Mitochondrial Dysfunction as a Novel Target for Neuroprotective Nutraceuticals in Ocular Diseases

The eyes require a rich oxygen and nutrient supply; hence, the high-energy demand of the visual system makes it sensitive to oxidative stress. Excessive free radicals result in mitochondrial dysfunction and lead to retinal neurodegeneration, as an early stage of retinal metabolic disorders. Retinal cells are vulnerable because of their coordinated interaction and intricate neural networks. Nutraceuticals are believed to target multiple pathways and have shown neuroprotective benefits by scavenging free radicals and promoting mitochondrial gene expression. Furthermore, encouraging results demonstrate that nutraceuticals improve the organization of retinal cells and visual functions. This review discusses the mitochondrial impairments of retinal cells and the mechanisms underlying the neuroprotective effects of nutraceuticals. However, some unsolved problems still exist between laboratory study and clinical therapy. Poor bioavailability and bioaccessibility strongly limit their development. A new delivery system and improved formulation may offer promise for health care applications.


Introduction
Retinal neurodegeneration is one of the major causes of visual impairment and is highly associated with atrophy or cell death of the retina in ocular diseases, such as glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR) [1]. The metabolic rate of the visual system is higher than that of others within the brain, and further disruptions in the metabolic homeostasis can lead to vulnerability of the retina [2] In this review, we endeavored to focus on the neuropathies of retinal cell types induced by metabolic impairment, as well as the potential neuroprotective nutraceuticals based on evidence from animal experiments and clinical studies.

The High-Energy Demands of the Retina
The process of visualizing information is achieved by transmitting light into electrical impulses, triggered mainly by rods and cones, through the optic nerve to the brain. Bipolar cells and amacrine cells collect the signals from photoreceptor cells and synapse with retinal ganglion cells (RGCs). RGCs are a type of neuron and propagate action potentials (AP) to axons. The axons form the optic nerve and project to the lateral geniculate nucleus (LGN), the medial geniculate body, and superior colliculus. There are photoreceptors [27]. In addition, the metabolism of photoreceptors becomes maximal to generate dark current at night [28]. Therefore, photoreceptors are the major site of ROS generation and show apoptosis in the early stages of diabetes [29] and atrophy in the later stages of AMD [17]. In brief, the high energy requirement and electrophysiological function of retinal cells make them more vulnerable to oxidative stress.

Nutraceuticals as Neuroprotectants for Retinal Neurodegeneration
Metabolic impairments cause numerous retinal manifestations in chronic progression. This results in irreversible damage of retinal cells if oxidative stress is not reduced with treatment. Some clinical medications or surgeries are approved to treat ocular diseases. However, these therapies are used for the late stages of disease progression. There is a clear need for new strategies to act at the molecular or cellular target to prevent the development of the disorder. Mitochondrial dysfunction has been shown to be one of the early events in retinal neurodegeneration [30,31]. Targeting the mitochondrial function brings the promise of new options. Nutraceuticals are believed to target multiple pathways and attenuate the progression of neuronal destruction through mitochondrial dysfunction [32,33]. Therefore, nutraceuticals could be considered as positive neuroprotectants of retinal cells. Here, we summarize the retinopathy progression related to mitochondrial dysfunction and the effects of nutraceuticals with improved retinal neurodegeneration as below.

Glaucoma
Glaucoma is the second leading cause of blindness worldwide and is characterized by the progressive degeneration or loss of RGCs [34]. Most glaucoma cases are classified into primary open-angle glaucoma (POAG) and angle-closure glaucoma (ACG) caused by an increase in the intraocular pressure (IOP). Mitochondrial dysfunction in the trabecular meshwork may impair their cytoarchitecture and lead to alteration in the drainage of aqueous humor, further raising the IOP [16,35]. IOP-induced stress and strain are biomechanical factors of damage in the lamina cribrosa and adjacent tissues [34]. It also induces metabolic stress causing mitochondrial dysfunction in mouse RGCs [36]. In some individuals with normal range IOP, particularly in Asians, they are classified as normal-tension glaucoma (NTG) [37]. The pathogenic mechanism of NTG is not fully understood; the low pressure of cerebrospinal fluid in the optic nerve subarachnoid space may cause trans-lamina cribrosa pressure difference and compress the optic nerve [38]. Numerous biomarkers related to oxidative stress are reported to be significantly higher in glaucoma patients [39]. Therefore, recent scientific literature demonstrates that mitochondrial dysfunction and oxidative stress are both a cause and consequence and play a central role in the process of glaucoma [40].

Age-Related Macular Degeneration
AMD is a progressive ocular disease with loss of central vision and is a major cause of visual impairment in the developed world. It is clinically classified as early-stage (formation of drusen deposits between the Bruch's membrane and RPE) and late-stage AMD owing to atrophy of the RPE/photoreceptors (dry) or choroidal neovascularization (wet) [41]. Complement factor H (CFH) is an important component of drusen, indicating a local complement-activation at the RPE [42]. Oxidative stress suppresses the expression of CFH [43] and promotes complement system activation [44], abolishing its protective function from the lipid peroxidation product [45]. Smoking is believed to be the strongest risk for developing AMD [46] and leads to oxidative stress and complement activation, resulting in the endoplasmic reticulum (ER) stress-mediated lipid accumulation [47]. Initial RPE mitochondrial abnormalities have been revealed in AMD patients [48]. Furthermore, mtDNA damage is found in the macular and peripheral RPE of AMD human samples [49]. Therefore, AMD could be seen as a progressive neurodegenerative disease primarily causing damage to mtDNA and further affecting the mitochondrial function of RPE [50].

Diabetic Retinopathy
DR is one of the most common complications of diabetes and remains the leading cause of vision loss among working-age adults in developed countries [51]. It is traditionally characterized as a microvascular disease [52] and has recently been recognized as a disruption of the interdependence between multiple retinal cell-types, causing neurodegeneration at the endpoint [53,54]. Hyperglycemia and poor glucose control are fundamental in the development of DR [55,56]. RGCs, amacrine cells, and photoreceptors have an increased apoptotic rate at the early stages of DR development in humans [57][58][59]. This apoptotic death causes pericentral macular thinning of both the inner retinal layers and the nerve fiber layer (NFL), otherwise, hypertrophy (swelling) of Müller cells increases the thickness of the inner nuclear layer (INL) [60][61][62]. The detailed mechanisms of apoptosis in the development of DR are still not clear [63]. There are multiple factors involved in the pathogenesis, including AGEs, free radicals, excitotoxicity, and mitochondrial damage as mentioned above. Finally, hyperglycemia-induced metabolic stress may initiate a vicious cycle to amplify mitochondrial dysfunction, and further accelerate the apoptosis of retinal cells [64,65]. Severe loss of retinal cells causes failures in orchestrating intimate communication and promotes compensatory over-angiogenesis in proliferative DR [66,67] ( Figure 1).

Diabetic Retinopathy
DR is one of the most common complications of diabetes and remains the leading cause of vision loss among working-age adults in developed countries [51]. It is traditionally characterized as a microvascular disease [52] and has recently been recognized as a disruption of the interdependence between multiple retinal cell-types, causing neurodegeneration at the endpoint [53,54]. Hyperglycemia and poor glucose control are fundamental in the development of DR [55,56]. RGCs, amacrine cells, and photoreceptors have an increased apoptotic rate at the early stages of DR development in humans [57][58][59]. This apoptotic death causes pericentral macular thinning of both the inner retinal layers and the nerve fiber layer (NFL), otherwise, hypertrophy (swelling) of Müller cells increases the thickness of the inner nuclear layer (INL) [60][61][62]. The detailed mechanisms of apoptosis in the development of DR are still not clear [63]. There are multiple factors involved in the pathogenesis, including AGEs, free radicals, excitotoxicity, and mitochondrial damage as mentioned above. Finally, hyperglycemia-induced metabolic stress may initiate a vicious cycle to amplify mitochondrial dysfunction, and further accelerate the apoptosis of retinal cells [64,65]. Severe loss of retinal cells causes failures in orchestrating intimate communication and promotes compensatory over-angiogenesis in proliferative DR [66,67] (Figure 1).

Resveratrol
Resveratrol is a plant polyphenol found in grapes and red wine [68], and is reported to improve mitochondrial function by activating SIRT1 and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) [69]. It is also effective for age-related ocular diseases through
Resveratrol has numerous beneficial effects on anti-cancer, cardiovascular diseases, obesity, diabetes, and neurological disorders in humans. It is reported to be safe at a dose of 1 g or more per day, however, the major obstacle for clinical therapy is the rapid metabolism and poor bioavailability [81][82][83]. Until now, research on resveratrol has been limited to animal models and in vitro experiments in ocular diseases [70,84]. Case report observations have shown resveratrol based nutritional supplements have benefits to improve RPE functions in AMD patients [85]. In addition, a recent double-blind randomized control trial indicates resveratrol notably reduces muscle fat and improves mitochondrial function in diabetes type 2 (T2D) patients [86]. Further investigations into the retino-protective effects of resveratrol should include more clinical studies.

Quercetin
Quercetin is a dietary flavonoid compound found in fruits, vegetables and beverages [87]. It has a substantial antioxidant ability to scavenge ROS [88] and ameliorates mitochondrial dysfunction through an AMP-activated protein kinase (AMPK)/SIRT1 signaling pathway [89,90]. An increasing number of studies show that quercetin reduces ROS [91,92], mitochondrial membrane potential (∆Ψm) and has anti-apoptotic effects on RGCs [93]. Zhou and colleagues recently report quercetin alleviates the excitability of RGCs through increased miniature GABAergic neurotransmission and decreasing miniature glutamatergic neurotransmission [94]. On the other hand, some studies have demonstrated that quercetin has an inhibitory effect of heat shock protein 72 (HSP 72) in RGCs [95][96][97]. Quercetin has neuroprotective effects of retinal layers [98] and cytoprotective effects of photoreceptor, RPE and RGCs through inhibiting activity of AP-1 pathway [99] in light-induced retinal degeneration rodent models. It attenuates hyperglycemia [100] and dyslipidemia [101], and also has anti-retinal oxidative stress, anti-neuroinflammation and anti-apoptosis protective effects in diabetic animal models [102].
Clinical trials on quercetin have shown multiple effects, such as anti-inflammatory effects through the reduction in plasma C-reactive protein [103] or oxidative stress markers [104], anti-cancer effects, and cancer chemoprevention [105][106][107]. In a recent cohort study with a 15-year follow-up, dietary intake of quercetin was shown to reduce the prevalence of any AMD (OR: 0.76; 95% CI: 0.58, 0.99) [108]. However, the lack of clinical data limits its application in ocular diseases; thus, more clinical studies are required in the future.

Xanthophylls (Lutein and Zeaxanthin)
Lutein and zeaxanthin stereoisomer are oxygenated carotenoids (xanthophylls) and are present at the macula as macular pigments [109]. Xanthophylls cannot be synthesized in humans and their supplements depend on dietary sources. They are abundant in various foods such as spinach, egg yolk, and wolfberry [110]. Xanthophylls play a key role in ROS scavenging and have anti-inflammatory and neuroprotective functions [110][111][112]. They are cleaved by β,β-carotene 9 ,10 -oxygenase 2 (BCO2), however, inactivity of human BCO2 causes carotenoid accumulation [113]. This phenomenon may be an important mechanism for protecting the macula from short-wavelength light-induced damage [114]. Lutein has multiple benefits via anti-apoptosis [115], antioxidant [116] and reducing ER stress [117] in the retina. Recent studies demonstrate that xanthophylls could upregulate carotenoid metabolic genes and also improve mitochondrial biogenesis in primate animal models [118,119].
Many studies show multi-ingredient formulations for individuals could increase the concentrations of lutein or xanthophylls in the plasma and macular pigment (reviewed by Bernstein et al. [120]). Epidemiologic studies support lutein for the prevention of developing AMD in the early or intermediate stage [121,122]. It is also reported that lutein/zeaxanthin may be protective against late AMD [123]. A recent systematic review reported that there are at least 47 publications from 1946 to October 2016 and its conclusions show a strong relationship between lutein/zeaxanthin supplementation and evaluation of both macular pigment density and visual function [124]. There are controversial results of lutein and zeaxanthin in an Age-Related Eye Disease Study 2 (AREDS2) [125]. Their data showed that AREDS2 formulation in primary analyses did not prevent process of advanced AMD. However, when participants were limited to those with the lowest dietary intake of lutein + zeaxanthin, results of exploratory subgroup analyses showed a protective effect for progression to advanced AMD (HR: 0.74; 95% CI, 0.59-0.94; p = 0.01). These inconsistent results of xanthophylls in clinical trials need further design approaches to confirm their benefits.

Omega-3 Fatty Acid
Docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5), belonging to omega-3 (α-linolenic acid, n-3) polyunsaturated fatty acids (PUFAs), are required for membrane organization and cell integrity. PUFAs intake from dietary supplementation is essential as mammals lack the enzymes for its generation [126]. Omega-3 PUFAs improve the mitochondrial dysfunction by upregulating mitochondrial biogenesis, ATP production, and dissipating the proton gradient uncoupling proteins (UCPs) gene expression in vivo [127][128][129]. DHA is enriched in the retina, where it has both structural and neuroprotective functions, and is converted by lipoxygenase (LO) to 10,17S-docosatriene (neuroprotectin D1, NPD1) under oxidative stress conditions [130]. Furthermore, NPD1 inhibits pro-inflammatory and apoptotic gene expression, and consequently promotes the survival of photoreceptors [131]. Dietary manipulation of omega-3 PUFAs lowers IOP in aged rats and is associated with a significant increase in the outflow facility and a decrease in ocular rigidity [132]. Another study showed that a dietary combination of omega-3 and omega-6 PUFAs are more effective for preventing retinal cell structure and decreasing the glial cell activation [133]. An omega-3 fatty acid diet has been shown to have a retinal protective function in the AMD-like retinal lesions [134] and type 2 diabetic mice [135]. A recent study indicated that omega-3 PUFAs reduce lipofuscin granule formation and protect the photoreceptor layer. Its mechanism may involve an increase in the myelin basic protein (MBP), myelin proteolipid protein (MPP), myelin regulatory factor-like protein (MRFLP), and glial fibrillar acidic protein (GFAP) expression [136].
Omega-3 PUFAs intake is associated with a 30% decrease in the development of central geographic atrophy (CGA) and neovascular AMD [137]. Dietary supplementation with 4 g of omega-3 PUFAs for 6 months increases the serum omega-3 in patients with dry AMD by an average of 7.6%, however, there are no statistically significant changes in the retinal function of visual acuity or ERG [138]. The modified AREDS formulation also has a similar outcome in that the addition of omega-3 PUFAs in primary analyses did not reduce risk of progression to advanced AMD [125]. A recent AREDS2 study showed similar results, where omega-3 PUFAs did not demonstrate any significant benefit in the reduction in their risk for progression to late AMD in participants with CFH or age-related maculopathy susceptibility 2 (ARMS2) risk genotype [139]. Conversely, increasing dietary PUFA, rather than saturated FA, is associated with a reduced likelihood of the presence and severity of DR [140]. A Mediterranean diet with omega-3 PUFAs (≥500 mg/day) supplements also showed a 48% relatively reduced risk in the incidence sight-threatening DR in individuals with type 2 diabetes [141].

Curcumin
Curcumin is a polyphenol extracted from turmeric (Curcuma longa), which is used as a spice and as a traditional herbal medicine in Asia. It is a hydrophobic molecule and is almost insoluble in water (approximately 30 nM). Curcumin has strong free radical scavenging activity due to its functional groups and sequentially improves mitochondrial functions through the nuclear factor erythroid 2-related factor 2 (Nrf2) [142]. However, its poor solubility and low bioavailability have limited the clinical applications of curcumin (see a recent review in detail [143]). New strategies, including liposomes and nanoparticle carriers, or modified formulations may be an ideal approach to deliver curcumin to the lesions. For example, Davis et al. developed a curcumin nanocarrier combined with D-α-tocopherol polyethene glycol 1000 succinate (TPGS), a non-ionic surfactant, and Pluronic F127, a difunctional block copolymer surfactant, which increased curcumin solubility by 400,000 times and that enhanced curcumin transport across ocular barriers. A topically administered curcumin nanocarrier has neuroprotective effects of retinal cells in vitro and in vivo [144]. In addition, Cheng and colleagues recently reported a dual-drug delivery system which consisted of thermosensitive chitosan-gelatin-based hydrogel containing curcumin-loaded nanoparticles and latanoprost, which release medicine and was extended to 7 days. Treatment with curcumin-containing hydrogel effectively decreased the oxidative stress-mediated damage in trabecular meshwork cells [145].
Clinical application of curcumin has been broadly discussed in multiple malignant diseases [146]. However, there are few studies which report that curcumin has clinical benefits for eye disorders. Improved formulation may overcome this problem. One example is that oral administration of a curcumin-phospholipid delivery system is effective in the management of central serous chorioretinopathy (CSCR). The results show administration of curcumin significantly improves visual acuity and retinal thickness [147]. A recent review examined this issue and the authors categorize three broad formulation strategies to enhance bioavailability and metabolism of curcumin [148]. These well-designed formulations require more clinical trials to confirm their substantial benefits.

Crocetin
Crocetin is an apocarotenoid, which is found both in the saffron crocus (Crocus starus L.) and in gardenia fruit (Gardenia jasminoides Ellis) [149,150]. Saffron and its components (crocetin, crocins, and safranal) have therapeutic properties of liver, nervous and cardiovascular systems, including anti-oxidant, anti-inflammatory, and anti-apoptotic properties [151,152]. Crocetin rescues disruption of the ∆Ψm induced by tunicamycin or hydrogen peroxide (H 2 O 2 ) in vitro and has protective effects against retinal degeneration in vivo [153]. It is reported to inhibit oxidative stress via mitogen-activated protein kinases (MAPK), extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK), p38, and the redox-sensitive NF-κB and c-Jun pathway in an ischemia/reperfusion (I/R) mouse model [154]. A hydrophilic saffron extract standardized to 3% crocin reduces higher IOP values and activated microglia cells [155]. Saffron has been shown to have beneficial effects for ocular diseases in clinical studies (see a recent review [156]). However, a recent clinical study shows short-term saffron supplementation had no significant effects on the visual acuity and focal ERG in Stargardt disease/fundus flavimaculatus (STG/FF) patients with ATP binding cassette subfamily A member 4 (ABCA4) gene mutations [157].

Other Potential Nutraceuticals
Some potential nutraceuticals, like traditional Chinese medicine, have effects including neural and mitochondrial protection. Ginkgo biloba extract (GBE) contains the flavone glycosides and terpenoids, and showed stabilization and protection of mitochondrial function in Alzheimer's disease [158]. GBE has various effects of antioxidant, microcirculation and neuroprotection activities in ocular diseases [159,160]. A topical formulation of GBE reduces IOP elevation and accumulation of extracellular materials in dexamethasone-induced ocular hypertension rabbits [161]. GBE administration has been shown to improve pre-existing visual field damage [162] and visual acuity analyzed by Humphrey Visual Field (HVF) [163] in patients with NTG. However, there is a recent study that showed no effect on mean defect or contrast sensitivity in Chinese patients with NTG [164].
Danshen (Salvia miltiorrhiza) is used for treating hyperlipidemia, acute ischemia, and stroke in traditional Chinese medicine [165]. Danshen extracts increase the levels of glutathione (GSH) and reduce the levels of malondialdehyde (MDA) in the eye tissues of hyperglycemic rats [166]. Salvianolic acids are a natural compound extracted from Danshen and more than 10 different salvianolic acids have been identified to date. Salvianolic acid A and B are the most effective and abundant compounds [167]. Salvianolic acid A has anti-oxidative stress potential, through the activation of Nrf2 and hemeoxygenase-1 (HO-1) expressions in RPE cells [168]. It also has an anti-angiogenesis function through the downregulation of cylindromatosis (CYLD) signaling pathways in choroidal neovascularization (CNV) mice [169]. A recent study showed that salvianolic acid A improved the mitochondrial function of high glucose-injured Schwann cells and diabetic peripheral neuropathy (DPN) in KK-A y diabetic mice via upregulation of nuclear Nrf2 expression [170]. Salvianolic acid B has been reported to protect against oxidative injury through Nrf2 and glutaredoxin 1 (a thiol repair enzyme, Grx1) in primary human RPE cells [171]. A multiple-formula containing Danshen, notoginseng, and borneol (Compound Danshen Dripping Pill, CDDP) significantly improved the best corrected visual acuity and retinal pathogenesis in non-proliferative diabetic retinopathy (NPDR) patients [172]. Another study showed that CDDP significantly improved fluorescence fundus angiography and funduscopic examination parameters in NPDR patients [173].
Astragali Radix (Huangqi) is one of the most frequently used herbal medicines in traditional Chinese medicine and has a wide range of biological activities [174]. The extract of Astragalus mongholicus has been reported that protected oxidative damage through ameliorating activities of the mitochondrial complexes I, II, malate dehydrogenase and ∆Ψm [175]. Astragalus polysaccharides protects mitochondria by scavenging ROS, inhibiting mitochondrial permeability transition and increasing the activities of catalase (CAT), SOD, and glutathione peroxidase (GPx) [176]. Huang and colleagues further demonstrated that Astragalus polysaccharides restored the imbalance of mitochondrial fusion-fission processes, activation of mitophagy, and decrease in PGC-1α expression in vivo [177]. The active compounds astragaloside IV and formononetin extracted from huangqi have also been indicated to inhibit aldose reductase (AR) and hypoxia-induced neovascularization, respectively [178,179]. These data suggest that extracts from huangqi may have therapeutic benefits for DR. Major findings are summarized in the Table 1 (animal studies) and Table 2 (clinical trials).     Improvement of fluorescence fundus angiography and funduscopic examination (p < 0.001) [173] Odds ratios (OR), Confidence interval (CI), Relative risk (RR), Hazard ratio (HR).

Conclusions
In this review, we summarize the underlying mechanisms of high-energy consumption and metabolic homeostasis that play a decisive role in the retina. The loss of balance between energy production and free radicals quenching causes oxidative stress, and further leads to mitochondrial dysfunction. The structural and functional integrity of the mitochondrion is important for maintaining the organization of retinal cells. Retinal neurodegeneration is a pathogenic result of mitochondrial dysfunction and contributes to an early stage of progression in retinal metabolic disorders.
A large body of evidence demonstrates that nutraceuticals target mitochondrial function to restore the mitochondrial flexibility. Some are essential nutrients and have benefits for both forming the cellular structure and scavenging ROS. Otherwise, multiple nutraceuticals with the potential for improving mitochondrial integrity have been reported in animal studies. Unfortunately, poor bioavailability and bioaccessibility limit their current therapeutic use. A new delivery system and improved formulation may bridge the gap between laboratory study and clinical treatment [180]. Future clinical trials require an additional focus on the next generation of nutraceuticals to confirm their health benefits.