WFS1 gene delivery rescues visual function in a mouse model of Wolfram syndrome

doi:10.21203/rs.3.rs-7424550/v1

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WFS1 gene delivery rescues visual function in a mouse model of Wolfram syndrome

https://doi.org/10.21203/rs.3.rs-7424550/v1

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Wolfram syndrome is a rare childhood neurodegenerative disease characterized by diabetes followed by severe and rapid optic atrophy leading to blindness before the age of 20. Patients often develop other symptoms, such as deafness and neurological dysfunction. Wolfram syndrome is caused by mutations in the WFS1 gene, which encodes wolframin protein. Despite decades of intensive research, the complex mechanisms of optic neuropathy are not fully understood, and there are currently no therapies to prevent vision loss in Wolfram patients. Here, we showed that Wfs1 knockout mice generated by disruption of exon 8 of the Wfs1 gene develop progressive loss of visual acuity, optic disc pallor and severe optic nerve damage. We tested the efficiency of gene therapy using AAV2 to deliver human WFS1 to retinal ganglion cells in Wfs1 knockout mice. Our results provide the first evidence that intravitreal injection of human WFS1 has significant neuroprotective effects on retinal ganglion cells and their axons and slows the loss of visual acuity. These results demonstrate that WFS1 is able to provide both functional and structural protection to retinal ganglion cells in Wfs1 knockout mice and provide important evidence for the efficacy of WFS1 as a neuroprotective treatment for Wolfram syndrome. These results demonstrate the promising effects of gene therapy for Wolfram syndrome and encourage future research aimed at conducting clinical trials in patients.

Wolfram syndrome

WFS1 gene

retina

gene therapy

mouse

Wolfram syndrome (WS) (OMIM 222300) is a rare multisystemic disorder (1/160,000 in Europe; 1/100,000 in North America) featuring systematic early-onset type I diabetes mellitus and optic neuropathy that is variably associated with other neurological dysfunctions [8, 10, 55, 81]. Patients often develop second-decade sensorineural hearing impairment, diabetes insipidus and urinary dysfunctions. Psychiatric illness impairs the lives of most diagnosed patients and sometimes their relatives as heterozygous carriers [24, 72, 73].

WS is an autosomal recessive genetic disease caused by mutations in WFS1. However, we discovered that recessive mutations in WFS1 also cause nonsyndromic forms of inherited optic neuropathies (ION) [26]. In addition, beside WS and nonsyndromic recessive ION, there are dominant forms of severe to profound hearing loss, which can be associated with optic atrophy (OA) and glucose intolerance due to heterozygous mutations in WFS1, commonly referred to as WS-like diseases [20, 50, 58]. Thus, WFS1 mutations appear to underlie a spectrum of various dominant and recessive disorders, featuring common multimodal neurosensory involvement.

Eye fundus examination of WS patients revealed bilateral optic disc pallor as a consequence of optic nerve atrophy [9, 69]. Magnetic resonance imaging reveals visual system atrophy at the central level, with reduced signals in the optic nerves, chiasm and tracts [7, 25]. Optical coherence tomography (OCT) studies revealed retinal nerve fiber layer (RNFL) thinning in WS patients [7, 27, 85, 86].

Consequent to anatomic loss, visual impairment is diagnosed by a reduced visual field and pattern visual evoked potentials (P-VEP) [10, 16, 37, 71]. A progressive decrease in visual acuity associated with visual field scotomas leads to legal blindness.

WFS1 encodes a transmembrane endoplasmic reticulum (ER) protein called wolframin [29, 34]. Wolframin plays a role in Ca²⁺ homeostasis, as it can function as, or activate, an ER Ca²⁺ channel [38, 74] and modulate ER stress [22, 23]. In the human retina, wolframin is detected in retinal ganglion cell (RGC) somas and axons and in nonmyelinated proximal optic nerves (ONs) [65]. In pancreatic beta cells, the absence of Wolframin leads to a major deficit in insulin synthesis, which causes diabetes [30]. Glucose dysregulation affects the cell cycle of Wfs1-deficient beta cells and increases their degree of apoptosis as a consequence of an increased ER stress response [74]. However, today, how these functions are related is not known, given that structure function correlations are not available for Wolframin. The authors suggested a relationship between cytosolic Ca²⁺ disturbance, impaired mitochondrial dynamics and delayed neuronal development in WFS1-deficient neurons [15]. We demonstrated that WFS1 forms a complex with IP₃Rs and the neuronal calcium sensor (NCS1) and regulates the dynamic interaction between the ER and mitochondria at mitochondria-associated membranes (MAMs) [3].

In mice, wolframin is expressed in the retina, with a predominance in RGCs and the inner nuclear layer [65]. Several Wfs1 mutant rodents have been developed as disease models of Wolfram syndrome [30, 34, 41, 44, 53, 59, 60]. Impairment of visual function has been described in some cases [2, 12, 62]. A rat model of WS has been characterized by features of retinal gliosis, cataracts and optic nerve volume reductions, and disrupted myelin structure [53].

To address many of the unanswered questions related to the molecular and cellular pathophysiology of WFS1 optic atrophy and to develop models for testing RGC rescue, we analyzed the visual function of Wfs1-knockout (KO) mice generated by disrupting exon 8 of the Wfs1 gene (Wfs1^exon8del) and overexpressing human WFS1 in the eye via gene therapy to prevent cellular and axonal degeneration. In this study, we show a progressive loss of visual acuity and RGC damage in this model. Furthermore, we demonstrate the efficacy of the intravitreal delivery of AAV serotype 2 expressing human WFS1 to prevent optic atrophy in this mouse model of Wolfram syndrome.

Animals

Wfs1-deficient mice were generated by invalidation of exon 8 of the Wfs1 gene (Wfs1^exon8del) [35]. The mice were in the 129S6/SvEvTac × C57BL/6 genetic background. Therefore, wild-type and homozygous littermates were used in the present study. All experimental protocols were approved by the Institute National de la Santé et de la Recherche Médicale (Inserm; Montpellier, France), were consistent with the European directives, and complied with the ARVO Statement for the Use of Animals in Ophthalmic Research. They were carried out under the agreement of the Languedoc Roussillon Comity of Ethics in Animal Experimentation (CEEALR; nuCEEA-LR-12123). The animals were kept under a 12 h light‒dark cycle with food and water available ad libitum.

Blood glucose measurement

Blood glucose levels for the Wfs1^exon8del line were measured at night and the following morning after overnight fasting, following the protocol used for human patients. Briefly, a tip of the disinfected tail was cut off, and a drop of blood was applied to the test strip previously inserted into the Accu-Chek® Performa (Aviva Blood Glucose Meter System, Roche; Meylan, France). The mean ± SEM results in mg/dL for 3- and 6-month-old mice (n = 8–10) were then calculated and plotted.

Visual acuity and contrast sensitivity

We tested 3- and 6-month-old WT and KO mice for visual acuity and contrast sensitivity with the OptoMotry virtual-reality system (Cerebral Mechanics, Lethbride, Canada). They were placed on an elevated platform in the center of a chamber surrounded by LCD screens, with a recording camera installed over the platform. The screens displayed black and white stripes that created an illusion of an external rotating cylinder. The animals were untrained and were moving freely. During the test, the grating circulation was continuously centered on the mouse’s head. First, the stripes were moved at a constant speed of 2 rpm (12 degrees per second (d/s)), minimal spatial frequency (0.031 cycles per degree (c/d)) and maximal contrast (100%) for short adaptation. A positive response to the stimulus, called the optomotor reflex (OMR), was noted when the mouse moved her head in coherence with the movement of the encircling stripes. The OMR was examined with respect to either increasing spatial frequency or decreasing contrast, with the other factors remaining stable. When the frequency was changed, the thickness of the black stripes decreased. When the contrast was changed, the black stripes were paling. The software then changed the frequency (or contrast) back and forth with decreasing steps until the visual threshold was established via the staircase technique [19, 54, 57]. A minimum of 10 mice of each age, genotype and sex were examined. First, the test was performed clockwise, which should influence the left eye of the mouse more, and then counterclockwise, which should influence the right eye [19]. The mean threshold value from both eyes of each mouse was taken for analysis, with an unpaired two-tailed t test for statistical comparison between the groups.

Eye fundus

Pupillary dilatation was induced by topical application of 0.5% tropicamide (Mydriaticum; Laboratoires Théa; Clermont-Ferrand, France) to both eyes. The mice were anesthetized via the intraperitoneal injection of ketamine (45 mg/kg body weight; Merial, France) and xylazine (17 mg/kg body weight; Bayer Healthcare, Germany). Once asleep, the eyes were instilled with 0.4% oxybuprocaine hydrochloride (Cebesine; Laboratoire Chauvin, Bausch + Lomb; Montpellier, France), and the excess fluid was wiped off, ensuring ocular anesthesia and immobility. Fundoscopy was performed with a Micron III retinal imaging system (Phoenix Research Labs; Pleasanton, USA). During the exam, the eyes were covered with an ophthalmic gel (Lacryvisc; Alcon; Fort Worth, USA) to minimize light refraction and dry the eye. Pictures obtained with StreamPix 3 (Norpix; Montreal, Canada) were analyzed for retinal damage and optic disc (OD) pallor, with at least 5 pictures per eye.

Optic Coherence Tomography

Optic coherence tomography (OCT) of the mouse retina was performed with an EnVisu R2200 imaging system (Bioptigen, Leica Microsystems; Wetzlar, Germany) as previously described [32]. Briefly, the eye fundus of the mice was prepressed, and anesthesia and mydriasis were performed. The eyes were hydrated with Systene® Ultra eye drops (Alcon®, Novartis; Fort Worth, USA), and whiskers were set aside. Vertical and circular ring scanning was applied, with the latter collecting 100 vertical scans, always centered on the ON. The measurements were performed at 5 points on both sides of the ON, in proximity, using ImageJ macro developed with Volker Becker, PhD. The exam was performed for Wfs1 WT and KO mice at 6.5 months, with 12 and 15 animals per group, respectively.

Transmission electron microscopy

The myelinated optic nerves (ONs) of 7-month-old WT and KO mice (n = 6–7) were prepared for transmission electron microscopy (TEM) as described previously [12] with minor modifications. Briefly, freshly isolated tissues were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences; Hatfield, USA) in PHEM buffer (homemade, pH 7.0) for 1 h at room temperature (RT) and then incubated overnight at 4°C. The following morning, the tissues were transferred to 0.5% glutaraldehyde in PHEM. The samples were washed in PHEM buffer and subsequently fixed in 0.5% osmic acid (Electron Microscopy Sciences; Hatfield, USA) for 2 h in darkness at RT. Next, they were rinsed twice in PHEM buffer and dehydrated in a series of EtOH solutions at gradually increasing concentrations (30–100%; Sigma‒Aldrich, Merck; St. Louis, USA). Finally, ONs were embedded in EmBed 812 Resin (Electron Microscopy Sciences, Hatfield, USA), following the manufacturer’s protocol, with an automated microwave tissue processor for electronic microscopy (EM AMW; Leica; Wetzlar, Germany). After processing, 70-nm-thick transverse sections were cut at different sample sizes with an ultramicrotome (Ultracut E; Leica-Reichert; Wetzlar, Germany), collected and counterstained with 1.5% uranyl acetate (Electron Microscopy Sciences; Hatfield, USA) in 70% EtOH and lead citrate (Electron Microscopy Sciences; Hatfield, USA). The ON sections were then observed via transmission electron microscopy (TEM) (Tecnai G² F20 Spirit BioTWIN TEM; FEI, Thermo Fisher Scientific; Hillsboro, USA) and randomly imaged with an Eagle 4K HS camera, which is optimal for high-resolution data acquisition.

Calculations were performed for myelinated axons in a minimum of 20 images of 4.4∙ 10⁻⁻¹¹ m² per mouse for the total number of axons per surface and the percentage of damaged axons in relation to their size. The pictures were then reanalyzed with ImageJ via the G ratio calculator 1.0 plugin (CIF, University of Lausanne; Switzerland) for myelin thickness. Data were collected for the inner perimeter, area, and index of the circularity (IC) parameters.

Immunohistochemistry

Flat-mounted retinas were prepared as follows. The enucleated eyes were first pierced and immersed in 4% paraformaldehyde (PFA) in PBS (homemade from powder; Sigma‒Aldrich, Merck; St. Louis, USA) for 3h at 4°C. After washing in PBS, the retinas were gently isolated from the eyes and permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT. Then, they were saturated for 30–60 min in a buffer of 10% donkey serum, 0.1% Triton X-100 and 0.1% Tween-20 in PBS. RGC nuclear staining was performed with anti-Brn3a goat polyclonal antibodies (1/500; Santa Cruz Biotechnology, Heidelberg, Germany) in saturation buffer overnight at 4°C. Human WFS1 was visualized using human-specific WFS1 antibody (rabbit 1/500; Cell Signaling Technology; Danvers, USA). After washing in PBS, secondary, anti-goat donkey antibodies were applied (1/800, 1 h at RT; Thermo Fisher Scientific; Waltham, USA). Hoechst (1/10 000 in PBS; Gibco™, Thermo Fisher Scientific; Waltham, USA) was applied during the last 15 min of incubation for nuclear counterstaining. After washing, cover slips were mounted on the slides (fluorescence mounting medium; Dako, Agilent Technologies; Glostrup, Denmark), and they were left to dry at 4°C. Each retina was nicked at the side in 4–5 places to enable flat mounting in a flower shape. The retinas were observed under fluorescence (Axio Imager D1/AxioVision with AxioCam MRc; Zeiss; Oberkochen, Germany) for further analysis.

Quantitative PCR assay

Total RNA was isolated from snap-frozen retinas of 6.5-month-old WT and KO mice (1 per mouse). The RNeasy mini kit (Qiagen; Courtaboeuf, France) was used following the manufacturer’s protocol, after which the purified RNA was stored at -80°C until further use. To obtain mRNA, equal quantities of the purified RNA were reverse transcribed with the Superscript III First Strand Synthesis System Kit (Life Technologies; Saint-Aubin, France) following the standard protocol. The acquired DNA was diluted to 5 ng/µl. SYBR® Green detection-based quantitative PCR (qPCR) was performed with a LightCycler® 480 system (Roche; Meylan, France) with previously established gene-dependent optimal melting temperatures and 40 replication cycles for 10 ng of DNA in triplicate. The expression of the tested genes was referenced against that of the L27 housekeeping gene and assessed via the absolute advanced quantification method of the software. The primers used are listed in supplementary table S1. For statistical purposes, the expression levels in the KO retinas were normalized to those in the WT retinas, and the mean ± SEM was subsequently calculated from three independent experiments.

Intravitreal injection

Gene therapy (GT) of the Wfs1^exon8del strain was performed intravitreally at P30 ± 1 day. First, the transduction efficiency was tested with AAV-2/2-CMV-GFP, n = 4. It was injected exactly as the AAV-2/2-CMV-WFS1 construct, which harbors the functional human gene, in a volume of 2 µl per eye at a titration of 1,5 ∙ 10¹¹ VG/ml in PBS. The viral constructs used in this study were produced at Laboratoire d'Amplification de Vecteurs – CHU de Nantes.

The GT procedure started with the previously described methods of prepizing and anesthetizing. Once asleep, the mouse was placed on its side under a binocular loupe (M80, Leica Microsystems; Wetzlar, Germany), and the eye was covered with ophthalmic gel (Lacryvisc; Alcon®, Novartis; Fort Worth, USA). After a circular cover slip was installed, the eye was pierced with a sharp 34-gauge needle (Hamilton MicroSyringe; Reno, USA) for pressure equalization. A loaded Hamilton 5 µl syringe (Microliter™ #65; Hamilton MicroSyringe; Reno, USA) with a blunt 34-gauge needle was subsequently used for gene delivery. It was performed on the opposite side of the first hole, and the needle was kept inside until the eye color returned to normal; therefore, it was maintained for several minutes. Finally, the needle was slowly removed, and the procedure was repeated on the second eye. The mouse was allowed to recover on a heating plate at 37°C, with the eyes covered by the ophthalmic gel, and was kept in a separate cage until full recovery.

Fundus fluorescein angiography

A total of 0.5 µl/g of 10% fluorescein sodium (Laboratoires Théa; Clermont-Ferrand, France) was intraperitoneally injected into a sleeping mouse. Immediately after, a sequence of pictures was taken (StreamPix 3; Norpix; Montreal, Canada) with a red filter in Micron III (Phoenix Research Labs; Pleasanton, USA).

Statistical analysis

GraphPad Prism software (version 10) (GraphPad Software, San Diego, CA) was used to calculate, plot and analyze the data. All the data are presented as the means ± standard errors of the means (SEMs) with error bars. The number of samples (n) is given in each figure. Student's t test was used for the analysis of two unpaired sets of data. Significance is indicated as follows: ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Wfs1^exon8del KO mice exhibit hyperglycemia

As WS induces diabetes in humans, blood glucose levels were measured in the Wfs1^exon8del line (Fig. 1). It has already been reported that KO mice exhibit glucose intolerance and a tendency toward diabetes [31, 43]. To confirm that the diet provided did not influence this trait, the measurements were taken at night, after a day of normal activity. At 3 months, the WT mice had slightly lower blood glucose levels than the KO mice did (Fig. 1A), at 125.2 ± 5.2 mg/dL vs. 139.5 ± 10.0 mg/dL. At 6 months, however, the WT had a similar level of 129.9 ± 4.7 mg/dL to that at 3 months, whereas the KOs had an increase of 191.4 ± 29.7 mg/dL, which was almost significant (p = 0.036). This level was not high enough to indicate diabetes, but it appears that KO mice have hyperglycemia.

In the morning after overnight fasting, blood glucose levels decreased uniformly in all test groups (Fig. 1B). Specifically, 3-month-old WT mice presented 79.4 ± 5.4 mg/dL vs. 71.7 ± 4.5 mg/dL peripheral blood glucose in the KO. At 6 months, the results were 84.1 ± 4.2 mg/dL vs. 84.3 ± 6.1 mg/dL, again similar. Overall, the morning values in the KO groups were much less variable than the nighttime values were.

Wfs1^exon8del KO mice show progressive loss of visual acuity and contrast sensitivity

To determine whether visual acuity was affected in Wfs1 KO mice, vision was evaluated via a behavioral test in which the optokinetic tracking response was measured. Visual acuity may be measured either by changing the spatial frequency or the contrast. Because it is much harder to measure contrast thresholds, this test was performed only for 3- and 6-month-old mice. A small yet statistically significant difference between the genotypes was reported at 1 month, with a spatial frequency threshold of 0.415 ± 0.002 c/d (cycles per degree) for the WT mice and 0.399 ± 0.003 c/d for the KO mice (Fig. 2A). This difference increased over time, as the threshold for the 3-month-old WT mice increased to 0.421 ± 0.002 c/d (p = 0.016), whereas for the KO mice, it decreased to 0.378 ± 0.004 c/d (p < 0.001). At 6 months, the discrepancy was even more evident (p < 0.001), as the threshold for the WT mice slightly decreased to 0.407 ± 0.002 c/d (p < 0.001), whereas the decline was steep for the KO mice (0.318 ± 0.008 c/d, p < 0.001). Briefly, a lack of Wfs1 caused a progressive loss of visual acuity. In accordance with what was observed for frequency, the KO animals had a worse contrast threshold optomotor response than the WT controls did (Fig. 2B). Three-month-old WT mice were able to detect grating movement with a contrast as little as 6.42 ± 0.17%, whereas the KO stopped at 15.33 ± 1.21%, reaching a significant difference (p < 0.001). When the mice were 6 months old, the divergence was even greater (p < 0.001); hence the WT mice 8.71 ± 0.17% and the KO mice only 28.17 ± 1.07%. The performance of both genotypes decreased over time (WT: p < 0.001, KO: p < 0.001); however, the visual acuity decline, again, was much sharper for the KO animals. These results confirm that the absence of Wfs1 provoked a progressive loss of contrast sensitivity.

The sex of the animal may influence the results, and in view of obvious physiological variations between females and males of the studied line [51], we also analyzed the thresholds for each sex separately. Our results revealed that KO males were more affected than KO females were (Supplementary Fig. 1).

Wfs1 KO mice develop optic disc pallor

In vivo retinal morphology was assessed by noninvasive fundus and OCT imaging every 3 months for 6 months. Fluorescein angiography did not indicate microvascular leakage (Supplementary Fig. 2), which is often present in patients with diabetes [39] but rarely occurs in WS patients [61]. However, a major difference in optic disc (OD) coloration was detected between WT and KO mice. In WT, the eye fundus was gray, whereas in KO, it was much paler and often white (Fig. 3A). Manual scoring of this phenomenon, with 0 points for the gray OD, 2 points for the white OD, and 1 point in between, revealed a significant difference at 3 months (p₃ = 0.001, p₆ < 0.001; Fig. 3B). Specifically, the 3-month-old WT and KO eyes scored 0.13 ± 0.13 and 1.13 ± 0.13, respectively, whereas at 6 months, the scores were 0.05 ± 0.03 and 1.06 ± 0.16, respectively.

Decrease of Thinning of RGC/RNFL and RGC complex layers in Wfs1 KO mice

Retinal thinning, namely, the retinal nerve fiber layer (RNFL), has become a reliable indicator of WS progression [26, 28, 85]. Therefore, RGC/RNFL and RGC complex layer thicknesses were measured via optical coherence tomography (OCT) in WT and KO retinas (Fig. 4). OCT was performed for 6.5-month-old mice when the KO phenotype of impaired visual function was established. There was significant shrinkage of both the RGC/RNFL (Fig. 4A) and the RGC complex layers in the KO mice (Fig. 4B). Specifically, the thickness of the RGC/RNFL in WT retinas was (22.9 ± 0.4) ∙ 10^− 6 m, whereas it was (17.8 ± 0.3) ∙ 10^− 6 m for KO retinas, whereas the area of measurement was (1.29 ± 0.02) ∙ 10^− 8 m² and (1.01 ± 0.01) ∙ 10^− 8 m², respectively. For the RGC complex layer, the difference seemed smaller because a large part of this layer consists of components other than RGCs. Thus, in the WT retinas, the thickness was (57.9 ± 1.3) ∙ 10^− 6 m vs (56.2 ± 1.5) ∙ 10^− 6 m for the KOs, indicating a thinning of 2.9%. The results for the area of measurement were analogous: (3.24 ± 0.07) ∙ 10^− 8 m² vs (3.16 ± 0.08) ∙ 10^− 8 m². Taken together, the OCT scans demonstrated a loss of RGCs or their protrusions in the KO mice (Fig. 4C). An analysis of each sex separately revealed that the KO females seemed to be more affected than the KO males were (Supplementary Fig. 3).

Severe axonal damage in Wfs1 KO mice

To assess RGC axon loss and/or damage, following the OCT results, transmission electron microscopy (TEM) images of ON transverse sections were analyzed (Fig. 5). With respect to the number of axons per surface and thus their density (Fig. 5A), although this difference was not significant, there was a trend toward an increased number of small RGC axons in KO mice ON, whereas large axons appeared less abundant. The total axonal density of the KO mice seemed to be greater than that of the WT mice, but this difference was not significant. For careful assessment of the optic nerve structure, transmission electron microscopy (TEM) was applied to identify damage. Axons were considered damaged when they presented abnormally thin or thick discontinuous myelin with adjacent blebs, axons with spiraling myelin, and axons that seemed torn or completely empty. Altogether, damaged axons in the WTs were around 25.9%, while in the KOs, axons damaged represented 68%, indicating a drastic increase (Fig. 5B). When analyzed as a function of size, 41% and 64.3% of the large axons were damaged in WT and KO mice, respectively (Fig. 5B). Finally, there was 16.8% versus 68.2% damaged small axons in WT and KO mice, respectively (Fig. 5B). These results suggest that KO mice have much more axonal damage, favoring small axons, which seem more abundant than those in the WT phenotype, at the cost of large axons (Fig. 5C).

No RGC loss in WFS1 KO mouse retina

As we have previously shown for the Wfs1^exon2del mouse strain [12], there was no loss of RGCs in the retina of the KO mice at 6 months post-injection. The difference in the number of cells expressing the Brn3a marker in the RGC layer (Supplementary Fig. 4) was not significant between WT and KO mice. On average, there were 172 ± 15 and 218 ± 19 Brn3a + cells, respectively. Moreover, when hematoxylin and eosin (H&E) staining was performed, there was no apparent difference in either the abundance or density of cells in the RGC layer between the two genotypes (Supplementary Fig. 4). There was only a tendency to have fewer cells close to the ON in the KO retina (142 ± 5 vs. WT 171 ± 26, 112 ± 8 mm-1 vs. WT 120 ± 13 mm-1).

No ER stress was detected in WFS1 KO mouse retinas

The gene expression of Bip, Chop and Xbp1S increases in response to ER stress (Riggs et al, 2005; Ueda et al, 2005; Yamada et al, 2006) and was therefore investigated in Wfs1 KO mouse retinas. When referenced to L27 and normalized to WT, the mean expression levels of Bip, Chop and Xbp1S in KO mice were 1.13 ± 0.06, 1.27 ± 0.47 and 0.99 ± 0.20, respectively, with no significant difference between genotypes (Supplementary Fig. 5).

AAV2-WFS1 drives targeted transgene expression in RGCs

Our results revealed that Wfs1 KO mouse retinas present similar clinical features to those of patients with Wolfram syndrome, such as progressive loss of visual acuity, optic disc pallor, axonal damage and retinal thinning. With the objective of correcting these retinal defects, we evaluated a gene therapy approach using intravitreal delivery of an adeno-associated viral 2/2 (AAV2/2) vector that encodes the human WFS1 cDNA. One-month-old animals included in the study received an intravitreal injection of 2 µl per eye at a titer of 1.5 10¹¹viral genomes (vg)/ml of AAV2-WFS1 or AAV2-GFP. Fundus imaging performed 2 weeks after intravitreal viral injections showed robust GFP expression in the animals that received AAV2-GFP (Fig. 6A). There was no apparent injury to the retina or signs of bleeding or cataracts. Immunostaining of flat-mounted retinas for WFS1 at 1 month following intravitreal injection confirmed the presence of WFS1 in the retinal ganglion cells of animals that received AAV2-WFS1 (Fig. 6B). There was an abundance of WFS1-positive “threads”, which are thought to be RGC protrusions. It appears that the intravitreal injections led to successful transduction in the retina.

AAV2-WFS1 rescues optic disc pallor and optic nerve damage in Wfs1 KO mice

To evaluate the rescue efficacy of optic nerve atrophy, we performed fundoscopy at 6.5 months. Fundoscopy revealed healthier retinas in KO-rescue eyes with reduced optic disc pallor than in KO-PBS- or KO-untreated eyes (Fig. 7). However, OCT scanning revealed no significant differences between untreated and KO-treated eyes (Supplementary Fig. 6). To further evaluate the influence of gene therapy on axonal degeneration in Wfs1 KO mice, ultrastructural analysis of the optic nerve using TEM was performed 7 months after intravitreal injection (Fig. 8A). Axon density distributions were not obviously different between the WT, KO-Wfs1 and WFS1 gene therapy rescue groups (Fig. 8B, C, D). Degenerating axons were identified by the presence of condensed electron dense cytoplasm, myelin debris or empty, swollen axonal profiles. Before gene therapy, analysis of axons clearly showed a higher frequency of pathological ultrastructural changes in untreated KO-Wfs1 mice (68% ± 4.9), with 64.3% ± 7.3 in large axons, and 68.2% ± 4.7 in small axons, compared to wild-type mice (25.5% ± 4.1, with 41.0%± 4.0 in large axons and 16.8% ± 3.5 in small axons) (Fig. 8E, F). In the WFS1 gene therapy group, the distribution of healthy axons reached that observed in control mice (Fig. 8G). These results indicate that human WFS1 partially rescues the ultrastructural axonal damage induced by loss of Wfs1.

To analyze the myelin thickness in ON axons, we measured the index of circularity (IC), the inner diameter and the area (Fig. 9). The IC of the axon was calculated as the ratio between the observed myelinated axonal area and the area of a circle with the same perimeter [76]. There was a significant increase in the IC in the untreated KO mice, which was 0.645 ± 0.006 µm greater than that in the wild-type animals (0.542 ± 0.006 µm), indicating that the KO axons were closer to the perfect circle (Fig. 9A). In the PBS and WFS1 groups, this feature was lost, with values of approximately 0.550 µm. In terms of the inner diameter of the axons (Fig. 9B), there were smaller axons in the NI KO mice than in the healthy WT mice, since the mean inner diameter was 0.601 ± 0.014 µm and 0.972 ± 0.018 µm, respectively. In the PBS group, the values were similar for both genotypes (0.937 ± 0.019 µm vs 0.913 ± 0.015 µm), as if the balance was restored in the sham group. In the group of KO mice treated with WFS1, the inner diameter of the axons increased compared with that of untreated KO mice, reaching 0.851 ± 0.018 µm. The inner area, which depends on axonal shape and size, was analyzed in a similar way (Fig. 9C). The untreated WT mice had a mean inner ON axonal area of 0.404 ± 0.013 µm², whereas the KO mice had a reduced area of 0.207 ± 0.013 µm², just as for the inner diameter. Compared with untreated KO-Wfs1 mice, WFS1-treated KO-Wfs1 mice in the rescue group presented an increase in the inner area (0.332 ± 0.012 µm²) (Fig. 9C). Histograms of the frequency distribution of index of circularity, inner diameter and area of axons in the untreated and treated groups are shown in Fig. 10. For each parameter, the overlap between WT and treated KO mice suggests that the administration of human WFS1 can restore normal axonal circularity and the ratio of small to large axons, i.e., their shape and size.

AAV2-WFS1 prevents loss of visual acuity

We assessed the visual acuity of Wfs1 KO mice treated (at 1 month) or not treated via the optomotor test, which measures the integrity of the subcortical visual pathways. In the wild type, 2 months after treatment, no differences were detected in the treated, PBS-treated and untreated mice (treated 0.420 ± 0.003 c/d; untreated 0.416 ± 0.003 c/d; and PBS-treated 0.418 ± 0.004 c/d). In contrast, in the disease model, Wfs1 KO mice treated with AAV2-WFS1 showed higher visual acuity than untreated or PBS-treated Wfs1 KO mice did (Fig. 11A) (treated 0.407 ± 0.003 c/d, untreated 0.379 ± 0.004 c/d, and PBS-treated 0.385 ± 0.009 c/d). Three months later (5 months after treatment), untreated mice exhibited the expected pattern of deterioration in visual acuity (Fig. 11B). The VA of the NI WT mice was 0.414 ± 0.002 c/d, whereas that of the NI KO mice was 0.334 ± 0.007 c/d (p < 0.001). KO mice injected with WFS1 were protected from a decrease in visual acuity and reached a score of 0.387 ± 0.008 c/d. PBS appeared to have a slight protective effect, with a score of 0.367 ± 0.008 c/d for the KO mice compared with the wild-type mice (0.416 ± 0.005 c/d). Intraocular injection of WFS1 appeared to significantly slow vision loss in KO mice.

Wolfram syndrome is a severe disease affecting several systems in the body. The main characteristics are diabetes mellitus, optic atrophy, diabetes insipidus and deafness. Other signs may develop, such as neurological problems, urinary tract issues and respiratory problems, due to brainstem degeneration. Symptoms generally appear in childhood and progressively worsen over time. Most WS patients carry mutations in the WFS1 gene, which is highly expressed in the eye and in the brain. It encodes a protein located within the endoplasmic reticulum. For reasons that are still unclear, RGCs are preferentially affected, explaining the marked loss of vision in this disease. The exact mechanisms involved in the development of optic neuropathy are still largely unknown, let alone the therapeutic options used to help prevent vision loss. The development and validation of new therapies require disease-relevant animal models. Among the existing animal models of WS, the Wfs1^exon8del KO mouse used in this study mimics the clinical features of WS patients [2, 35, 42, 43, 56, 62, 78–80].

In this study, we show that Wfs1^exon8del KO mice exhibit hyperglycemia and progressive visual loss starting before the age of 3 months and that intravitreal injection of human WFS1 has significant neuroprotective effects on RGCs and their axons in this mouse model of Wolfram syndrome.

Since the first WS symptom was diabetes mellitus, we checked the glucose level in the peripheral blood. Consistent with previous results, we found that KO mice were hyperglycemic but not diabetic, as glucose was below 500 mg/dL, a standard for Akita mice, which are diabetic due to ER stress-induced β-cell destruction [14]. Furthermore, hyperglycemia only occurs with normal food intake, confirming the observed glucose intolerance [43].

Optic atrophy is one of the most striking phenotypes in human patients with WFS1 mutations, and our analysis of Wfs1^exon8del mutant mice confirmed and clarified this phenomenon only. The optomotor test revealed progressive visual acuity loss in the KO mice from 3 months of age in response to changes in both spatial frequency and contrast, a similar phenomenon reported in WS patients [61]. These findings are consistent with the reduced contrast sensitivity and visual acuity loss observed by Ahuja et al. [2]. In our study, we show for the first time a significant difference in optic disc coloration between WT and KO mice at 3 months. Indeed, the fundus of KO mice has a much paler optic disc than that of WT mice, which is due to damage to optic nerve fibers. This optic nerve degeneration is also evidenced by a significant reduction in the thickness of the RNFL and RGC complex and abnormal myelination of RGC axons in KO mice. Recent studies suggest that the loss of Wfs1 function in a mouse model of Wolfram syndrome primarily leads to the disruption of myelin in the optic nerve[62]. These observations are thought to underlie the axonal dysfunction of RGCs responsible for optic nerve damage [62]. Furthermore, OCT analysis of RGC degeneration in a cohort of patients with WS revealed rapid deterioration of visual function and structural changes in the RNFL beginning in the early stage [7, 85, 86]. Interestingly, OCT imaging in patients has suggested that axonal degeneration of retinal ganglion cells precedes the atrophy of cell bodies by approximately one decade [7].

We did not observe a significant increase in ER stress in the retina of 6.5-month-old KO mice, although there was an apparent trend toward increased BiP expression in the retinas of mutant mice. Our results are consistent with those of Ahuja et al. [2]and the lack of statistical significance may be explained by the small sample size. It is also possible that increased ER stress occurs preferentially in RGCs or even only in their axons, as seems to be the case in Ahuja et al. [2].

WS is currently treated with supportive care. The identification of new therapeutic approaches to combat WS, particularly vision loss, is an active area of research. The aim of our work was to delay the deterioration of the retina caused by the loss of function of WFS1. It is thought that even if some regenerative neuronal potential is lost in early childhood, some plasticity remains. It is therefore possible that RGCs can be protected and form functional synapses after appropriate treatment. A promising therapeutic approach to treat visual impairment in WS is the transfer of wild-type WFS1 into RGCs using AAV technology. Several factors make the eye an ideal organ for gene therapy, including its accessibility, immune privilege, small size and compartmentalization [13, 83]. The eye is one of the few organs for which adeno-associated virus (AAV)-based gene therapy has received FDA approval, and it remains a target of great interest, as demonstrated by the success of RPE65 clinical trials [6, 18, 45, 63, 66]. In the field of optic neuropathies, several phase III clinical trials are now underway for the m.11778G > A mutation in MT-ND4, which is the most common mutation responsible for LHON diseases (RESCUE (NCT02652767), REVERSE (NCT02652780) and REFLECT (NCT03293524)). The administration of a single unilateral intravitreal injection of an adeno-associated virus serotype 2 (AAV2) expressing a wild-type ND4 transgene resulted in a reproducible increase in visual acuity in LHON patients [11, 48, 77, 84].

In a previous study, we showed that OPA1-targeted gene therapy can treat dominant optic atrophy in a preclinical model [64].

In this work, we developed a new therapeutic vector, AAV2/2-CMV-WFS1, and validated its therapeutic efficacy in vivo. We administered AAV2 intravitreally to deliver human WFS1, which promoted WFS1 overexpression in mouse RGCs. We evaluated the efficacy of the therapeutic vector in Wfs1 KO mice and demonstrated that Wfs1 expression in RGCs mediated by AAV2/2 is able to protect against optic nerve damage and preserve visual function. Treatment was initiated at one month of age in mice, corresponding to approximately 10 years in humans, when vision loss in Wolfram syndrome typically begins. We observed robust transgene expression in RGCs and their protrusions both in vivo and in vitro, accompanied by rescue of VA, OD pallor and axonal damage in the KO mice. Even though the therapy did not stop the disease completely, a marked progression delay was achieved. No signs of uveitis or inflammation of the retina or uvea were observed during treatment. Transgene expression was first observed via fundoscopy after AAV2/2-GFP injection and then postmortem examination of flat-mounted retinas. Both GFP and human WFS1 were present in RGCs as well as their protrusions. AAV2/2 was used because it is the most commonly used serotype and has preferential tropism for RGCs. Compared with lentiviral vectors, it has superior safety and allows long-term expression of the transgene. Moreover, AAV2 is also the serotype used in Luxturna, the first AAV gene therapy for ocular use to receive approval in the United States and Europe. AAV-2/2-CMV-WFS1 markedly improved the OMR in KO mice even though a complete cure was not achieved. The ability of WFS1 injection to slow the progression of the disease as early as 3 months was particularly interesting. The injection of PBS resulted in signs of improvement. This makes sense given its ability to recruit neurotrophic factors to the retina [47, 70], which can lead to a reparative response. Indeed, intravitreal injection itself has been shown to increase RGC survival and slow RGC degeneration [47, 70]. The potential inflammation associated with injection may stimulate RGC regeneration[70].

The efficacy of the AAV-2/2-CMV-WFS1 vector was further demonstrated by fundoscopy and TEM. In 6-month-old mice, the OD pallor disappeared, while the RGC axons were much less damaged, and their shape and diameter were normal. Therefore, it seems that optic neuropathy was largely prevented because axons have high energy demands, and the presence of WFS1 allows them to be met without stress-related damage. In contrast, OCT revealed no apparent change in retinal thickness between treated and untreated mice. This may be due to the small sample size or too low a dose of GT. Nevertheless, as stated by Grenier et al.[26], the prerequisites for OA are the presence of OD pallor and a decrease in VA, which was reported in KO mice and rescued with the AAV-2/2-CMV-WFS1 vector. On the basis of the promising results obtained in this study, ocular WFS1 gene therapy represents an additional strategy for treating WS. It is conceivable that the various symptoms of WS could be effectively treated by a combination of WFS1 ocular gene therapy and one of the other therapeutic approaches that are either in preclinical development or in clinical trials. In WS, multiple avenues of potential treatment are being explored, including ER calcium stabilizers [21, 40, 49], molecules regulating ER stress [1, 17, 36, 46, 75] or mitochondrial function[5, 82], but none of them specifically focus on restoring the visual system. One promising strategy is to repurpose existing diabetes drugs, such as GLP1 receptor agonists, for the treatment of Wolfram syndrome [52]. In particular, a GLP-1 receptor agonist, liraglutide, has been shown to prevent the development of glucose intolerance and delay the progression of hearing and visual loss in a Wfs1 mutant rat model [33, 67, 68, 75]. In addition, visual improvement has been described in one WS patient treated with idebenone, a mitochondrial respiration activator [4]. To date, there are two clinical trials in progress for WS in which patients are being treated: one with dandrolene, which is an inhibitor of ryanodine receptors in the ER (ClinicalTrials.gov identifier: NCT02829268), and the other with sodium valproate, a regulator of ER stress (linicalTrials.gov identifier: NCT03717909).

In conclusion, WFS1 gene replacement therapy appears safe and effective in Wfs1 KO mice in vivo. These preclinical results are essential to the design of a clinical trial and suggest that a gene therapy strategy could be beneficial for improving visual function in WS patients. Importantly, these experiments were carried out on mice. Nevertheless, these findings represent a step forward for potential therapies for WS patients and provide a framework for future WS clinical trials.

AAV Adeno

Associated Virus

Ethics approval and consent to participate

This study adhered to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. All experimental protocols were carried out under the agreement of the Languedoc Roussillon Comity of Ethics in Animal Experimentation (CEEALR; nuCEEA-LR-12123).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Funding

JJ was supported by a grant from CBS2 doctoral School, University of Montpellier. This work was supported by Inserm, the Region Occitanie, Retina France, AFM Telethon, the Wolfram Syndrome French Association and the Snow Foundation.

Author Contribution

The project was conceived by CD and MP. The methodology was developed by CD, MP, ES and AM. The experiments were performed by JJ, MP, ES, MQ and CC. SK provides animals. JJ prepared the figures. CD wrote the manuscript together with JJ and MP. All the authors read and approved the final version of the manuscript.

Acknowledgement

We dedicate this work to Christian Hamel, who died prematurely on August 15, 2017. The authors thank the different technical platforms of the Institute for Neurosciences of Montpellier, especially the RAM-Neuro, an animal core facility supervised by Denis Greuet, the imaging facility MRI, a member of the France Bio-Imaging National Infrastructure supported by the French National Research Agency (ANR-10-INBS-04, “Investments for the Future’), and the CPV vector core and preclinical analytics core from the TarGeT lab, INSERM UMR 1089, Nantes University. We are indebted to the technical support of Hassan Boukhaddaoui and Chamroeun Sar from MRI.

Availability of data and materials

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

Abreu D, Urano F (2019) Current Landscape of Treatments for Wolfram Syndrome. Trends Pharmacol Sci 40:711–714. 10.1016/j.tips.2019.07.011
Ahuja K, Vandenabeele M, Nami F, Lefevere E, Van Hoecke J, Bergmans S, Claes M, Vervliet T, Neyrinck K, Burg T et al (2024) A deep phenotyping study in mouse and iPSC models to understand the role of oligodendroglia in optic neuropathy in Wolfram syndrome. Acta Neuropathol Commun 12: 140 10.1186/s40478-024-01851-7
Angebault C, Fauconnier J, Patergnani S, Rieusset J, Danese A, Affortit CA, Jagodzinska J, Megy C, Quiles M, Cazevieille C al (2018) ER-mitochondria cross-talk is regulated by the Ca(2+) sensor NCS1 and is impaired in Wolfram syndrome. Sci Signal 11. 10.1126/scisignal.aaq1380
Bababeygy SR, Wang MY, Khaderi KR, Sadun AA (2012) Visual improvement with the use of idebenone in the treatment of Wolfram syndrome. J Neuroophthalmol 32:386–389. 10.1097/WNO.0b013e318273c102
Bababeygy SR, Wang MY, Khaderi KR, Sadun AA (2012) Visual Improvement With the Use of Idebenone in the Treatment of Wolfram Syndrome. J Neuroophthalmol 32:386–389. 10.1097/WNO.0b013e318273c102
Bainbridge JWB, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, Georgiadis A, Mowat FM, Beattie SG, Gardner PJ et al (2015) Long-term effect of gene therapy on Leber's congenital amaurosis. N Engl J Med 372: 1887–1897 10.1056/NEJMoa1414221
Barboni P, Amore G, Cascavilla ML, Battista M, Frontino G, Romagnoli M, Caporali L, Baldoli C, Gramegna LL, Sessagesimi Eet al et al (2022) The Pattern of Retinal Ganglion Cell Loss in Wolfram Syndrome is Distinct From Mitochondrial Optic Neuropathies. Am J Ophthalmol 241:206–216. 10.1016/j.ajo.2022.03.019
Barrett TG, Bundey SE (1997) Wolfram (DIDMOAD) syndrome. J Med Genet 34:838–841. 10.1136/jmg.34.10.838
Barrett TG, Bundey SE, Fielder AR, Good PA (1997) Optic atrophy in Wolfram (DIDMOAD) syndrome. Eye (Lond) 11 (Pt. 6882–888. 10.1038/eye.1997.226
Barrett TG, Bundey SE, Macleod AF (1995) Neurodegeneration and diabetes: UK nationwide study of Wolfram (DIDMOAD) syndrome. Lancet 346:1458–1463. 10.1016/s0140-6736(95)92473-6
Biousse V, Newman NJ, Yu-Wai-Man P, Carelli V, Moster ML, Vignal-Clermont C, Klopstock T, Sadun AA, Sergott RC, Hage Ret al et al (2021) Long-Term Follow-Up After Unilateral Intravitreal Gene Therapy for Leber Hereditary Optic Neuropathy: The RESTORE Study. J Neuroophthalmol 41:309–315. 10.1097/WNO.0000000000001367
Bonnet Wersinger D, Benkafadar N, Jagodzinska J, Hamel C, Tanizawa Y, Lenaers G, Delettre C (2014) Impairment of visual function and retinal ER stress activation in Wfs1-deficient mice. PLoS ONE 9:e97222. 10.1371/journal.pone.0097222
Boye SE, Boye SL, Lewin AS, Hauswirth WW (2013) A comprehensive review of retinal gene therapy. Mol Ther 21:509–519. 10.1038/mt.2012.280
Bugger H, Boudina S, Hu XX, Tuinei J, Zaha VG, Theobald HA, Yun UJ, McQueen AP, Wayment B, Litwin SE al (2008) Type 1 Diabetic Akita Mouse Hearts Are Insulin Sensitive but Manifest Structurally Abnormal Mitochondria That Remain Coupled Despite Increased Uncoupling Protein 3. Diabetes 57:2924–2932. 10.2337/db08-0079
Cagalinec M, Liiv M, Hodurova Z, Hickey MA, Vaarmann A, Mandel M, Zeb A, Choubey V, Kuum M, Safiulina D et al (2016) Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome. PLoS Biol 14: e1002511 10.1371/journal.pbio.1002511
Cillino S, Anastasi M, Lodato G (1989) Incomplete Wolfram syndrome: clinical and electrophysiologic study of two familial cases. Graefes Arch Clin Exp Ophthalmol 227:131–135. 10.1007/BF02169784
Danielpur L, Sohn YS, Karmi O, Fogel C, Zinger A, Abu-Libdeh A, Israeli T, Riahi Y, Pappo O, Birk R et al (2016) GLP-1-RA Corrects Mitochondrial Labile Iron Accumulation and Improves beta-Cell Function in Type 2 Wolfram Syndrome. J Clin Endocrinol Metab 101: 3592–3599 10.1210/jc.2016–2240
Deng C, Zhao PY, Branham K, Schlegel D, Fahim AT, Jayasundera TK, Khan N, Besirli CG (2022) Real-world outcomes of voretigene neparvovec treatment in pediatric patients with RPE65-associated Leber congenital amaurosis. Graefes Arch Clin Exp Ophthalmol 260:1543–1550. 10.1007/s00417-021-05508-2
Douglas Rm A, Nm S, Bd MG, Tj T, Ww P Gt (2005) Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system. Vis Neurosci 22:677–684. 10.1017/S0952523805225166
Eiberg H, Hansen L, Kjer B, Hansen T, Pedersen O, Bille M, Rosenberg T, Tranebjaerg L (2006) Autosomal dominant optic atrophy associated with hearing impairment and impaired glucose regulation caused by a missense mutation in the WFS1 gene. J Med Genet 43:435–440. 10.1136/jmg.2005.034892
Fischer TT, Nguyen LD, Ehrlich BE (2021) Neuronal calcium sensor 1 (NCS1) dependent modulation of neuronal morphology and development. FASEB J 35:e21873. 10.1096/fj.202100731R
Fonseca SG, Fukuma M, Lipson KL, Nguyen LX, Allen JR, Oka Y, Urano F (2005) WFS1 is a novel component of the unfolded protein response and maintains homeostasis of the endoplasmic reticulum in pancreatic beta-cells. J Biol Chem 280:39609–39615. 10.1074/jbc.M507426200
Fonseca SG, Ishigaki S, Oslowski CM, Lu S, Lipson KL, Ghosh R, Hayashi E, Ishihara H, Oka Y, Permutt MA al (2010) Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells. J Clin Invest 120:744–755. 10.1172/JCI39678
Furlong RA, Ho LW, Rubinsztein JS, Michael A, Walsh C, Paykel ES, Rubinsztein DC (1999) A rare coding variant within the wolframin gene in bipolar and unipolar affective disorder cases. Neurosci Lett 277:123–126. 10.1016/s0304-3940(99)00865-4
Galluzzi P, Filosomi G, Vallone IM, Bardelli AM, Venturi C (1999) MRI of Wolfram syndrome (DIDMOAD). Neuroradiology 41:729–731. 10.1007/s002340050832
Grenier J, Meunier I, Daien V, Baudoin C, Halloy F, Bocquet B, Blanchet C, Delettre C, Esmenjaud E, Roubertie A et al (2016) WFS1 in Optic Neuropathies: Mutation Findings in Nonsyndromic Optic Atrophy and Assessment of Clinical Severity. Ophthalmology 123: 1989–1998 10.1016/j.ophtha.2016.05.036
Hilson JB, Merchant SN, Adams JC, Joseph JT (2009) Wolfram syndrome: a clinicopathologic correlation. Acta Neuropathol 118:415–428. 10.1007/s00401-009-0546-8
Hoekel J, Chisholm SA, Al-Lozi A, Hershey T, Tychsen L, Washington University Wolfram Study G (2014) Ophthalmologic correlates of disease severity in children and adolescents with Wolfram syndrome. J AAPOS 18:461–465e461. 10.1016/j.jaapos.2014.07.162
Hofmann S, Philbrook C, Gerbitz K-D, Bauer MF (2003) Wolfram syndrome: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product. Hum Mol Genet 12:2003–2012
Ishihara H, Takeda S, Tamura A, Takahashi R, Yamaguchi S, Takei D, Yamada T, Inoue H, Soga H, Katagiri Het al et al (2004) Disruption of the WFS1 gene in mice causes progressive beta-cell loss and impaired stimulus-secretion coupling in insulin secretion. Hum Mol Genet 13:1159–1170. 10.1093/hmg/ddh125
Ivask M, Hugill A, Kõks S (2016) RNA-sequencing of WFS1-deficient pancreatic islets. Physiol Rep 4. 10.14814/phy2.12750
Jagodzinska J, Sarzi E, Cavalier M, Seveno M, Baecker V, Hamel C, Pequignot M, Delettre C (2017) Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo. J Vis Exp: Doi. 10.3791/55865
Jagomae T, Seppa K, Reimets R, Pastak M, Plaas M, Hickey MA, Kukker KG, Moons L, De Groef L, Vasar Eet al et al (2021) Early Intervention and Lifelong Treatment with GLP1 Receptor Agonist Liraglutide in a Wolfram Syndrome Rat Model with an Emphasis on Visual Neurodegeneration, Sensorineural Hearing Loss and Diabetic Phenotype. Cells 10. 10.3390/cells10113193
Koks S (2023) Genomics of Wolfram Syndrome 1 (WFS1). Biomolecules 13. 10.3390/biom13091346
Koks S, Soomets U, Paya-Cano JL, Fernandes C, Luuk H, Plaas M, Terasmaa A, Tillmann V, Noormets K, Vasar E et al (2009) Wfs1 gene deletion causes growth retardation in mice and interferes with the growth hormone pathway. Physiol Genomics 37: 249–259 10.1152/physiolgenomics.90407.2008
Kondo M, Tanabe K, Amo-Shiinoki K, Hatanaka M, Morii T, Takahashi H, Seino S, Yamada Y, Tanizawa Y (2018) Activation of GLP-1 receptor signalling alleviates cellular stresses and improves beta cell function in a mouse model of Wolfram syndrome. Diabetologia 61:2189–2201. 10.1007/s00125-018-4679-y
Langwinska-Wosko E, Broniek-Kowalik K, Szulborski K (2012) A clinical case study of a Wolfram syndrome-affected family: pattern-reversal visual evoked potentials and electroretinography analysis. Doc Ophthalmol 124:133–141. 10.1007/s10633-011-9308-8
Liu G, Li D, Pasumarthy MK, Kowalczyk TH, Gedeon CR, Hyatt SL, Payne JM, Miller TJ, Brunovskis P, Fink TL et al (2003) Nanoparticles of compacted DNA transfect postmitotic cells. J Biol Chem 278: 32578–32586 10.1074/jbc.M305776200
Lois N, McCarter RV, O’Neill C, Medina RJ, Stitt AW (2014) Endothelial Progenitor Cells in Diabetic Retinopathy. Front Endocrinol (Lausanne) 5. 10.3389/fendo.2014.00044
Lu S, Kanekura K, Hara T, Mahadevan J, Spears LD, Oslowski CM, Martinez R, Yamazaki-Inoue M, Toyoda M, Neilson A al (2014) A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome. Proc Natl Acad Sci U S A 111:E5292–5301. 10.1073/pnas.1421055111
Lu S, Kanekura K, Hara T, Mahadevan J, Spears LD, Oslowski CM, Martinez R, Yamazaki-Inoue M, Toyoda M, Neilson A al (2014) A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome. Proc Natl Acad Sci U S A 111:E5292–5301. 10.1073/pnas.1421055111
Luuk H, Koks S, Plaas M, Hannibal J, Rehfeld JF, Vasar E (2008) Distribution of Wfs1 protein in the central nervous system of the mouse and its relation to clinical symptoms of the Wolfram syndrome. J Comp Neurol 509:642–660. 10.1002/cne.21777
Luuk H, Plaas M, Raud S, Innos J, Sutt S, Lasner H, Abramov U, Kurrikoff K, Koks S, Vasar E (2009) Wfs1-deficient mice display impaired behavioural adaptation in stressful environment. Behav Brain Res 198:334–345. 10.1016/j.bbr.2008.11.007
Luuk H, Plaas M, Raud S, Innos J, Sütt S, Lasner H, Abramov U, Kurrikoff K, Kõks S, Vasar E (2009) Wfs1-deficient mice display impaired behavioural adaptation in stressful environment. Behav Brain Res 198:334–345. 10.1016/j.bbr.2008.11.007
Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr., Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM al (2008) Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 358:2240–2248. 10.1056/NEJMoa0802315
Mahadevan J, Morikawa S, Yagi T, Abreu D, Lu S, Kanekura K, Brown CM, Urano F (2020) A soluble endoplasmic reticulum factor as regenerative therapy for Wolfram syndrome. Lab Invest 100:1197–1207. 10.1038/s41374-020-0436-1
Morgan JE (2004) Circulation and axonal transport in the optic nerve. Eye 18:1089–1095. 10.1038/sj.eye.6701574
Newman NJ, Yu-Wai-Man P, Carelli V, Moster ML, Biousse V, Vignal-Clermont C, Sergott RC, Klopstock T, Sadun AA, Barboni Pet al et al (2021) Efficacy and Safety of Intravitreal Gene Therapy for Leber Hereditary Optic Neuropathy Treated within 6 Months of Disease Onset. Ophthalmology 128:649–660. 10.1016/j.ophtha.2020.12.012
Nguyen LD, Fischer TT, Abreu D, Arroyo A, Urano F, Ehrlich BE (2020) Calpain inhibitor and ibudilast rescue beta cell functions in a cellular model of Wolfram syndrome. Proc Natl Acad Sci U S A 117:17389–17398. 10.1073/pnas.2007136117
Niu Z, Feng Y, Hu Z, Li J, Sun J, Chen H, He C, Wang X, Jiang L, Liu Y al (2017) Exome sequencing identifies a novel missense mutation of WFS1 as the cause of non-syndromic low-frequency hearing loss in a Chinese family. Int J Pediatr Otorhinolaryngol 100:1–7. 10.1016/j.ijporl.2017.06.008
Noormets K, Kõks S, Kavak A, Arend A, Aunapuu M, Keldrimaa A, Vasar E, Tillmann V (2009) Male mice with deleted Wolframin (Wfs1) gene have reduced fertility. Reprod Biol Endocrinol 7:82. 10.1186/1477-7827-7-82
Panfili E, Frontino G, Pallotta MT (2023) GLP-1 receptor agonists as promising disease-modifying agents in WFS1 spectrum disorder. Front Clin Diabetes Healthc 4:1171091. 10.3389/fcdhc.2023.1171091
Plaas M, Seppa K, Reimets R, Jagomae T, Toots M, Koppel T, Vallisoo T, Nigul M, Heinla I, Meier R et al (2017) Wfs1- deficient rats develop primary symptoms of Wolfram syndrome: insulin-dependent diabetes, optic nerve atrophy and medullary degeneration. Sci Rep 7: 10220 10.1038/s41598-017-09392-x
Prusky GT, Alam NM, Beekman S, Douglas RM (2004) Rapid Quantification of Adult and Developing Mouse Spatial Vision Using a Virtual Optomotor System. Invest Opthalmology Visual Sci 45:4611. 10.1167/iovs.04-0541
Rando TA, Horton JC, Layzer RB (1992) Wolfram syndrome: evidence of a diffuse neurodegenerative disease by magnetic resonance imaging. Neurology 42:1220–1224. 10.1212/wnl.42.6.1220
Raud S, Sutt S, Luuk H, Plaas M, Innos J, Koks S, Vasar E (2009) Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABA(A) receptors in Wfs1-deficient mice. Neurosci Lett 460:138–142. 10.1016/j.neulet.2009.05.054
Redfern WS, Storey S, Tse K, Hussain Q, Maung KP, Valentin J-P, Ahmed G, Bigley A, Heathcote D, McKay JS (2011) Evaluation of a convenient method of assessing rodent visual function in safety pharmacology studies: effects of sodium iodate on visual acuity and retinal morphology in albino and pigmented rats and mice. J Pharmacol Toxicol Methods 63:102–114. 10.1016/j.vascn.2010.06.008
Rendtorff ND, Lodahl M, Boulahbel H, Johansen IR, Pandya A, Welch KO, Norris VW, Arnos KS, Bitner-Glindzicz M, Emery SB al (2011) Identification of p.A684V missense mutation in the WFS1 gene as a frequent cause of autosomal dominant optic atrophy and hearing impairment. Am J Med Genet A 155A:1298–1313. 10.1002/ajmg.a.33970
Richard EM, Brun E, Korchagina J, Crouzier L, Affortit C, Alves S, Cazevieille C, Mausset-Bonnefont AL, Lenoir M, Puel JL al (2023) Wfs1(E864K) knock-in mice illuminate the fundamental role of Wfs1 in endocochlear potential production. Cell Death Dis 14:387. 10.1038/s41419-023-05912-y
Riggs AC, Bernal-Mizrachi E, Ohsugi M, Wasson J, Fatrai S, Welling C, Murray J, Schmidt RE, Herrera PL, Permutt MA (2005) Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis. Diabetologia 48:2313–2321. 10.1007/s00125-005-1947-4
Rigoli L, Di Bella C (2012) Wolfram syndrome 1 and Wolfram syndrome 2. Curr Opin Pediatr 24:512–517. 10.1097/MOP.0b013e328354ccdf
Rossi G, Ordazzo G, Vanni NN, Castoldi V, Iannielli A, Di Silvestre D, Bellini E, Bernardo L, Giannelli SG, Luoni M et al (2023) MCT1-dependent energetic failure and neuroinflammation underlie optic nerve degeneration in Wolfram syndrome mice. Elife 12: 10.7554/eLife.81779
Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A, Wittes J, Pappas J, Elci O, McCague S al (2017) Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390:849–860. 10.1016/S0140-6736(17)31868-8
Sarzi E, Seveno M, Piro-Megy C, Elziere L, Quiles M, Pequignot M, Muller A, Hamel CP, Lenaers G, Delettre C (2018) OPA1 gene therapy prevents retinal ganglion cell loss in a Dominant Optic Atrophy mouse model. Sci Rep 8:2468. 10.1038/s41598-018-20838-8
Schmidt-Kastner R, Kreczmanski P, Preising M, Diederen R, Schmitz C, Reis D, Blanks J, Dorey CK (2009) Expression of the diabetes risk gene wolframin (WFS1) in the human retina. Exp Eye Res 89:568–574. 10.1016/j.exer.2009.05.007
Sengillo JD, Gregori NZ, Sisk RA, Weng CY, Berrocal AM, Davis JL, Mendoza-Santiesteban CE, Zheng DD, Feuer WJ, Lam BL (2022) Visual Acuity, Retinal Morphology, and Patients' Perceptions after Voretigene Neparovec-rzyl Therapy for RPE65-Associated Retinal Disease. Ophthalmol Retina 6:273–283. 10.1016/j.oret.2021.11.005
Seppa K, Jagomae T, Kukker KG, Reimets R, Pastak M, Vasar E, Terasmaa A, Plaas M (2021) Liraglutide, 7,8-DHF and their co-treatment prevents loss of vision and cognitive decline in a Wolfram syndrome rat model. Sci Rep 11:2275. 10.1038/s41598-021-81768-6
Seppa K, Toots M, Reimets R, Jagomae T, Koppel T, Pallase M, Hasselholt S, Krogsbaek Mikkelsen M, Randel Nyengaard J, Vasar E al (2019) GLP-1 receptor agonist liraglutide has a neuroprotective effect on an aged rat model of Wolfram syndrome. Sci Rep 9:15742. 10.1038/s41598-019-52295-2
Seynaeve H, Vermeiren A, Leys A, Dralands L (1994) Four cases of Wolfram syndrome: ophthalmologic findings and complications. Bull Soc Belge Ophtalmol 252:75–80
Shum JWH, Liu K, So K-F (2016) The progress in optic nerve regeneration, where are we? Neural Regen Res 11:32–36. 10.4103/1673-5374.175038
Simsek E, Simsek T, Tekgul S, Hosal S, Seyrantepe V, Aktan G (2003) Wolfram (DIDMOAD) syndrome: a multidisciplinary clinical study in nine Turkish patients and review of the literature. Acta Paediatr 92:55–61. 10.1111/j.1651-2227.2003.tb00469.x
Swift M, Swift RG (2005) Wolframin mutations and hospitalization for psychiatric illness. Mol Psychiatry 10:799–803. 10.1038/sj.mp.4001681
Swift RG, Polymeropoulos MH, Torres R, Swift M (1998) Predisposition of Wolfram syndrome heterozygotes to psychiatric illness. Mol Psychiatry 3:86–91. 10.1038/sj.mp.4000344
Takei D, Ishihara H, Yamaguchi S, Yamada T, Tamura A, Katagiri H, Maruyama Y, Oka Y (2006) WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum. FEBS Lett 580:5635–5640. 10.1016/j.febslet.2006.09.007
Toots M, Seppa K, Jagomae T, Koppel T, Pallase M, Heinla I, Terasmaa A, Plaas M, Vasar E (2018) Preventive treatment with liraglutide protects against development of glucose intolerance in a rat model of Wolfram syndrome. Sci Rep 8:10183. 10.1038/s41598-018-28314-z
Vaitkeviciene I, Vaitkevicius R, Paipaliene P, Zekonis G (2006) Morphometric analysis of pulpal myelinated nerve fibers in human teeth with chronic periodontitis and root sensitivity. Med (Kaunas) 42:914–922
Vignal-Clermont C, Yu-Wai-Man P, Newman NJ, Carelli V, Moster ML, Biousse V, Subramanian PS, Wang AG, Donahue SP, Leroy BP al (2023) Safety of Lenadogene Nolparvovec Gene Therapy Over 5 Years in 189 Patients With Leber Hereditary Optic Neuropathy. Am J Ophthalmol 249:108–125. 10.1016/j.ajo.2022.11.026
Visnapuu T, Plaas M, Reimets R, Raud S, Terasmaa A, Kõks S, Sütt S, Luuk H, Hundahl CA, Eskla K, -Let al et al (2013) Evidence for impaired function of dopaminergic system in Wfs1-deficient mice. Behav Brain Res 244:90–99. 10.1016/j.bbr.2013.01.046
Visnapuu T, Raud S, Loomets M, Reimets R, Sütt S, Luuk H, Plaas M, Kõks S, Volke V, Alttoa A al (2013) Wfs1-deficient mice display altered function of serotonergic system and increased behavioral response to antidepressants. Front Neurosci 7:132. 10.3389/fnins.2013.00132
Waszczykowska A, Zmyslowska A, Braun M, Ivask M, Koks S, Jurowski P, Mlynarski W (2020) Multiple Retinal Anomalies in Wfs1-Deficient Mice. Diagnostics (Basel) 10. 10.3390/diagnostics10090607
Wolfram DJ, Wagner HP (1938) Diabetes mellitus and simple optic atrophy among siblings: report of four cases. 13: 715–718
Yang Y, Sauve AA (2016) NAD(+) metabolism: Bioenergetics, signaling and manipulation for therapy. Biochim Biophys Acta 1864:1787–1800. 10.1016/j.bbapap.2016.06.014
Yu-Wai-Man P (2016) Genetic manipulation for inherited neurodegenerative diseases: myth or reality? Br J Ophthalmol: Doi. 10.1136/bjophthalmol-2015-308329
Yu-Wai-Man P, Newman NJ, Carelli V, Moster ML, Biousse V, Sadun AA, Klopstock T, Vignal-Clermont C, Sergott RC, Rudolph Get al et al (2020) Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy. Sci Transl Med 12. 10.1126/scitranslmed.aaz7423
Zmyslowska A, Fendler W, Niwald A, Ludwikowska-Pawlowska M, Borowiec M, Antosik K, Szadkowska A, Mlynarski W (2015) Retinal Thinning as a Marker of Disease Progression in Patients With Wolfram Syndrome. Diabetes Care 38:e36–e37. 10.2337/dc14-1898
Zmyslowska A, Waszczykowska A, Baranska D, Stawiski K, Borowiec M, Jurowski P, Fendler W, Mlynarski W (2019) Optical coherence tomography and magnetic resonance imaging visual pathway evaluation in Wolfram syndrome. Dev Med Child Neurol 61:359–365. 10.1111/dmcn.14040

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WFS1 gene delivery rescues visual function in a mouse model of Wolfram syndrome

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Abstract

Figures

Introduction

Materials and methods

Animals

Blood glucose measurement

Visual acuity and contrast sensitivity

Eye fundus

Optic Coherence Tomography

Transmission electron microscopy

Immunohistochemistry

Quantitative PCR assay

Intravitreal injection

Fundus fluorescein angiography

Statistical analysis

Results

Wfs1 KO mice develop optic disc pallor

Decrease of Thinning of RGC/RNFL and RGC complex layers in Wfs1 KO mice

Severe axonal damage in Wfs1 KO mice

No RGC loss in WFS1 KO mouse retina

No ER stress was detected in WFS1 KO mouse retinas

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Supplementary Files

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