o) 14 months of age. Islet mass in rats at (p) 3, (q) 7 and (r) 14 months of age. The data were compared using two-way ANOVAs followed by Tukey’s HSD tests; #p < 0.05, ## < 0.01 between genotypes, *p < 0.05, **p < 0.01 within genotype (vs baseline and vs 3 months age). The data are presented as the mean ± SEM, n = 6–8.
Figure 3
Figure 3
Expression of XBP1 and BiP in islet of Langerhans. Immunofluorescence analysis of endoplasmic reticulum (ER) stress markers (a,e,c,g) BiP and (b,f,d,h) XBP1 in islets of Langerhans (dotted lines) from 3- and 7-month-old rats. At 3 months, the expression of (a) BiP and (b) XBP1 in control rats and (e,f) Wfs1-ex5-KO232 rats was comparable. By 7 months, the expression of (c) BiP and (d) XBP1 remained at basal level in WT rats, whereas the expression of these ER stress markers was clearly elevated in (g,h) the islets of Langerhans of Wfs1-ex5-KO232 rats. (i – l) Quantification of signal intensity of ER stress markers. Levels of (k) BiP and (l) XBP1 were increased in islets of Langerhans from 7-month-old Wfs1-ex5-KO232 rats. (m,n,o,p) mRNA analysis of ER stress markers BiP and spliced Xbp1 in lysates of isolated islets of Langerhans. Expression level of BiP was not altered in (m) 3-month- or (o) 7-month-old Wfs1-ex5-KO232 rats compared to expression in wild-type rats. Xbp1 splicing was increased in (n) 3-month- and (p) 7-month-old Wfs1-ex5-KO232 rats compared to expression in WT rats. The data were compared using t-tests; ***p < 0.001, **p < 0.01, *p < 0.05 between genotypes. The data are presented as the mean ± SEM, n = 4 to 9. Cell nuclei (blue) were counterstained with DAPI. Scalebar: 100 µm.
Figure 4
Figure 4
Development of cataracts and the localization and expression of glial fibrillary acidic protein and phospho-IRE1α in retinas. Dissected lenses from (a–c) wild-type (WT) and (d–f) Wfs1-ex5-KO232 rats. (d) 3-month-old Wfs1-ex5-KO232 lenses displayed greater signs of developing cataracts (arrowhead), with a prevalence of 2/5, compared to controls (0/4). (e) In 7-month-old knock-out rats, various degrees of cataract progression were seen, with a prevalence of 3/5. (f) All observed 14-month-old Wfs1-ex5-KO232 rat lenses (3/3) expressed clear signs of cataracts. Expression and immunolocalization of (g,i) glial fibrillary acidic protein (GFAP) and the endoplasmic reticulum stress marker (h,j) phospho-IRE1α in 15-month-old rats. (g) Retinas of WT rats showed GFAP expression in astrocytes at the ganglion cell layer (GCL). (i) Retinas displayed a clear upregulation of GFAP in Müller glial processes (arrowhead). Phospho-IRE1α in (h) WT rats showed a uniform immunoreactivity over the entire retina, whereas in (j) Wfs1-ex5-KO232 retina, a robust increase was observed. (g–j) Nuclei were counterstained with DAPI (blue). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scalebar: 100 µm.
Figure 5
Figure 5
Ultrastructure of the optic nerve in 15-month-old wild-type and Wfs1-ex5-KO232 rats. Low-magnification image shows axons in (a) wild-type (WT) and (b,c) Wfs1-ex5-KO232 rats. (a) Axons are tightly packed and show orderly appearance in WT rats. (b) Axons are more disorganized and space between axons is increased in Wfs1-ex5-KO232 rats. Axons in knock-out rats show (d) hypermyelination as indicated by the thickened myelin sheath. (e,f) Higher magnification images from (b) and (c) show necrotic fibres in Wfs1-ex5-KO232 rats. (g) Axonal compression and (h,i) demyelination, with extensive vacuolization is clearly evident in the Wfs1-ex5-KO232 nerve. Images are representative, 4 animals per genotype were analysed.
Figure 6
Figure 6
In vivo T2-weighted RARE MR imaging of the optic nerve. Wild-type (WT) and Wfs1-ex5-KO232 rats were anaesthetized and imaged at 8 months (WT, n = 6; Wfs1-ex5-KO232, n = 6) and 15 months (WT, n = 6; Wfs1-ex5-KO232, n = 7). The optic nerve was segmented manually by an observer blinded to the genotypes of the rats using IDK-SNAP (V 3.6.0). Left: By 15 months, Wfs1-ex5-KO232 rats showed optic nerve atrophy (optic nerve = optic nerve, chiasm and tract); Right: 3D renderings of the optic nerve (WT, black; Wfs1-ex5-KO232, red) generated using IDK-SNAP and Paraview (V 4.4.0). The data were analysed using a completely randomized ANOVA followed by Fisher’s LSD post hoc tests (see text for details); **p < 0.01 between genotypes. The data are presented as the mean ± SEM.
Figure 7
Figure 7
Localization of WFS1 protein in the medullas. Coronal sections of the medullas at the level of inferior olive (bregma −12.60 mm) were stained with anti-WFS1 antibody. WFS1 staining was localized mainly to the linear nucleus (LI) and inferior olive (IO). WFS1 staining was less intense in the medulla of 15-month-old (b) Wfs1-ex5-KO232 rats than in that of (a) wild-type (WT) rats of the same age. (c) WFS1 showed a similar expression pattern with that of the IO neuronal marker FOXP2. (d) Higher magnification of the IO shows the cytoplasmic localization of WFS1. (e) WFS1 and FOXP2 were expressed in the same set of cells in the IO. (f) Real-time PCR analysis of the ER stress marker BiP showed an increase in ER stress in the ventral medullas of 7-month-old Wfs1-ex5-KO232 rats but not in those of 3-month-old animals. (g) Expression of the ER stress chaperone Chop was similar between genotypes at both time points. (h) Expression of Wfs1 mRNA was similar in 3- and 7-month-old Wfs1-ex5-KO232 and WT rats. (i) Expression of Foxp2 was similar in 3-month-old and decreased in 7-month-old Wfs1-ex5-KO232 rats compared to expression in WT animals. The data were compared using two-way ANOVAs followed by Tukey’s HSD tests; *p < 0.05, **p < 0.01 between genotype. The data are presented as the mean ± SEM, n = 8 to 10.
Figure 8
Figure 8
Quantitative MRI analysis of brainstem volume of Wfs1-ex5-KO232 rats. The medulla was manually traced by an observer blinded to the genotypes of the rats from T2 images using ITK-SNAP software. The volumes of the segmented structures were calculated as volume per slice from bregma level −9.48 to −15.48 mm. (a) The total volume of the medulla and (b) volume of the extraparenchymal space (EPS). Volumes of individual MRI slices from (c) 8-month- and (d) 15–month-old animals. The volume of the extraparenchymal space is increased at the level of the inferior olive (IO) in Wfs1-ex5-KO232 rats at 15 months. Line shows localization of IO (bregma −12.00 until −14.76 mm). Representative T2-weighted MR images of the medulla of wild-type (WT) and Wfs1-ex5-KO232 rats are taken at the level of the IO (bregma approx. −12.63 mm). (e,f) There are no significant changes in medullary volume at 8 months of age. At 15 months of age, the ventral surface of the medulla appear more concave (arrowhead) in (h) Wfs1-ex5-KO232 rats than in (g) WT littermates. (h) The area of the extraparenchymal space surrounding the medulla is also increased in Wfs1-ex5-KO232 rats. The data were compared using two-way ANOVAs followed by Fisher’s LSD tests; *p < 0.05, **p < 0.01 between genotypes. The data are presented as the mean ± SEM, n = 6–7.

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