Fucoxanthin protects retinal ganglion cells and regulates Parkin-mediated mitophagy in experimental glaucoma

Abstract

Objective

Glaucoma is a common neurodegenerative disease resulting in irreversible blindness. This study investigates whether fucoxanthin can safeguard retinal ganglion cells (RGCs) by modulating Parkin-mediated mitophagy in experimental glaucoma.

Methods

An experimental glaucomatous model was induced in Sprague-Dawley rats via translimbal laser photocoagulation. Intraocular pressure (IOP) was monitored using a Tonolab tonometer. RGC survival was evaluated through FluoroGold labelling. Retinal and optic nerve samples were analysed at 3 days and 2 weeks post-IOP elevation for mitochondrial morphology and gene/protein expression using immunohistochemistry and molecular assays.

Results

Results demonstrated that mitophagy was acutely overactivated in the short term and impaired over the long term in ocular hypertensive rats. Fucoxanthin intravitreal administration enhanced RGC survival and Bcl-2 expression while reducing Bax and glial fibrillar acidic protein levels. During acute IOP elevation, fucoxanthin curtailed Parkin expression and mitophagosome formation, mitigating excessive mitophagy. Under prolonged IOP elevation, it elevated mitophagy-related proteins and restored mitophagy function, contributing to damaged mitochondrial clearance.

Conclusion

Fucoxanthin exerts neuroprotective effects in experimental glaucoma by modulating Parkin-mediated mitophagy. This highlights the therapeutic potential of maintaining mitophagy homeostasis for glaucoma treatment.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Dysfunctional mitophagy has been linked to glaucoma. Fucoxanthin plays a protective role in neuronal death and ameliorates mitophagy dysfunction in non-neuronal cells. However, reports on its benefits for glaucoma are still limited.

WHAT THIS STUDY ADDS

• Our study found a novel neuroprotective role for fucoxanthin in preserving retinal ganglion cells in experimental glaucoma. By regulating Parkin-mediated mitophagy, fucoxanthin prevents excessive mitophagy during acute intraocular pressure elevation and restores mitophagy function during sustained stress.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Maintaining mitophagy homeostasis through Parkin signalling may be a promising therapeutic target for glaucoma. Further research is warranted to explore fucoxanthin as a supplementary neuroprotective agent for glaucoma treatment.

Introduction

Glaucoma is a prominent global cause of blindness, characterised by progressive degeneration of retinal ganglion cells (RGCs) and their axons, culminating in irreversible vision loss.¹ Despite advances in understanding the disease, the precise mechanisms underlying RGC loss remain elusive. Currently, intraocular pressure (IOP) elevation is the only modifiable risk factor.² However, even with effective IOP-lowering interventions, glaucomatous neuropathy can progress, necessitating alternative therapeutic approaches. Increasing evidence implicates mitochondrial dysfunction in RGCs and their axons as a pivotal contributor to glaucoma pathogenesis,³ with defective mitophagy identified as a key driver of RGC loss.4,7

Mitophagy, a selective form of autophagy, plays a critical role in maintaining cellular homeostasis by removing damaged or excess mitochondria. This process is orchestrated by Parkin, an E3 ubiquitin ligase, which is recruited to the outer mitochondrial membrane (OMM) upon mitochondrial damage and depolarisation of the mitochondrial membrane potential. Parkin ubiquitinates OMM proteins, facilitating the recruitment of downstream effectors such as optineurin to promote mitophagy.^{8 9} Studies suggest that enhancing Parkin-mediated mitophagy can protect RGCs in experimental glaucoma models, presenting a promising therapeutic target.^{7 10}

Fucoxanthin, a marine carotenoid abundant in brown algae, exhibits diverse pharmacological properties, including antioxidant, anti-inflammatory and neuroprotective effects.¹¹ Recent studies have demonstrated its ability to mitigate oxidative stress and neuronal loss in models of neurodegenerative diseases,¹² restore mitophagy in cells under oxidative stress¹³ and prevent glutamate-induced cytotoxicity in cultured RGCs.¹⁴ However, the effects of fucoxanthin on RGCs in the context of elevated IOP and glaucomatous damage remain largely unexplored.

This study aims to illuminate the neuroprotective effects of fucoxanthin in glaucoma and to clarify its underlying mechanisms, focusing on the regulation of Parkin-mediated mitophagy. By investigating how fucoxanthin influences mitophagy homeostasis, we strive to identify new therapeutic strategies for glaucoma management.

Materials and methods

Animal models

Male Sprague-Dawley rats aged 7–8 weeks and weighing 250±10 g, free from any ocular abnormalities, were selected for this study. The animals were housed under controlled conditions with a 12-hour light/dark cycle and had unrestricted access to water and a standard laboratory diet. They were acclimatised for at least 3 days before the experiments commenced. They were divided into a control group (27 eyes), a glaucomatous group (27 eyes), a glaucomatous-fucoxanthin group (27 eyes) and a glaucomatous-vehicle control group (27 eyes) randomly. All animal procedures adhered strictly to the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research and were approved by the Animal Ethics Committee of Ningbo University, China (Approval No. 10478).

Induction of experimental glaucoma

An experimental glaucomatous model was established by performing translimbal laser photocoagulation on the trabecular meshwork, as previously described.¹⁵ Rats were anaesthetised using general anaesthesia, while proparacaine hydrochloride eye drops served as topical anaesthesia. An argon laser (532 nm wavelength, 300 mW power, 0.5 s per burn, 50 μm spot size; Visulas Trion, Carl Zeiss Meditec AG, Jena, Germany) was employed to create approximately 100–120 burns targeting the trabecular meshwork circumferentially at the limbus of each eye.

Measurement of IOP

IOP was measured under general anaesthesia using a handheld rebound tonometer (TonoLab; Tiolat Oy, Helsinki, Finland). Baseline IOP readings were obtained prior to laser treatment, followed by measurements at 1, 3 and 7 days after the laser application. To ensure consistency, all IOP measurements were conducted between 07:00 and 08:00 by the same operator. A slit-lamp biomicroscope was used at each timepoint to examine the anterior chamber for signs of inflammation or haemorrhage, and rats exhibiting such complications were excluded (two rats were disused due to inflammation of the anterior chamber). For rats not designated for early sacrifice, laser treatment was repeated after 1 week to maintain elevated IOP.

Intravitreal injection procedure

Intravitreal injections were performed following general anaesthesia and pupil dilation with phenylephrine hydrochloride and tropicamide (Shenyang Xinqi Pharmaceutical Co, China) 3 days prior to establishing the experimental glaucoma model. A 26G needle was used to premark the injection site, and a microsyringe was employed to deliver 5 μL of fucoxanthin (0.5 μM) or vehicle control into the vitreous cavity over a 1-min period. The syringe was held in position for an additional minute to minimise reflux along the injection tract. A careful technique was employed to avoid damage to the lens or retina. Postprocedure, eyes were evaluated under a slit lamp for lens puncture or inflammation, and retinal integrity was assessed using an indirect ophthalmoscope. Any rats with evidence of lens injury or retinal damage were excluded from further analysis.

FluoroGold retrograde labelling of RGCs

To trace RGCs, 3% FluoroGold (dissolved in 10% dimethyl sulfoxide; Sigma-Aldrich, St. Louis, Missouri, USA) was bilaterally microinjected into the superior colliculi (2 μL per injection) of anaesthetised rats 4 days before the first laser treatment. Two weeks after IOP elevation, the eyes were enucleated and fixed in 4% paraformaldehyde for 1.5 hours. Corneas and lenses were removed using corneal scissors and forceps, and neural retinas were dissected and flattened. Flat-mounted retinas were imaged using a fluorescence microscope (Leica). RGC density was quantified by averaging cell counts in the central (1–2 mm from the optic disc), middle (2–3 mm) and peripheral (3–4 mm) retinal regions.

Quantitative PCR (qPCR) analysis

Total RNA was extracted from RGC-enriched retinal tissue in each experimental group using Trizol reagent (Invitrogen, Carlsbad, California, USA). Complementary DNA was synthesised, and target gene expression was analysed by qPCR using the SYBR Green PCR kit (Takara, Tokyo, Japan). Amplification was performed under the following conditions: denaturation at 95°C for 15 s, followed by annealing/extension at 60°C for 30 s over 45 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The 2^−ΔΔCt method was applied to calculate relative expression levels.

Western blot analysis of protein expression

Retinal and optic nerve tissues (n=3 per group) were lysed in radioimmunoprecipitation assay buffer (Beyotime, China) and homogenised on ice via ultrasonication. Protein concentrations were normalised, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked and incubated overnight at 4°C with specific primary antibodies, including rabbit antiglial fibrillar acidic protein (GFAP) (1:10 000), rabbit anti-Parkin (1:500), rabbit antioptineurin (1:200), rabbit anti-Bcl-2 (1:500), rabbit antilysosomal-associated membrane protein 1 (LAMP1) (1:1000), rabbit anti-Bax (1:1000), rabbit antimicrotubule-associated protein 1A/1B-light chain 3 (LC3) (1:1000) and rabbit anti-GAPDH (1:10 000) (all from Abcam). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch). Protein bands were detected using enhanced chemiluminescence (Weiao Biotechnology Co, Shanghai, China) on a Tanon-4600 Imaging System (Tanon, China) and quantified using ImageJ software (NIH).

Immunohistochemistry analysis

Frozen retinal sections (7 μm thick) were prepared for immunohistochemistry. Three sections from each retina (n=3 per group) were permeabilised with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min and blocked with 5% bovine serum albumin in PBS for 1 hour. Sections were incubated overnight at 4°C with primary antibodies, including mouse anti-GFAP (1:200; Abcam) and rabbit anti-Parkin (1:200; Abcam). After washing, sections were treated with Alexa Fluor 488-conjugated secondary antibodies (1:200; Life Technologies) for 1 hour, counterstained with Hoechst 33 342 (1 μg/mL; Life Technologies) and imaged using a confocal microscope (Nikon Eclipse Ti).

Transmission electron microscopy of retinal tissues

Optic nerve head samples (n=3 per group) were dissected on ice and immediately fixed in 2.5% glutaraldehyde (Ted Pella, Redding, California, USA) in 0.1 M phosphate buffer. Fixed tissues were washed, postfixed in 1% osmium tetroxide at 4°C for 2 hours, washed again, dehydrated in graded ethanol and embedded in epoxy resin. Ultrathin sections of unmyelinated optic nerves were prepared and observed using a transmission electron microscope to assess mitochondrial morphology and structural integrity. Mitochondrial health was assessed using a scoring system based on the appearance of cristae in ultrastructural analyses. The number of mitochondria, autophagosomes and mitophagosomes was quantified in each transmission electron microscopy image and subsequently converted to counts based on the corresponding scale bars.

Statistical analysis

All data were collected from three independent experiments and are presented as mean±SD. Statistical comparisons were performed using one-way analysis of variance followed by Bonferroni post-hoc tests. A p value of <0.05 was considered statistically significant.

Results

Fucoxanthin enhances RGC survival and regulates apoptosis-related proteins in glaucomatous rats

This study aimed to investigate the neuroprotective properties of fucoxanthin in glaucomatous rats. To model the condition, intravitreal injections were administered 3 days prior to inducing elevated IOP via translimbal laser photocoagulation. As anticipated, laser-treated eyes displayed significantly elevated IOP compared with their untreated contralateral controls. Importantly, no significant differences in IOP were observed between vehicle-treated and fucoxanthin-treated groups, as illustrated in online supplemental table 1.

To assess RGC survival, we performed retrograde labelling using FluoroGold injections into the superior colliculi. In untreated normal rat retinas, RGC densities were consistent across regions, with averages of 2448 cells/mm² centrally, 2290 cells/mm² in the mid-periphery and 1867 cells/mm² peripherally (n=6; figure 1A,D,G). At 2 weeks following IOP elevation, laser-treated eyes exhibited pronounced RGC loss, with reduced densities of 1788 cells/mm² centrally, 1577 cells/mm² in the mid-periphery and 1195 cells/mm² peripherally (n=6; figure 1B,E,H). These reductions corresponded to losses of 26.96%, 31.14% and 35.99%, respectively (n=6; p<0.01; figure 1J). However, fucoxanthin-treated retinas showed significantly improved RGC survival compared with the vehicle-treated group. Specifically, RGC densities increased to 2015 cells/mm² centrally, 1781 cells/mm² in the mid-periphery and 1373 cells/mm² peripherally after fucoxanthin treatment (n=6; figure 1C,F,I), reflecting increases of 12.70%, 12.94% and 14.90%, respectively, relative to vehicle-treated controls (n=6; p<0.01; figure 1J).

Figure 1. Effect of fucoxanthin on RGC survival in ocular hypertensive rat retinas. The retinal flat mounts of blank control rats (A, D and G), vehicle-treated ocular hypertensive rats (B, E and H) and fucoxanthin-treated ocular hypertensive rats (C, F and I). Quantitative analysis of RGC survival (J) (n=6; data are expressed as mean±SD; **p<0.01; scale bar=100 µm (A–I)). (K) Proposed mechanism by which fucoxanthin mitigates RGC loss in glaucomatous rats by modulating Parkin-mediated mitophagy (created by biorender.com). LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1A/1B-light chain 3; NC, normal control; OHT, ocular hypertension; OHT+FX, fucoxanthin-treated ocular hypertension; RGC, retinal ganglion cell.

To further explore the effects of fucoxanthin, we analysed the expression of GFAP, a marker of Müller cell activation. Ocular hypertensive retinas demonstrated significantly elevated GFAP messenger RNA (mRNA) levels at both 3 days and 2 weeks post-IOP elevation compared with contralateral controls (p<0.01; figure 2C). Western blot analysis corroborated these findings, showing markedly increased GFAP protein expression at both time points (p<0.01; figure 2B). Notably, fucoxanthin treatment significantly reduced GFAP mRNA and protein levels at both 3 days and 2 weeks relative to the vehicle-treated group (p<0.01; figure 2B,C). Immunohistochemistry further confirmed a reduction in GFAP immunoreactivity in Müller cells following treatment with fucoxanthin (figure 3E).

Figure 2. Impact of fucoxanthin on apoptotic protein expression and Müller glial activation in glaucomatous rats. In untreated ocular hypertensive retinas, GFAP (B) and Bax (D) protein levels were elevated, while Bcl-2 (F) levels were reduced at both 3 days and 2 weeks post-IOP elevation. Fucoxanthin treatment resulted in a decrease in GFAP levels at both time points compared with vehicle-treated groups (B). Bax levels initially increased at 3 days but decreased at 2 weeks following fucoxanthin treatment (D), whereas Bcl-2 expression showed an increase at 14 days (F). At the mRNA level, ocular hypertensive retinas exhibited increased GFAP (C) and Bax (E) mRNA levels, with GFAP elevated at both 3 days and 2 weeks, and Bax increased at 2 weeks. Conversely, Bcl-2 (G) mRNA was decreased at both time points. Fucoxanthin treatment led to a reduction in GFAP mRNA at 3 days and 2 weeks, a decrease in Bax mRNA at 2 weeks and an increase in Bcl-2 mRNA at both 3 days and 2 weeks compared with the vehicle-treated group (n=3; data are expressed as mean±SD; *p<0.05; **p<0.01). BAX, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillar acidic protein;mRNA, messenger RNA; NC, normal control; OHT, ocular hypertension; OHT+FX, fucoxanthin-treated ocular hypertension.

Figure 3. Effect of fucoxanthin on mitophagy marker expression in glaucomatous rats. In untreated ocular hypertensive retinas, mRNA levels of Parkin (A), optineurin (B) and LAMP1 (D) increased at 3 days but decreased at 2 weeks, while LC3 (C) mRNA levels increased at both 3 days and 2 weeks. Following fucoxanthin treatment, mRNA levels of Parkin (A), optineurin (B), LC3 (C) and LAMP1 (D) decreased at 3 days but increased at 2 weeks compared with the vehicle-treated group (n=3; data are expressed as mean±SD; *p<0.05; **p<0.01). (E) Immunofluorescence analysis of Parkin and GFAP expression in vehicle-treated and fucoxanthin-treated ocular hypertensive retinas. Compared with vehicle-treated ocular hypertensive retinas (a–d), GFAP immunoreactivity decreased, whereas Parkin immunoreactivity increased, particularly in the retinal nerve fibre layer of fucoxanthin-treated ocular hypertensive retinas (e–h). Scale bar=50 µm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCL, ganglion cell layer; GFAP, glial fibrillar acidic protein; INL, inner nuclear layer; IPL, inner plexiform layer; LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1A/1B-light chain 3; mRNA, messenger RNA; NC, normal control; OHT, ocular hypertension; OHT+FX, fucoxanthin-treated ocular hypertension; ONL, outer nuclear layer; OPL, outer plexiform layer.

To evaluate the effects of fucoxanthin on apoptosis, we assessed the expression of Bax and Bcl-2, key markers of cell death and survival, respectively. Laser-induced IOP elevation resulted in increased Bax protein levels and decreased Bcl-2 protein levels at both 3 days and 2 weeks compared with untreated controls (p<0.01; figure 2D,F). qPCR analysis revealed similar trends, with elevated Bax mRNA levels at 2 weeks and reduced Bcl-2 mRNA levels at both 3 days and 2 weeks post-treatment (p<0.01; figure 2E,G). These findings suggest that elevated IOP activates Müller cells and promotes apoptotic pathways. However, fucoxanthin treatment significantly mitigated these effects, reducing Bax mRNA levels at 2 weeks and increasing Bcl-2 mRNA levels at both 3 days and 2 weeks compared with vehicle-treated controls (p<0.01; figure 2E,G). Protein analysis further revealed a decline in Bax expression at 2 weeks and an increase in Bcl-2 expression at the same time point following fucoxanthin treatment (p<0.01; figure 2D,F). Collectively, these results indicate that fucoxanthin provides robust neuroprotection for RGCs by suppressing apoptosis and Müller cell activation in glaucomatous rats.

Fucoxanthin modulates mitophagy in the retina of glaucomatous rats

Given the observed protective effects of fucoxanthin on RGCs, we investigated its potential role in regulating mitophagy, a critical cellular process for maintaining mitochondrial health.

In ocular hypertensive retinas, the mRNA expression of Parkin, a key regulator of mitophagy, increased at 3 days but decreased significantly at 2 weeks post-IOP elevation compared with controls (p<0.05; figure 3A). Optineurin, a downstream effector of Parkin, exhibited a similar trend (p<0.05; figure 3B). Additionally, LAMP1 mRNA levels, indicative of lysosomal activity, increased at 3 days but declined by 2 weeks (p<0.01; figure 3D), while LC3 mRNA levels were elevated at both time points (p<0.01; figure 3C).

In the fucoxanthin-treated group, Parkin and optineurin mRNA levels were significantly reduced at 3 days but increased at 2 weeks relative to vehicle-treated controls (p<0.01; figure 3A,B). Similar trends were observed for LC3 and LAMP1 mRNA levels, with decreases at 3 days followed by increases at 2 weeks (p<0.01; figure 3C,D). Immunohistochemical analysis confirmed enhanced Parkin immunoreactivity, particularly in the ganglion cell layer, after fucoxanthin treatment (figure 3E). These findings suggest that fucoxanthin restores mitophagy homeostasis, promoting mitochondrial quality control and RGC survival in glaucomatous rats.

Fucoxanthin improves mitochondrial health and enhances mitophagy in the optic nerve

To evaluate mitochondrial integrity in the optic nerve, we used ultrastructural analysis and a mitochondrial health grading scale based on cristae morphology. In ocular hypertensive optic nerves, mitochondria displayed marked structural disruptions, including swollen and fragmented cristae. Mitochondrial health scores were significantly lower in ocular hypertensive optic nerves compared with controls at both 3 days and 2 weeks post-IOP elevation (p<0.01; figure 4E). Moreover, transmission electron microscopy revealed an increased number of mitochondria, autophagosomes and mitophagosomes in ocular hypertensive optic nerves (p<0.01; figure 4F,G,H).

Figure 4. Effects of fucoxanthin on mitochondrial morphology and mitophagy. (A) Mitochondrial health was assessed using a scoring system based on the appearance of cristae in ultrastructural analyses. (B) NC group, where green arrows indicate healthy mitochondria. (C) OHT group, characterised by yellow arrows pointing to unhealthy mitochondria. (D) OHT+FX group, with red arrows indicating mitophagosomes. Relative to the control group (B), ocular hypertensive optic nerves (C) demonstrated a significant increase in the number of mitochondria (F), autophagosomes (G) and mitophagosomes (H) at both 3 days and 2 weeks post-treatment. Treatment with fucoxanthin resulted in a higher mitochondrial health score (E) and a greater number of mitochondria (F) at 3 days and 2 weeks compared with the vehicle-treated group. Notably, the number of autophagosomes (G) was reduced at 3 days but increased at 2 weeks in the fucoxanthin-treated group (n=3; data are expressed as mean±SD; *p<0.05; **p<0.01). Scale bar=500 nm (B–D). IOP, intraocular pressure; NC, normal control; OHT, ocular hypertension; OHT+FX, fucoxanthin-treated ocular hypertension.

Fucoxanthin treatment significantly improved mitochondrial health scores and increased mitochondrial numbers at both time points compared with vehicle-treated controls (p<0.01; figure 4E,F). Interestingly, while autophagosome and mitophagosome numbers were higher in ocular hypertensive optic nerves, fucoxanthin treatment reduced these structures at 3 days but promoted their accumulation at 2 weeks (p<0.01; figure 4G,H).

Western blot analysis further revealed dynamic changes in mitophagy-related proteins. Parkin protein expression was elevated at 3 days but reduced at 2 weeks in ocular hypertensive optic nerves (p<0.01; figure 5B). Optineurin levels increased at both time points, while the LC3-II/LC3-I ratio and LAMP1 expression showed patterns consistent with impaired mitophagy (p<0.05; figure 5C–E). Fucoxanthin treatment reversed these alterations, reducing Parkin and optineurin expression at 3 days and enhancing their levels at 2 weeks (p<0.01; figure 5B,C). LC3-II/LC3-I ratios and LAMP1 levels followed similar trends (p<0.01; figure 5D,E). These results suggest that fucoxanthin modulates Parkin-mediated mitophagy, contributing to improved mitochondrial integrity and neuroprotection in the optic nerve.

Figure 5. Impact of fucoxanthin on mitophagy-related proteins in glaucomatous rats. Compared with the control group, the protein levels of Parkin (B) and LAMP1 (E) were significantly elevated at 3 days but decreased at 2 weeks. The protein levels of optineurin (C) and the ratio of LC3-II/LC3-I (D) were increased at both 3 days and 2 weeks in the ocular hypertensive optic nerves. In the fucoxanthin-treated group, the protein expression of Parkin (B), optineurin (C), LAMP1 (E) and the ratio of LC3-II/LC3-I (D) was notably reduced at 3 days and increased at 2 weeks compared with the vehicle-treated group (n=3; data are expressed as mean±SD; *p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC3, microtubule-associated protein 1A/1B-light chain 3; LAMP1, lysosomal-associated membrane protein 1; NC, normal control; OHT, ocular hypertension; OHT+FX, fucoxanthin-treated ocular hypertension.

Discussion

The results of this study reveal a strong association between ocular hypertension, the accumulation of damaged mitochondria and increased mitophagosomes, which collectively contribute to the loss of RGCs. In the context of acute IOP elevation, fucoxanthin demonstrated significant neuroprotective effects. It suppressed GFAP expression, reduced levels of Parkin and associated mitophagy-related proteins and decreased the abundance of autophagosomes and mitophagosomes. These effects played a critical role in mitigating RGC damage by preventing excessive mitophagy. Conversely, in the setting of prolonged IOP elevation, fucoxanthin exhibited additional benefits by reducing RGC loss, improving mitochondrial health and enhancing the expression of Parkin and other proteins essential for Parkin-mediated mitophagy. Notably, fucoxanthin also improved the efficiency of the mitophagy pathway, restoring mitochondrial homeostasis and protecting RGCs from progressive damage.

The pathophysiology of glaucomatous neuropathy is closely linked to the activation of glial cells, which release inflammatory cytokines that exacerbate retinal damage following optic nerve injury.¹⁶ In our study, we observed a significant upregulation of GFAP expression in ocular hypertensive retinas, indicative of retinal cell stress and damage. Apoptosis, a key mechanism driving RGC loss, is heavily influenced by the balance between the proapoptotic (eg, Bax) and antiapoptotic (eg, Bcl-2) members of the Bcl-2 protein family. This balance regulates mitochondrial outer membrane permeabilisation, a pivotal step in initiating cell death.¹⁷ Under normal physiological conditions, the levels of Bax and Bcl-2 remain relatively stable. However, glaucoma-induced neurodegeneration disrupts this equilibrium, favouring apoptotic pathways and accelerating RGC loss. Consistent with prior studies,¹⁸ our findings demonstrate elevated Bax expression and decreased Bcl-2 levels in ocular hypertensive rats, underscoring the role of mitochondrial dysfunction and apoptosis in glaucoma.

Mitophagy, a selective form of autophagy that removes damaged mitochondria, is critical for maintaining mitochondrial quality and cellular homeostasis. Upon mitochondrial damage, PTEN-induced putative kinase1 (PINK1) accumulates on the OMM, recruiting Parkin from the cytosol. This recruitment activates Parkin’s ligase function, resulting in the ubiquitination of mitochondrial proteins and promoting mitophagy.⁹ Evidence suggests that enhancing Parkin activity can stimulate mitophagy and protect RGCs from glaucomatous damage.¹⁹ Optineurin, a key mitophagy receptor, binds ubiquitinated mitochondrial proteins and interacts with LC3 to facilitate autophagosome formation. Enhanced optineurin binding amplifies mitophagy activity, whereas mutations in optineurin impair mitophagy and lead to the accumulation of dysfunctional mitochondria.²⁰

In our study, we observed an upregulation of Parkin and downstream optineurin mRNA levels at 3 days following IOP elevation, followed by a decline at 2 weeks. These findings suggest that Parkin overexpression protected against RGC loss, improved mitochondrial health and partially restored mitophagy function in early neurodegeneration events in ocular hypertensive rats. This protective effect may be attributed to Parkin’s role in promoting mitophagy and reducing mitochondrial damage at an early stage, thereby enhancing cell survival rates and stress resistance. Consequently, the increased expression of Parkin at 3 days might represent a rapid adaptive response to cellular stress induced by ocular hypertension, aiming to mitigate mitochondrial dysfunction and apoptosis. However, with the accumulation of damage induced by elevated IOP (14 days of high IOP), compensatory mechanisms begin to fail. Concurrently, further impairment of RGCs leads to a decline in Parkin expression, indicating impaired mitophagy function.^{4 10} LC3, a marker of autophagosomes, exists in two forms: LC3-I (cytosolic) and LC3-II (lipidated). During autophagy, LC3-I is converted to LC3-II, which associates with autophagosome membranes. The LC3-II/LC3-I ratio is widely used as a marker of autophagic activity. Our results showed an elevated LC3-II/LC3-I ratio in ocular hypertensive optic nerves, indicating increased autophagosome formation. However, this increase may not solely reflect enhanced autophagy but could also result from impaired autophagosome clearance due to defective lysosomal function.²¹

To investigate this further, we examined LAMP1, a lysosomal marker. LAMP1 expression increased at 3 days post-IOP elevation but decreased at 2 weeks, while the LC3-II/LC3-I ratio remained elevated. Transmission electron microscopy revealed a higher number of unhealthy mitochondria and mitophagosomes in ocular hypertensive optic nerves, suggesting reduced autophagosome turnover and impaired mitochondrial recycling. Consistent with our findings, Coughlin et al⁶ reported that in the DBA/2J mouse model of glaucoma, increased autophagosomes and mitochondria were accompanied by decreased LAMP1 expression, indicating defective mitophagy. Similarly, Dai et al⁴ observed an accumulation of autophagosomes and mitophagosomes in ocular hypertensive optic nerves, coupled with an elevated LC3-II/LC3-I ratio and reduced LAMP1 levels. These observations suggest that while mitophagy is initially upregulated to counteract stress, prolonged IOP elevation overwhelms this compensatory mechanism, leading to impaired mitophagy and progressive RGC damage.

Fucoxanthin, a natural carotenoid, has shown potential neuroprotective effects in neurodegenerative diseases. Zhang et al²² demonstrated that fucoxanthin reduces Bax expression, enhances Bcl-2 levels and mitigates apoptosis, thereby improving brain function in models of traumatic brain injury. Similarly, our study showed that fucoxanthin suppresses GFAP and Bax expression, promotes Bcl-2 upregulation and significantly enhances RGC survival in glaucomatous rats. Prior studies further suggest that fucoxanthin stabilises mitochondrial membranes and reduces apoptosis in models of glutamate excitotoxicity.¹⁴ Collectively, these findings highlight fucoxanthin’s ability to regulate apoptotic pathways and preserve mitochondrial integrity, emphasising its therapeutic potential in glaucomatous neuropathy.

Emerging research also supports the role of fucoxanthin in improving mitochondrial function. Fucoxanthin has been shown to enhance mitochondrial activity during oxidative stress in retinal pigment epithelial cells²³ and dexamethasone-induced atrophy in C2C12 myotubes.²⁴ Additionally, it restores mitophagy-related gene expression in models of inflammation and promotes mitophagy through the mitochondrial outer membrane protein BNIP3/NIX signalling in high glucose (HG)-treated cardiomyocytes.^{13 25} In the present study, we observed that fucoxanthin modulates mitophagy during both acute and chronic IOP elevation. During early-stage IOP elevation, fucoxanthin reduced excessive mitophagy by downregulating Parkin, optineurin and mitophagosome levels, thereby protecting RGCs from overactivation of mitophagy pathways. In contrast, during sustained IOP elevation, fucoxanthin enhanced Parkin expression and mitophagy-related proteins, restoring mitophagy balance and mitigating mitochondrial dysfunction (figure 1). These findings align with prior evidence showing that Parkin overexpression partially rescues mitophagy deficits in glaucomatous models.⁴

Conclusions

In conclusion, our study identifies a novel neuroprotective role for fucoxanthin in preserving RGCs in experimental glaucomatous rats. By regulating Parkin-mediated mitophagy, fucoxanthin prevents excessive mitophagy during acute IOP elevation and restores mitophagy function during sustained stress. These findings suggest that targeting mitophagy homeostasis through Parkin signalling could represent a promising therapeutic strategy for glaucoma and other neurodegenerative diseases. Future studies are warranted to further explore the therapeutic potential of fucoxanthin as a neuroprotective agent in clinical settings.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.