Mutations in the WFS1 gene encoding the Wolframin1 protein (WFS1) cause the classical form of the rare condition Wolfram Syndrome (WS). WFS1 plays critical roles in regulating endoplasmic reticulum (ER) stress, calcium homeostasis and mitochondrial function and is broadly expressed across several organs (1). It can also directly modulate major physiological systems like the Renin-Angiotensin-Aldosterone and Kinin-Kallikrein systems (RAAS, KKS) that regulate critical functions, including blood pressure, fluid balance, and inflammatory response (1–3). WS is therefore a multi-systemic condition and hallmarks include diabetes, optic nerve atrophy, deafness and progressive neurodegeneration. Consequently, prognoses are extremely poor with a median lifespan of 30-40 years and effective disease modifying treatments are urgently needed (4). Drug repurposing efforts have identified various molecular pathways, like the ER stress response and calcium signaling, for pharmacological intervention (5). For instance, chemical chaperones like valproate and dantrolene have demonstrated protective effects in animal models of WS and are now being explored in clinical trials (6–9). The prototypic Sigma1 receptor agonist PRE-084 and AMX0035, a combination of two chaperones (4-phenylbutyric acid and tauroursodeoxycholic acid, Amylyx Pharmaceuticals) also provided symptomatic benefit in pre-clinical models (10, 11). Notably, AMX0035, although originally approved for the treatment of amyotrophic lateral sclerosis in the USA, is now being evaluated in a Phase II clinical trial with WS patients (12).

Drugs targeting the metabolic incretin system have proven particularly efficacious; the incretins are hormones secreted by the gut in response to nutrient ingestion (13). They include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), each of which have specific receptors. Incretins regulate glycemic homeostasis via their additive actions on insulin and glucagon and also provide trophic support to pancreatic beta cells (14, 15). Incretin mimetics like GLP-1 receptor (GLP-1R) agonists (for e.g., Liraglutide, Semaglutide), were initially developed for the treatment of Type 2 diabetes mellitus (T2DM) and obesity but have demonstrated therapeutic benefit across several domains in WS. For instance, our group has extensively tested Liraglutide (LIRA) in an established loss-of-function rat model of WS (Wfs1 coding exon 5 knock-out, Wfs1KO) to show that treatment can substantially delay the onset of glucose intolerance and loss of vision (16–18). Therapeutic benefits were also evident in aged animals where treatment began after symptom onset; KO rats displayed improved performance on cognitive tasks and had reduced ER stress, neuroinflammation, optic nerve atrophy and retinal ganglion cell death (18, 19). Similarly, the long-lasting agonist dulaglutide had both a preventive and curative effect on glucose intolerance in a mouse model of WS (20).

We have additionally shown that 16-month long preventive LIRA treatment commencing at 2 months of age was not only well-tolerated, but also conferred significant protection against the progression of hyperglycaemia and vision loss in Wfs1KO rats (16). Consequently, GLP-1 agonists are already being used by the majority of paediatric WS patients for symptom management (21–23).

While the underlying basis of incretin agonism’s therapeutic effects remain to be fully elucidated, several—likely overlapping—mechanisms of action have been reported. These include promoting proliferation and repair, protecting against oxidative stress and excitoxicity-induced apoptosis (24–26), and regulating cellular processes like inflammation, ER stress and autophagy (27, 28). For instance, the GLP-1R agonist exenatide protected iPSC-derived WFS1-deficient beta cells and cerebellar neurons from ER stress and mitigated mitochondrial dysfunction and the production of reactive oxygen species (20). Similarly, while Wfs1 deficiency can directly impair components of the autophagy lysosomal pathway (29), GLP-1R agonists have been shown to restore lysosomal function (30, 31).

Incretin agonism may also drive the “resensitization” of peripheral and central insulinogenic signaling pathways and a resultant decrease in pro-inflammatory factors (32). This would also explain the treatment-induced benefits observed in conditions like Alzheimer’s and Parkinson’s Disease (AD, PD), as impaired cerebral metabolism and insulin responsiveness have been implicated in several neurodegenerative pathologies (33).

In lieu of this, reports of highly potent dual GLP-1/GIP agonists are relevant, given that these also appear to be better tolerated by patients (34). These are unimolecular peptides that provide targeted and balanced agonism of both the GLP-1 and GIP receptors, thereby addressing both insulin resistance and deficiency (35). Their synergistic pharmacology has yielded superior metabolic outcomes in rodent and non-human primate models and human trials of obesity and T2DM than selective GLP-1 mono-therapy (36). Dual agonists have also demonstrated stronger therapeutic potential in neurodegenerative disorders (37–39); in a mouse model of PD, the dual agonist DA-CH5 had better blood-brain barrier (BBB) penetration than a comparable LIRA dose and reduced glial activation and apoptotic signaling to a greater extent (38). Similarly, another dual agonist DA-JC4, attenuated memory impairments and levels of phosphorylated tau protein aggregates in a rat model of AD (35).

Accordingly, we assessed the efficacy of long-term dual agonist treatment in a rat model of WS and show that dual agonists have pleotropic therapeutic effects and represent a new class of potential disease-modifying therapies for WS.

While no cure currently exists for the progressive condition WS, incretin mimetics have emerged as a promising therapeutic option. Studies have primarily reported on mono-agonists targeting the GLP-1 receptor, as these have demonstrated robust anti-obesogenic and -hyperglycaemic effects in both pre-clinical and clinical settings. However, increased efficacy and patient compliance are limited by gastrointestinal complications associated with higher doses (52).

Accordingly, we show here that long-term dual-incretin agonist treatment demonstrated significant therapeutic effect across multiple phenotypic domains in a rat model of WS. To begin with, results from our acute treatment paradigm confirmed previous reports of the enhanced efficacy of dual agonists relative to mono-agonists (36, 53, 54). Both of the tested dual agonists DA-CH5 and DA-JC4, were able to normalize glucose intolerance and glucose-stimulated secretory response in symptomatic 5-month-old Wfs1 KO rats. Conversely, treatment with an equimolar dose of the mono-agonist Liraglutide only induced weight loss ( Supplementary Figure 2B ). Previous studies using this model have shown that an almost four-fold higher dose is needed for Liraglutide to mediate its anti-diabetic and neuroprotective effects, further underscoring the superior potency of dual agonists (16, 18, 19). This is particularly relevant given that longitudinal studies have indicated significant variability in treatment response to mono-agonists: for instance, approximately 50% of Wfs1 KO rats developed insulin-dependent diabetes despite lifelong Liraglutide treatment that commenced in the pre-symptomatic phase (16). The increased potency of dual-incretin agonists like DA-CH5 may derive from them being more physiologically appropriate and harnessing the complementary actions of GLP-1 and GIP. Although hyperglycaemia can severely impair GIP’s insulinotropic effects (55), these can be restored by using GLP-1’s preserved insulinotropic and glucagonostatic effects to normalize blood sugar. GIP receptor agonism can then induce insulin secretion by 1) direct β cell stimulation, 2) potentiating glucagon release and 3) triggering paracrine α-β cell communication, thereby delivering a stronger final synergistic effect (56–59). Increased efficacy is also driven by the differential action of dual agonists on GLP-1 and GIP receptor trafficking and recycling (58, 59).

Crucially, the same therapeutic benefit was noted in our long-term treatment paradigm despite treatment commencing when animals were 8 months old i.e., further along the disease course. This is particularly relevant given our previous observations that Liraglutide treatment was unable to rescue overt glucose intolerance in older Wfs1 KO rats (16, 18, 19). Continuous DA-CH5 therapy reversed manifest glucose intolerance in KO animals and maintained normoglycemia as they aged, underscoring the superior efficacy of dual agonists. Intriguingly, and in contrast to the acute treatment paradigm, no significant changes were noted in serum insulin and c-peptide levels; SAL- and DA-CH5-treated KO rats had similar glucose-stimulated secretory profiles at both the 10.5- and 12-month timepoints. However, the C-peptide index (C-peptide: blood glucose at 30 min following glucose administration) significantly improved in treated KO animals, particularly at the 10.5-month timepoint, indicating the presence of functional insulin. DA-CH5-associated glycaemic improvements may therefore at least partially be driven by improved β-cell function and/or insulin processing, particularly since WFS1 plays a critical role in proinsulin (insulin precursor) folding and processing (60). For instance, Finan et al. reported that diabetic rats treated with a dual agonist produced less insulin in response to glycemic challenge than those treated with the mono-agonist liraglutide, indicating an improvement in insulin sensitivity (36).

Alternatively, reduced insulin secretion may simply be the result of a prolonged normoglycemic state and decreased β cell stimulation as suggested by Irwin et. al., who reported a similar phenomenon in obese mice. Sub-chronic co-administration of GIP and GLP-1 analogues progressively reduced non-fasting plasma glucose levels but had no effect on plasma insulin (61).

The benefits of DA-CH5 treatment were also evident at the tissue level, as therapy normalized the increased pancreatic mass seen in KO rats. We speculate that this increase may have been driven by the exocrine pancreas, as it comprises most of the organ (~90% of mass) (62). Furthermore, we observed no evidence of pathological hyperplasia, oedema, or connective tissue proliferation. Disruptions in the exocrine pancreas may also underlie the frequent gastrointestinal complaints by WS patients (1), as proper digestive function and nutrient ingestion are tightly regulated by crosstalk between the exocrine pancreas and small intestine. Interestingly, Morikawa et al. also recently reported significant hypervascularization and immune cell infiltration in the pancreatic islets of Wfs1 KO mice, which may also contribute to increased tissue mass (63, 64).

Closer analysis of the endocrine islet revealed that DA-CH5 treatment significantly increased occupied area, indicating its curative potential. Of note, unlike % area, islet mass may not fully reflect the magnitude of the degenerative process, as it is calculated using pancreas mass (43). Values may therefore be skewed if pancreata are significantly heavier as in the KO-SAL group. Treatment also normalized intra-islet morphological and compositional alterations; notably, alterations were observed across all constituent cell types. Insulin+ β cell density was significantly reduced, while Glucagon+ α and SST+ δ cell densities were increased in SAL-treated KO rats. Gorgogetias et al. also reported reduced β cell and increased α cell densities in a mouse model of WS, confirming that the intricate regulatory feedback loops between endocrine hormones are markedly disturbed in WS and there is significant intra-islet stress (20). This was supported by our observation of significantly increased GLP-1+ cell density in islets from KO rats; similar alterations have been previously reported in animal models of and human patients with T2DM. For instance, Campbell et al. reported that islets from donors with T2DM had significantly more GLP-1+ α cells (65). Indeed, while physiological α-cell expression of GLP-1 has been reported in both murine and human islets, expression is increased under conditions of metabolic stress (65–69).

In WS, a cascade of events may therefore converge on the final insulin-dependent diabetic phenotype seen in patients; the loss of functional WFS1 and associated ER stress eventually leads to β-cell failure, compromised insulin synthesis and the breach of a “pro” to “anti-regenerative” glucose threshold (70). While α and δ cell densities may initially increase to help maintain glucose homeostasis, these increases, coupled with already low insulin levels, contribute to hyperglucagonemia. Ultimately, persistent and uncontrolled hyperglycaemia and associated cellular stress prevent β-cell recovery and compensatory proliferation, culminating in a permanently disturbed metabolic microenvironment. By reducing glycaemic load, DA-CH5 therapy may therefore give β cells a chance to regenerate, as seen here: β cell density and mass in KO rats were increased following DA-CH5 treatment. Treatment can improve insulin sensitivity and thereby lessen the “secretory burden” on β cells, enable the proliferation of surviving cells, and also encourage γ-aminobutyric acid (GABA)-induced trans differentiation of α to β-like cells (36, 71). Incretin agonism, as with DA-CH5, can also directly stimulate GABA formation, which itself has anti-inflammatory, anti-apoptotic and insulinogenic effects (72–77).

DA-CH5 treatment also conferred protection against visual impairments, as acuity progressively declined in SAL-treated KO rats, while DA-CH5-treated rats showed a significant improvement at 10.5 months. Although there was a deterioration at 12 months, this may have been influenced by cataract progression. Behavioural assessments of visual acuity were supplemented by immunohistological analysis of Brn3a+ RGC density; however, assessing vision is challenging given species-specific differences in retinal organization. For instance, rodents do not have a well-defined fovea and rod-cone distribution follows a characteristic dorsal-ventral gradient across the retina. However, these differences were accounted for by performing density analyses across the whole retina and regional quadrants (78). As with the optometry task, RGC density was indeed better preserved in DA-CH5 treated KO rats, confirming that therapy was protective. Regional analyses indicated that density reduction in KO rats was particularly pronounced in the superior-distal quadrant ( Supplementary Figure 4C ); intriguingly, this regional specificity was also observed for NeuN+ cells in an independent cohort of 3-month-old animals ( Supplementary Figure 4B ). Although not statistically significant, the result suggests that vision loss in WS may begin relatively early and set the stage for further degeneration. Indeed, electrophysiological retinal impairments were detectable by the age of 4 months in a mouse model of WS (79). The same study reported that RGC loss follows secondary to myelin thinning and axonal degeneration within the optic nerve; the same sequence has been noted in our rat model and explains the progressive worsening on functional assessments of visual acuity (16, 18). Finally, DA-CH5 therapy had no effect on WS-associated hearing loss; cochlear hearing levels were impaired in KO rats at 8 months as previously observed, and remained stable in both SAL and DA-CH5 treatment groups (16). As with vision however, progressive hearing loss is difficult to model in animals, given that it is also variable in human patients; onset can range from birth to adolescence (16, 80, 81). Early diagnosis and intervention are therefore crucial to prevent or at least mitigate irreversible damage.

The limitations of the present study include the lack of information on insulin sensitivity as an insulin tolerance test was not performed. Additionally, the long-term paradigm did not include a treatment-arm with the GLP-1 mono-agonist Liraglutide. As previously mentioned, this was because our acute paradigm revealed that at an equimolar dose, liraglutide yielded no glycemic normalization. This precluded a direct comparison with DA-CH5 in the subsequent longitudinal study. Future studies may therefore consider including independent treatment-arms with GLP-1 and GIP mono-agonists. An additional point to consider is that DA-CH5 is not structurally identical to commercially retailed dual agonists being considered for clinical trials with WS patients e.g., Tirzepatide (82). Unfortunately, we were unable to include these in the present study as these were not available for exploratory pre-clinical testing.

To conclude, the work presented is the first to demonstrate the superior therapeutic benefit of dual incretin agonists via meaningful improvements in various in vivo outcomes in a rat model of WS. To illustrate, while our previous longitudinal studies have shown that long-term Liraglutide treatment has pleiotropic effects, we show here that the same effects can be achieved with a much lower dose of DA-CH5. Furthermore, while the therapeutic protection conferred against vision was comparable, DA-CH5 had superior efficacy in normalizing WS-associated glucose intolerance, even in older animals with advanced disease. This has implications for a broad spectrum of metabolic disorders, particularly since dual agonism can prevent glycaemic volatility and safeguard against hypoglycaemia via GIP’s differential action on α cells (83). Dual agonists like DA-CH5 have also demonstrated superior neuroprotection in models of neurodegenerative diseases because they have been specifically designed to optimize BBB penetration (35, 38, 84). Accordingly, the data presented here suggest that dual incretin agonism may be an effective therapeutic strategy for patients with WS, particularly given the potent rescue effects observed for glucose intolerance and visual acuity. Further, commercially available candidates like Tirzepatide, owing to their pharmacokinetic properties, are associated with fewer gastrointestinal effects and need to be injected less frequently, which likely increases patient compliance. Long-term clinical trials that also allow monitoring of the impact of dual agonists on neuro-ophthalmologic outcomes in WS patients are therefore warranted.

Our data additionally emphasize the need for early diagnosis to allow intervention within a suitable window. Finally, our results highlight the translational value of validated animal models for rapidly identifying potential disease-modifying compounds and longitudinally tracking their effects. We recommend that future studies investigate the protective effects of prophylactic treatment with dual incretin agonists.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Estonian Project Authorization Committee for Animal Experiments (No 204, 11th November 2019). The study was conducted in accordance with the local legislation and institutional requirements.

Study conception and design: TJ, NG, EV, MaPl. Data acquisition and analysis: TJ, NG, KS, RR, MaPa, MaPl. Manuscript original draft compilation, subsequent editing, and figure preparation: TJ, NG, KS, RR, MaPa, MiPl, AK, EV, MaPl. All authors contributed to the article and approved the submitted version.