Natural history of retinal degeneration in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases with a common set of symptoms including cognitive and motor decline and vision loss. Naturally occurring sheep models of CLN5 and CLN6 disease display the key clinical features of NCL, including a progressive loss of vision. We assessed retinal histology, astrogliosis, and lysosomal storage accumulation in CLN5 affected (CLN5−/−) and CLN6 affected (CLN6−/−) sheep eyes and age-matched controls at 3, 6, 12, and 18 months of age to determine the onset and progression of retinal pathology in NCL sheep. The retina of CLN5−/− sheep shows progressive atrophy of the outer retinal layers, widespread gliosis, and accumulation of lysosomal storage in retinal ganglion cells late in disease. In contrast, CLN6−/− retina shows significant atrophy of all retinal layers, progressive gliosis, and earlier accumulation of lysosomal storage. This study has highlighted the differential vulnerability of retinal layers and the time course of retinal atrophy in two distinct models of NCL disease. This data will be valuable in determining potential targets for ocular therapies and the optimal timing of these therapies for protection from retinal dysfunction and degeneration in NCL.

Retinal pathology in sheep with CLN6 disease. Total retinal thickness was analysed in both the central and peripheral retina of control and CLN6 −/− sheep. In control sheep, the central retina grew initially to reach a peak thickness of 219 ± 2.6 µm at 12 months of age. The central retina of CLN6 −/− sheep also grew but reached a peak of 201 ± 3.9 µm at 6 months of age (Fig. 5a, b). Peripheral retina of control sheep remained relatively stable between 3 and 18 months of age. Conversely, CLN6 −/− sheep had significantly thicker peripheral retina at 3 months of age, showed a sharp decline between 3 and 6 months of age, and was significantly thinner (96 ± 1.6 µm) than control (117 ± 1.5 µm) peripheral retina at 18 months of age (Fig. 5c, d).
Looking at individual layers of the central retina, inner retinal layers (NFL, GCL, IPL and INL) were significantly thicker in CLN6 −/− animals compared to controls early in disease, but were significantly thinner by end stage disease at 18 months of age (Fig. 6a). A similar pattern was observed in the outer retinal layers, with the exception of the RPE which remained relatively unchanged between 3 and 18 months. The proportion of total retinal thickness taken up by each retinal layer was comparable between control and CLN6 −/− sheep at 3 and 18 months of age with the only notable difference being the ONL, which makes up 16% of total thickness in control sheep and only 8% of total thickness in CLN6 −/− sheep at 18 months of age (Fig. 6b). The number of ONL rows was significantly lower in CLN6 −/− sheep compared to controls from 6 months of age onwards, with a diseased average of only 1.4 ± 0.08 rows evident by 18 months of age (Fig. 6c).
Accumulation of AF signal was minimal in control central retina across all ages. Sparse AF puncta were observed in the control retina at 12 and 18 months of age however these did not co-localise with LAMP1. In the CLN6 −/− retina, AF signal was evident from 3 months of age but did not co-localise with LAMP1 at this age. By 6 months of age there was faint AF signal in LAMP1-positive ganglion cell bodies in the CLN6 −/− retina, which became stronger and more distinct at 12 and 18 months of age (Fig. 7, arrowheads).
GFAP immunoreactivity was confined to the quiescent elongated astrocytes of the NFL in the central retina of control sheep and showed minor increases in intensity with age (see Supplementary Fig. S2). Whilst GFAP signal was also initially localised to the same NFL cells in the CLN6 −/− retina, these cells had irregularly shaped

Discussion
This study details the progression of retinal degeneration from pre-symptomatic to end-stage disease in two distinct sheep models of NCL; CLN5 disease in Borderdale sheep and CLN6 disease in South Hampshire sheep. Total retinal thickness, individual layer thickness, gliosis, and accumulation of autofluorescent storage material were assessed in post-mortem eyes from diseased sheep and age-matched healthy controls. Four ages (3, 6, 12 and 18 months) were selected to represent four distinct clinical disease stages, being pre-symptomatic, early symptomatic, symptomatic and end-stage disease respectively. Total retinal thickness was assessed in both the central and peripheral retina, with the central retina showing an earlier and more distinct difference between control and affected sheep. In both breeds the central retina was thicker in affected animals compared to controls at 3 months of age. This may be due to early compensatory mechanisms or inflammatory edema in the retina of affected animals, however requires more investigation. Compensatory mechanisms may include upregulated mRNA or protein expression of neuroprotective markers and could be assessed by molecular or histological analysis of pre-symptomatic sheep retina. Although early increases in retinal thickness have not been observed in other animal models of NCL, they have been observed in several degenerative diseases including age-related macular degeneration, optic neuritis, diabetic retinopathy, and leber congenital amaurosis [13][14][15][16] . In many of these diseases the early increase in retinal thickness has been attributed to inflammatory edema which occurs in response to photoreceptor cell stress prior to retinal atrophy [13][14][15][16] . Although early upregulation of GFAP was not observed in affected sheep retina, it would be useful in future to assess these retina for other signs of inflammation such as microglial infiltration, or upregulation of Müller cell specific markers or inflammatory cytokines.
There was early growth of the central retina in control Borderdale sheep leading to thicker retina in control compared to CLN5 −/− sheep from 6 months onwards, however growth was slower in control South Hampshire sheep. Despite initial differences in peripheral retina thickness between control and affected sheep of both breeds, the thicknesses were similar from 6 months of age until a significant drop in thickness in CLN5 −/− and CLN6 −/− sheep at end-stage disease. While total retinal thickness is informative for tracking global changes in the retina, it is important to consider the structure and function of individual layers of the retina and how these change over the course of disease. This is particularly pertinent in CLN5 −/− retina at end stage disease where total thickness is comparable to controls, however almost 40% of this total thickness is taken up by the NFL.   In both breeds, lysosomal storage occurs primarily in ganglion cell bodies, however it appears earlier in the retina of CLN6 −/− sheep compared to CLN5 −/− sheep. Minor amounts are observed in the CLN6 −/− retina at 6 months of age, which then progresses until ganglion cell bodies appear full of storage by end stage disease. This is in keeping with early studies of the CLN6 −/− sheep retina, which showed atrophy of photoreceptor cell bodies and inner and outer segments, and accumulation of autofluorescent storage material primarily in ganglion cells 10,11 .
In the control sheep retina at all ages studied, GFAP immunoreactivity was primarily confined to horizontally elongated cell bodies and processes in the NFL, reminiscent of quiescent astrocytes commonly found in the healthy retina 17,18 . However, in affected sheep NFL resident astrocytes had an altered morphology and, from 6 months of age, GFAP-positive cells were evident throughout the retina. Müller cells typically do not express GFAP, but upregulate its expression in response to pathological processes in the retina 19 . GFAP-expressing Müller cells were evident in the outer retina of affected animals from 6 months of age, indicating the initiation of reactive gliosis. Early and progressive increases in GFAP immunoreactivity and activation of Müller cells has also been observed in the retina of several mouse models of NCLs 20-23 . A significant reduction in ERG a-and b-waves in CLN5 and CLN6 affected sheep occurs from 9 to 11 months of age, suggesting dysfunction of photoreceptor and bipolar cells 12 . This coincides with the age when clinical signs of visual deterioration, such as reduced menace response and pupillary light reflex, also begin to occur in affected sheep 12,24 . By 15 to 17 months of age ERG responses are abolished in the majority of affected sheep and they can no longer navigate through a maze 12,24 . The retinal pathology presented in this study aligns with these clinical features as the number of ONL rows, indicative of photoreceptor cell body numbers, show significant decline from 6 months of age and diminish over the disease course.
Post-mortem data from retina tissue of NCL patients is rare, however what is known about the retina of CLN5 and CLN6 patients is summarised in Table 1. The results presented here from CLN6 sheep align with what is observed in CLN6 patients as all retinal layers in affected sheep show progressive atrophy and accumulation of lysosomal storage in ganglion cells. In CLN5 sheep, no ganglion cell loss was observed as it is in human CLN5, www.nature.com/scientificreports/ however accumulation of lysosomal storage and outer retinal atrophy in sheep aligns with pathology in CLN5 patients. Visual impairment often presents in early to mid-disease in CLN5 and classical CLN6 patients, with abolished ERGs reported between 7 and 10 years of age in CLN5, and visual failure between 3 and 6 years in CLN6 25,26 . The sheep eye is increasingly being recognised as a good model for studying human retinal disease and for testing ocular therapies [28][29][30][31] , due to its similarities in globe size and retinal structure to humans. There are several different potential approaches for delivering corrective therapies to the retina in NCL, including intravitreal, subretinal, suprachoroidal, periocular, or topical administration. Of these, intravitreal delivery is one of the least invasive as it involves injection of the drug to the vitreous cavity of the eye, but has less target specificity than a subretinal or suprachoroidal delivery route. While the intravitreal route is not specifically targeted to the outer retina and has barriers such as the inner limiting membrane (ILM) to overcome there are several reasons to consider this approach for retinal diseases caused by soluble protein deficiencies. For example, in CLN5 disease if only inner retinal cells were corrected through intravitreal therapy they could secrete functional CLN5 protein to other retinal cells, a mechanism known as cross-correction 32 . In addition, given the fragility of the diseased retina it is likely the ILM is disrupted and more permeable in diseased eyes, and in fact others have shown better viral vector transduction in diseased eyes compared to wild-type 33 . Intravitreal drug delivery is commonplace in clinical settings for a range of ocular diseases [34][35][36] , and retinal dysfunction and pathology was successfully ameliorated up to 18 months of age following a single intravitreal injection of CLN5 gene therapy in CLN5 −/− sheep 37 . Biotechnology company Neurogene Inc has recently announced the planned initiation of a Phase I/II clinical trial for CLN5 disease utilising both intracerebroventricular and intravitreal delivery routes 38 and a clinical trial is underway for the intravitreal delivery of the CLN2 enzyme replacement therapy Brineura (ClinicalTrials.gov identifier: NCT05152914).
The outlook for treating the retinal component of CLN6 disease is more complicated as we have shown that all layers of the retina significantly degenerate in CLN6 affected sheep, and CLN6 is a membrane bound protein meaning cross-correction is unlikely to be of benefit. Indeed, a recent trial of intravitreal gene therapy in CLN6 −/− sheep showed some attenuation of pathology but no rescue of visual function 37 . A combination of approaches such as suprachoroidal and intravitreal may be appropriate in this case as suprachoroidal would target outer retinal cells with a lower risk of retinal detachment, and the intravitreal route would target inner retinal cells. The general consensus on timing of treatment for degenerative diseases is often 'the earlier the better' . Our results in NCL sheep have reaffirmed this, as atrophy of retinal layers and accumulation of lysosomal storage start becoming evident by 6 months of age. In addition, early treatment is preferable if using the intravitreal approach as the ILM is still developing and therefore more permeable in younger animals 39,40 .

Conclusions
This systematic study of the progression of retinal degeneration in distinct sheep models of CLN5 and CLN6 NCL has highlighted the differential vulnerability of retinal cell types in each disease and the time course of degeneration. In CLN5 sheep, the outer retina is most severely affected, with significant thinning of the outer nuclear layer evident from 6 months of age. Conversely in CLN6 sheep, all retinal layers show significant degeneration from 12 months of age. Lysosomal storage accumulation in retinal ganglion cells was a common feature in both sheep models, which is in keeping with observations of post-mortem human retina. This histological data, in addition to assessment of sheep eye globe dimensions, further validates the use of sheep to study the retinal component of NCL and potential ocular therapies to attenuate retinal dysfunction and degeneration.

Methods
Animals. Borderdale and South Hampshire sheep were diagnosed at birth 8,9 and maintained at the Lincoln University research farm under US National Institutes of Health guidelines for the care and use of animals in research and the NZ Animal Welfare Act (1999). Homozygous affected ewes from both breeds (CLN5 −/− , n = 2-4, CLN6 −/− , n = 2-5) were sacrificed at 3, 6, 12, and 18 months of age along with age-matched healthy heterozygous flockmates as controls (CLN5 +/− n = 2-4, CLN6 +/− n = 2-4). All experimental protocols were approved by the Lincoln University Animal Ethics Committee. All studies are reported in accordance with the ARRIVE Essential 10 guidelines.
Tissue processing. Sheep were sacrificed by penetrating captive bolt to the cervical spine followed by rapid exsanguination. Eye globes were enucleated at the time of death, fixed in 10% formalin for a minimum of 2 h, and sent to Gribbles Veterinary Pathology, Christchurch, NZ for post-fixation in Bouin's solution (Sigma-Aldrich, St Louis, MO, USA, HT10132) for 4 h, followed by wax embedding. Retinal paraffin sections were cut at 3 µm, mounted and a subset stained with Hematoxylin and Eosin (H + E) histological stain by Gribbles Veterinary Pathology for analysis of retinal thickness and layer differentiation. www.nature.com/scientificreports/