Oral levodopa rescues retinal morphology and visual function in a murine model of human albinism

Abstract Albinism is a group of disorders characterized by pigment deficiency and abnormal retinal development. Despite being a common cause for visual impairment worldwide, there is a paucity of treatments and patients typically suffer lifelong visual disability. Residual plasticity of the developing retina in young children with albinism has been demonstrated, suggesting a post‐natal window for therapeutic rescue. L‐3, 4 dihydroxyphenylalanine (L‐DOPA), a key signalling molecule which is essential for normal retinal development, is known to be deficient in albinism. In this study, we demonstrate for the first time that post‐natal L‐DOPA supplementation can rescue retinal development, morphology and visual function in a murine model of human albinism, but only if administered from birth or 15 days post‐natal age.

2013, is: "Can a treatment be developed to improve vision for people with albinism?" In order to develop a targeted and effective treatment for the visual disability associated with albinism, the role of the pigment synthesis pathway in normal ocular development needs to be interrogated (McKay, 2018). Normal ocular pigmentation and development are dependent upon tyrosinase (TYR) enzyme, (Beermann et al., 1990) which catalyses the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA), phaeomelanin, eumelanin and dopamine (DA) (Hearing & Jimenez, 1987). DA both induces and limits neurite elongation in the developing retina, by coupling with D1 and D2 dopaminergic receptors, respectively (Eisenhofer et al., 2003). L-DOPA also acts on OA1 (an orphan G-protein-coupled receptor) to upregulate pigment epithelium-derived growth factor, a key regulator of retinal development (Lopez, Decatur, Stamer, Lynch, & McKay, 2008). This pathway is disrupted in albinism, resulting in foveal hypoplasia and visual impairment. It is hypothesized that retinal development arrests prematurely in albinism (McAllister et al., 2010;Wilson, Mets, Nagy, & Kressel, 1988).
However, it has been demonstrated that retinal development is not arrested in young children with albinism, but is ongoing, albeit at a reduced rate and magnitude (Lee et al., 2015). This suggests that intervention(s), which take advantage of this post-natal period of neuroplasticity, may promote normal retinal development and improve visual function in albinism. It is also notable that the onset of nystagmus in children with albinism is typically between 2 and 6 months of age, again suggesting a period of plasticity and potential therapeutic window in the developing visual system (Gottlob, 1997). This is a feasible concept, as albinism can be diagnosed in early infancy (Apkarian, 1992).
One possible intervention is oral L-DOPA supplementation. In the developing murine retina, L-DOPA levels increase throughout pre-and post-natal development until the end of the first postnatal month (Roffler-Tarlov, Liu, Naumova, Bernal-Ayala, & Mason, 2013). The post-natal peak of L-DOPA expression coincides with bipolar cell and rod photoreceptor development, and the peak of synaptogenesis in the murine retina (Xu & Tian, 2004). L-DOPA is known to be deficient in the developing retina in murine OCA (Roffler-Tarlov et al., 2013). Oral L-DOPA replacement in pregnant albino mouse mothers results in the accumulation of L-DOPA in the retinal pigment epithelium of the foetuses (Roffler-Tarlov et al., 2013). Normally, L-DOPA is synthesized from L-tyrosine by two enzymes, tyrosinase (TYR) and tyrosine hydroxylase (TH). TYR is necessary for the subsequent conversion of L-DOPA to melanin, within ocular melanosomes (Lavado, Jeffery, Tovar, de la Villa, & Montoliu, 2006). Ectopic expression of TH in the retinal pigment epithelium (RPE) in the absence of functioning TYR enzyme rescues the retinal abnormalities and visual function in albino mice in the absence of melanin synthesis (Lavado et al., 2006). This suggests that lack of L-DOPA or one of its metabolic derivatives is key to the abnormal retinal development and function seen in albinism (Lavado et al., 2006). However, post-natal modulation of retinal morphology and function using oral L-DOPA in albinism has never been demonstrated and is key to translation of this potential therapeutic to human trials.
The aim of this study was to investigate whether post-natal retinal development, morphology and visual function in albinism can be modulated and improved through oral L-DOPA supplementation, if administered during the critical period of neuroplasticity, in a murine model of human OCA.

| MATERIAL S AND ME THODS
All experiments were performed in accordance with the ARVO Statement for the use of animals in ophthalmic and vision research, in accordance with the NC3Rs ARRIVE guidelines, and after approval was obtained from Animal Welfare and Ethical Review Body, University of Southampton (project licence number: P474F5E4D) ("ARVO Statement for the use of animals in ophthalmic & vision research"; NC3Rs Reporting Guidelines Working Group, 2010). All possible steps were taken to avoid animal suffering at each stage of the experiment.

Significance
In this study, we demonstrate for the first time that abnormal retinal development, morphology and visual function can be rescued post-natally using oral L-dihydroxyphenylalanine (L-DOPA) supplementation, if administered during the critical period of neuroplasticity, in a murine model of human albinism. By modelling the known post-natal period of plasticity seen in the retina in human albinism and rescuing visual function, we show that oral L-DOPA replacement in human albinism shows promise as a therapeutic intervention during development.
TA B L E 1 Summary of the numbers of mice included in each of the treatment arms

| Treatment of mice with L-DOPA
Albino and pigmented mice were supplemented with 1 mg/ml L-DOPA (Sigma) dissolved in the drinking water with ascorbate (2.5 mg/ml to prevent oxidation) for 28 days, at three different time points (Birth, 15 and 28 days post-natal age [PNA]), as per

| Electroretinogram recordings (ERG)
Electroretinogram recordings were performed with the Phoenix Micron III image-guided focal ERG system designed for rodents. were carried out with an interval time of 120 s, from which average A-and B-wave amplitudes and implicit times were calculated.

| Optical coherence tomography (OCT)
Mice were anesthetized, and their pupils dilated as described above.
Artificial tears (Systane Ultra, Alcon) were used throughout the procedure to maintain corneal moisture and clarity. Animals were

| Statistics and modelling
A linear mixed model, implemented in STATA™ (Copyright 1996-2014, was used to analyse the differences between untreated albino (CALBs) and pigmented (B6) mice with regard to A-and B-wave amplitudes, A-and B-wave latencies and thickness measurements obtained for each retinal layer. The model included fixed effects for mouse strain, age, eye and the interaction between mouse strain and age (mouse × age). The covariance structure was independent. All models included a random intercept and slope for each mouse thereby accounting for repeated measures over time.
Post hoc estimates of the marginal effects of mouse strain on A-and Note. a Based on an L-DOPA milk/plasma concentration of 0.3 (Witkovsky, 2004).
A-and B-wave amplitudes, A-and B-wave latencies and retinal layer thickness measurements for each retinal layer were calculated.
To determine the statistical significance of a predictor or interaction term in each model, we considered type 1 error rate as <0.05 (p < 0.05).
Spearman's rank correlation was conducted in order to assess the relationship between retinal layer OCT thickness measurements and A-and B-wave ERG amplitudes, using a Bonferroni correction for multiple comparisons.

| Oral L-DOPA is tolerated well in treated mice
L-dihydroxyphenylalanine is known to be deficient in the developing retina in OCA (Roffler-Tarlov et al., 2013). In this study, we have demonstrated for the first time that it is possible to modulate post-natal retinal morphology and rescue function and in albino (CALBs) mice, through the administration of a 28-day course of oral L-DOPA. Oral L-DOPA supplementation at the dosages administered in this study was tolerated very well, with no signs of toxicity (e.g., erect tails, piloerection, ataxia, lacrimation, increased activity, clonic convulsions, increased irritability and head and body tremors) observed in any of the treated mice.
Interestingly, this therapeutic window corresponds with the post- There were no significant differences in A-and B-wave latencies, between untreated B6 mice and untreated CALBs (see Table 3 for mean values). A-wave latencies were significantly decreased in CALBs supplemented with L-DOPA from birth (z = −2.64, p = 0.008) and 15 days PNA (z = −2.13, p = 0.033), but not in CALBs treated after 28 days PNA (Table 3). L-DOPA treatment became less effective with increasing age. Interestingly, significant decreases in A-wave latencies  It is known that L-DOPA supplementation increases dopamine (DA) levels in both the pigmented and albino mouse neural retina, through the conversion of DOPA to DA by intrinsic aromatic amino acid decarboxylase. DA has both synaptic and paracrine actions within the neural retina, which includes the modulation of photoreceptor activity and enhancement of light-driven cone input (Masson, Mestre, & Blin, 1993;Witkovsky, 2004). Potentially, L-DOPA supplementation could trigger reductions in A-and B-wave latencies on ERG recordings, through the enhancement of dopaminergic signalling within the neural retina (Schorderet & Nowak, 1990;Witkovsky, 2004). In the next phase of our work, we are planning to compare these ERG results, to visual acuity results obtained by assessing the optomotor response in awake mice, for the purposes of future translational research studies (Prusky & Douglas, 2004). & (d) mice treated with L-DOPA from birth, 15 and 28 days PNA, in comparison with untreated controls. The upper plots for each panel show the A-and B-wave amplitudes plotted with respect to post-natal age. Each point represents a single value from each ERG examination. The lines of best fit (trend lines) are shown in navy for the control mice, and maroon, green and orange for the mice treated from 28 days PNA, 15 days PNA and birth, respectively. The lower plots for each panel are marginal effects plots summarizing the average differences in A-and B-wave amplitudes, between each of the three treatment groups in comparison to untreated controls (y = 0), with respect to post-natal age. The error bars are representing the 95% confidence intervals. By calculating partial derivatives of the interaction term from the linear mixed model, the significant differences between each of the three treatment groups and the control groups were estimated at six specified time points: 28, 35, 42, 56, 82 and 112 days PNA. B6, C57BL/6 pigmented mice; CALBs, C57BL/6J-c2J OCA mice; ERG, electroretinogram; OCA, oculocutaneous albinism; PNA, post-natal age

| Post-natal L-DOPA supplementation increases retinal nerve fibre layer (RNFL) thickness in albino and pigmented mice
Optical coherence tomography (OCT) is a non-invasive imaging method that provides high-resolution cross-sectional images of in vivo retinal morphology (Adachi et al., 2016;Gabriele et al., 2011).
Retinal OCT imaging has been correlated with histological, ultrastructural and electroretinography findings and has the significant advantage of facilitating longitudinal evaluation of disease progression (Adachi et al., 2016;Spaide & Curcio, 2011;Vajzovic et al., 2012).
The effects of a 28-day course of oral L-DOPA supplementation on in vivo retinal development and morphology were investigated using OCT imaging (Leica Envisu R2200 Mouse Imaging System) ( Figure 3).

| L-DOPA supplementation decreases the combined ganglion cell layer-inner plexiform layer (GCL-IPL) thickness in albino and pigmented mice
The GCL-IPL thickness measurements were significantly greater in untreated albino (CALBs) mice, in comparison with untreated pigmented (B6) mice (z = 2.58, p = 0.010) (see Table 3 for mean values).

| L-DOPA supplementation increases inner nuclear layer (INL) thickness in albino and pigmented mice
There were no significant differences in INL thickness measurements between untreated pigmented (B6) and albino (CALBs) mice (Table 3).
Significant decreases in INL thickness measurements also became evident with increasing age, in B6 mice treated from 28 days PNA (z = −2.00, p = 0.046) ( Figure S1). These observations may be consistent with previously reported descriptions of the patterns of retinal cell addition and production in the INL of albino rats (Ilia & Jeffery, 2000).
In these studies, it has been demonstrated that cellular proliferation

| L-DOPA supplementation increases outer plexiform layer (OPL) thickness in albino and pigmented mice
Outer plexiform layer thickness measurements were significantly greater in untreated pigmented (B6) in comparison with untreated albino (CALBs) mice (z = 3.25, p = 0.001) (see Table 3 for mean values). In the OPL, cone pedicles and rod spherules are synaptic upon various bipolar cell and horizontal cell types. It has previously been demonstrated that there is a failure of 30% of the rod population to develop in albino mice (Ilia & Jeffery, 2000). Given this reduction in rod numbers, there is likely to be a decrease in the number of synapses between rod photoreceptors, horizontal and bipolar cells in the OPL, which could potentially manifest as a reduction in OPL thickness measurements in the CALBs (Ilia & Jeffery, 2000). OPL thickness measurements were significantly increased in CALBs treated from 15 (z = 3.6, p < 0.001) and 28 days PNA (z = 2.47, p = 0.013), and B6 mice supplemented from 28 days PNA (z = 3.82, p < 0.001) (Table 3, Figure S2). Interestingly, although OPL thickness measurements were initially decreased in CALBs treated from birth, significant increases emerged with increasing age (z = 3.93, p < 0.001) ( Figure S2). In contrast, significant decreases in OPL thickness measurements emerged with increasing age, in B6 mice treated from 28 days PNA (z = −3.86, p < 0.001) ( Figure S2). This suggests that L-DOPA supplementation in CALBs is potentially increasing the number of rod photoreceptors together with their associated synapses in the OPL, ultimately manifesting as an increase in OPL thickness measurements. However, this increase appears to be only sustained in CALBs treated before 28 days PNA, that is, during the period when L-DOPA levels normally peak in the developing mouse eye (Roffler-Tarlov et al., 2013). There were no significant differences in OPL thickness measurements in B6 mice treated from birth and 15 days PNA.

| L-DOPA supplementation increases outer nuclear layer (ONL) thickness in albino and pigmented mice
There were no significant differences between ONL thickness measurements in untreated pigmented (B6) and albino (CALBs) mice (see Table 3 for mean values). ONL thickness measurements were significantly increased in CALBs supplemented with L-DOPA from birth (z = 1.97, p = 0.049) and 15 days PNA (z = 3.9, p < 0.001), and B6 mice supplemented from 15 days PNA (z = 2.86, p = 0.004) (Table 3, Figure   S2). In contrast, ONL thickness measurements were significantly decreased in CALBs supplemented from 28 days PNA (z = −2.29, p = 0.022) (Table 3, Figure S2). Treatment became less effective with increasing age, until a tendency towards significantly decreased ONL thickness measurements emerged in CALBs treated from birth (z = −3.15, p = 0.002) and 15 days PNA (z = −5.2, p < 0.001) ( Figure   S2). This suggests that L-DOPA supplementation during the expected peak of post-natal DOPA expression can increase rod photoreceptor numbers in albinism, although this effect is not sustained (Ilia & Jeffery, 2000). There were no significant differences in ONL measurements in B6 mice treated from birth and 28 days PNA.

| Photoreceptor inner segment (IS) length is unaffected in albino and increased in pigmented mice by L-DOPA supplementation
Inner segment length measurements were significantly lower in untreated pigmented (B6) mice in comparison with untreated albino (CALBs) mice (z = −6.80, p < 0.001) (see Table 3 for mean values).
The IS normally forms the junction between the OS and the synapse, and the reduction in IS length measurements in untreated CALBs may reflect either a reduction in the number of rod photoreceptors or defective synaptogenesis, the peak of which normally coincides with the peak of DOPA expression in the developing mouse eye (Xu & Tian, 2004). Unexpectedly, there were no significant differences in IS length measurements in CALBs supplemented with L-DOPA from birth, 15 or 28 days PNA (Table 3, Figure S2). Conversely, IS length measurements were significantly increased in B6 mice supplemented with L-DOPA from birth (z = 2.6, p = 0.009), 15 (z = 5.16, p < 0.001) and 28 days PNA (z = 8.45, p < 0.001) (Table 3, Figure S2). Treatment became less effective with increasing age, until a tendency towards significantly reduced IS length measurements emerged in B6 mice treated from 28 days PNA ( Figure S2). If L-DOPA treatment increases the number of rod photoreceptors and their associated synapses in albinism, then one might expect to see this manifest morphologically, as an increase in photoreceptor IS length measurements in treated mice.
Perhaps as L-DOPA is being administered towards the end of the developmental period, there is not a large enough effect on rod photoreceptor numbers in order for it to manifest as a significant change in photoreceptor IS length measurements on OCT examinations.

| L-DOPA supplementation increases photoreceptor outer segment (OS) length in albino and pigmented mice
There were no significant differences between photoreceptor outer segment (OS) length measurements recorded from untreated pigmented (B6) mice and untreated albino (CALBs) mice (see Table 3 for mean values). OS length measurements were significantly increased in CALBs supplemented with L-DOPA from 15 days PNA (z = 5.98, p < 0.001), but not in CALBs treated after 28 days PNA (Table 3, Figure   S3). Although OS length measurements were initially decreased in CALBs treated from birth, significant increases emerged with increasing age (z = 5.29, p < 0.001) ( Figure S3). The OS length measurements were also significantly increased in B6 mice treated from 15 (z = 6.01, p < 0.001) and 28 days PNA (z = 2.06, p = 0.04) (Table 3, Figure S3).
Treatment became less effective with increasing age, until a tendency towards significantly decreased OS length measurements emerged in B6 mice treated from birth, 15 and 28 days PNA ( Figure S3). L-DOPA upregulates pigment epithelium-derived growth factor (PEDF), as part of normal retinal development, and this is necessary for the normal organization of photoreceptor OS morphology (Jablonski, Tombran-Tink, Mrazek, & Iannaccone, 2000). It is possible that in the absence of PEDF in albinism, photoreceptor OS morphology becomes disorganized, and its ability to elongate is impaired (Jablonski et al., 2000). Potentially, L-DOPA supplementation is stabilizing the morphology of the photoreceptor OS in albinism, in addition to increasing rod photoreceptor numbers (Jablonski et al., 2000). However, this effect would only be sustained while L-DOPA is present. Following cessation of L-DOPA supplementation, an increased rate of cell death might be expected, as the photoreceptor outer segments undergo a continual turnover process.

| L-DOPA supplementation increases photoreceptor end tips (ETPRS) thickness in albino and pigmented mice
There were no significant differences between ETPRS thickness measurements recorded from untreated pigmented (B6) mice and untreated albino (CALBs) mice (see Table 3 for mean values). ETPRS thickness measurements were significantly increased in CALBs supplemented with L-DOPA from birth (z = 3.43, p = 0.001) and 15 days PNA (z = 3.97, p < 0.001), and B6s treated from 15 days PNA (z = 10.77, p < 0.001) (Table 3, Figure S3). Treatment became less effective with increasing age, until a tendency towards significantly decreased ETPRS thickness measurements emerged in CALBs treated from birth (z = −6.74, p < 0.001), 15 (z = −7.42, p < 0.001) and 28 days PNA (z = −3.29, p = 0.001), and B6 mice treated from 26 days PNA ( Figure S3). These observations would be consistent with our hypothesis that L-DOPA supplementation is temporarily stabilizing the morphology of the photoreceptor outer segments. An initial increase followed by a decrease in ETPRS thickness measurements would be expected, in mice that received a course of L-DOPA supplementation.

| L-DOPA supplementation increases retinal pigment epithelium (RPE) thickness in albino mice and decreases RPE thickness in pigmented mice
Retinal pigment epithelium thickness measurements were significantly greater in untreated pigmented (B6) mice in comparison with untreated albino (CALBs) mice (z = 9.14, p < 0.001) (see Table 3 for mean values). Normally, melanin is synthesized from L-DOPA in the RPE, by melanosomes, which are located in the apically in the RPE cells. Melanin synthesis peaks early in foetal life (coinciding with ocular development) and diminishes with age (Sparrow, Hicks, & Hamel, 2010). This process is impaired/absent in albinism and appears to be manifesting morphologically as a decrease in RPE thickness measurements. RPE thickness measurements were significantly increased in CALBs supplemented with L-DOPA from 15 days PNA (z = 2.4, p = 0.016) (Table 3, Figure S3). Significant increases in RPE thickness measurements also became evident with increasing age, in CALBs supplemented with L-DOPA from birth (z = 2.76, p = 0.006) ( Figure S3). L-DOPA supplementation would not increase melanin biosynthesis, as there is no functional tyrosinase present in albinism. Perhaps, L-DOPA is having direct trophic effects on the RPE, through the synthesis of PEDF (Jablonski et al., 2000).
Conversely, there was a significant decrease in RPE thickness measurements in B6 mice treated from 15 days PNA (z = −3.56, p < 0.001) (  Figure S3). Treatment effect size decreased with increasing age, until significance was lost by 3 months PNA in B6 mice treated from 15 days PNA (z = 3.17, p = 0.002) ( Figure S3).

| Retinal layer optical coherence tomography (OCT) measurements are predictive morphological biomarkers for visual function in albino mice
Retinal nerve fibre layer (RNFL) OCT thickness measurements have been shown to be reliable surrogate biomarkers for visual function in humans with conditions such as multiple sclerosis (Fisher et al., 2006). The length of the photoreceptor outer segment (OS) has also been correlated with visual acuity in humans with albinism (Thomas  Figure S4 to view the individual data, colour coded based on treatment group. ETPRS, photoreceptor end tips; OCA, oculocutaneous albinism; OCT, optical coherence tomography; OPL, outer plexiform layer; OS, photoreceptor outer segment; RNFL, retinal nerve fibre layer; RPE, retinal pigment epithelium correlated significantly with increased A-and B-wave ERG amplitudes (Figures 4 and 5, Figure S4).

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N S
H.L. conceived and designed the experiments, acquired, analysed and interpreted the data, drafted the manuscript and supervised the project; J.S. and H.G. performed the experiments, acquired and analysed the data and reviewed the manuscript; J.E.S. provided technical support and conceptual advice, critically revised the manuscript and helped supervise the project; and A.L. provided technical support and conceptual advice, reviewed the manuscript and helped supervise the project. All authors discussed the results and contributed to the final manuscript.

DATA ACCE SS I B I LIT Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.