Role of the AMPK signalling pathway in the aetiopathogenesis of ocular diseases

Background AMP-activated protein kinase (AMPK) plays a precise role as a master regulator of cellular energy homeostasis. AMPK is activated in response to the signalling cues that exhaust cellular ATP levels such as hypoxia, ischaemia, glucose depletion and heat shock. As a central regulator of both lipid and glucose metabolism, AMPK is considered to be a potential therapeutic target for the treatment of various diseases, including eye disorders. Objective To review all the shreds of evidence concerning the role of the AMPK signalling pathway in the pathogenesis of ocular diseases. Method Scientific data search and review of available information evaluating the influence of AMPK signalling on ocular diseases. Results Review highlights the significance of AMPK signalling in the aetiopathogenesis of ocular diseases, including cataract, glaucoma, diabetic retinopathy, retinoblastoma, age-related macular degeneration, corneal diseases, etc. The review also provides the information on the AMPK-associated pathways with reference to ocular disease, which includes mitochondrial biogenesis, autophagy and regulation of inflammatory response. Conclusion The study concludes the role of AMPK in ocular diseases. There is growing interest in the therapeutic utilization of the AMPK pathway for ocular disease treatment. Furthermore, inhibition of AMPK signalling might represent more pertinent strategy than AMPK activation for ocular disease treatment. Such information will guide the development of more effective AMPK modulators for ocular diseases.


Introduction
Evolution of eukaryotes has rendered them with an excellent cellular energy sensor -AMP-activated protein kinase (AMPK). Among unicellular eukaryotes, yeast possesses Snf1, an orthologue of catalytic α subunit of mammalian AMPK. 1 Snf1(sucrose Non-fermenting1) switch between alternative carbon source utilization pathways (gluconeogenesis and respiration) when deprived of glucose while AMPK senses energy availability by directly binding to ATP and AMP or ADP. 2 In response to the signalling cues that causes energy stress such as glucose depletion, hypoxia, ischaemia and heat shock AMPK is activated. 3 Activated AMPK then shifts the metabolism more towards catabolism than anabolism via modulation in the phosphorylation status of enzymes involved in glucose-, lipid-and mitochondrial-homeostasis. This regulation of metabolism is mediated via phosphorylationdependent activation and inactivation of enzymes, transcription factors, co-activators and co-repressors.
Being central regulator of both lipid and glucose metabolism, AMPK is considered to be a potential therapeutic target for various diseases, including eye disorders. AMPK has also been proven to regulate ageing (known as pro-longevity kinase) in various species via its coordination with mTOR and sirtuins. 4 Although the role of an AMPK signalling pathway is well documented with reference to several diseases including type II diabetes mellitus, obesity and cancer, its role in the aetiopathogenesis of eye diseases is not highlighted yet. So, the present review is focused on the information available for AMPK signalling pathway and associated eye diseases. Furthermore, the information provided can be useful for the therapeutic intervention of various ocular disorders.

Significance of AMPK as a potential therapeutic target for different disorders
Because of the widespread and distinct expression of AMPK in almost all tissues, tissue-specific manipulation of AMPK signalling can be beneficial for a variety of disorders. Earlier studies have already established that activation of AMPK increases insulin sensitivity in type 2 diabetes. 5 During chronic inflammation AMPK activity decreases in response to LPS, TNF-α, high-fat diet in a variety of tissues such as adipose tissue, skeletal muscles and macrophages. With the subsequent decrease in LPS levels or diet-induced weightloss, AMPK activity increases. AMPK activity also increases in response to lipolytic flux induced by exercise or fasting. Pharmacological activation of AMPK inhibits proinflammatory cytokine production in macrophages and reduces insulin resistance. AMPK also inhibits the inflammation of adipose via the regulation of RAAS signalling. Therapeutic exploitation of these facts has already shown the potential of AMPK signalling for the treatment of many metabolic diseases such as obesity, atherosclerosis, non-alcoholic fatty liver disease and type 2 diabetes. 6,7 Owing to its central role in energy regulation and the inhibition of cell growth, the suppressive effects of AMPK in various cancers includes lung, liver, breast, prostate and colorectal cancers. 8,9 Activation of AMPK in astrocytes attenuates oxidative stress in response to hypoxia which suggests its anticipatory role in cerebral ischaemic/ hypoxic diseases. 10 AMPK activating agents such as resveratrol and atorvastatin have shown therapeutic potential in the treatment of stroke. Metformin-mediated AMPK activation induces VEGF expression, reduces the infarct size and boosts neurologic outcomes in vivo. Exposure of metformin following stroke induces angiogenesis to mitigate ischaemic injury via alteration of microglia/macrophage polarization. 11 Pharmacologic activation of AMPK plays a critical role in vascular calcification via induction of Runx2 signalling, eNOS signalling and autophagy. During the regulation of vascular smooth muscle cell differentiation, AMPK also attenuates ER stress and DRP1-mediated mitochondrial fission. 5

Structure of AMPK: Subunits and their functions
Both the Snf1 and AMPK are heterotrimers, containing a catalytic subunit and two regulatory subunits. In yeast, Snf1 itself is catalytic subunit which forms a complex with regulatory subunits Snf4 (orthologue of regulatory γ subunit of AMPK) and a member of Sip1/Sip2/Gal83 family of proteins (orthologue of regulatory β subunit of AMPK). 12 The catalytic α subunit and regulatory β subunit of AMPK each has two isoforms (α1, α2 and β1, β2, respectively), while γ subunit has three isoforms γ1, γ2 and γ3. 13 Different isoforms of PRKA gene encode each of these subunits, and when present in complex, they regulate metabolism variably in response to changing AMP, ADP and ATP levels. 14 Each combination of these subunits into a functional heterotrimeric complex is possible, which may function differently under different physiologic conditions. [15][16][17] α subunit possesses the catalytic activity and an activating amino acid Thr172 within its activation loop. AMPK is also activated by binding of AMP and or ADP to the cystathione-β-synthase domains present in the γ subunit. However, the binding of ATP to γ subunit competitively inhibits binding of both the ADP and AMP. Thus, AMPK senses the alterations in the fractions of ATP/AMP and ATP/ADP, not the total nucleotide levels. 18

Activation of AMPK by super kinases
The super kinases activate AMPK via phosphorylation of Thr172 in α subunit. The three known AMPK super kinases are LKB1, CAMKK2 and TGFβ-activated kinase (TAK1/ MAP3K7). 19,20 LKB1 or STK11, a serine-threonine kinase, is a tumour suppressor which explains its central role in metabolism and tumorigenesis. LKB1 is the major kinase that activates AMPK and AMPK-related kinases under the conditions of energy deprivation, low ATP levels and during mitochondrial malfunctioning. Inactive LKB1 remains localized to the nucleus, and upon low energy conditions, its cytoplasmic translocation triggers the formation of the heterotrimeric complex with its regulatory partners MO25 and STRADα. This complex then induces phosphorylation-dependent activation of AMPK and other AMPK-related kinases. 19,21 LKB1-AMPK regulates mTOR signalling, the central pathway involved in cell growth regulation. AMPK and mTOR have opposing effects; AMPK stimulates catabolism, whereas mTOR stimulates anabolic reactions. When energy levels are low, AMPK phosphorylates tuberous sclerosis complex 2 (TSC2) (tumour suppressor) which is an upstream regulator of mTORC1 and also facilitates subsequent inhibitory phosphorylation of Thr479 in α subunit by GSK3. 22 Phosphorylation of Thr479 in α subunit by GSK3 leads to decreased AMPK kinase activity. TSC2 inhibits mTORC1 and glycogen synthesis. 23 A component of mTORC1, Raptor is also phosphorylated by AMPK at two conserved sites (Ser722 Ser792) which inhibits mTORC1 activity. LKB1 and AMPK both stimulate p53 activity. 24 Sestrin2, the downstream target of p53 also activates AMPK, inhibits mTORC1 and induces apoptosis. 25 CAMKK2 (Ca 2+ /CaM-dependent kinase kinase 2), another super kinase activates AMPK in Ca 2+ -dependent manner. Ca 2+ act as a second messenger (ligand), and bind to its receptor calmodulin (CaM). In cells deficient in LKB1 or conditions of energy starvation, a flux of calcium ions either from intracellular storage or extracellular space saturates the CaM receptors. This induces a conformational change in CaM receptors and activates downstream target proteins, Ca 2+ /CaM-dependent kinases. These are further phosphorylated by CAMKK2 which phosphorylates Thr172 in α subunit of AMPK. The resulting complex of Ca 2+ /CaM, CAMKK2 and AMPK α and β subunits activates effector proteins required for cellular homeostasis. 26 However, regulatory γ subunit (senses AMP: ATP ratio) is not required for CAMKK2-mediated activation of AMPK. This explains that the intracellular Ca 2+ -dependent AMPK activation by CAMKK2 is independent of imbalance in AMP: ATP ratio. Previously, it was shown that CaMKK2 forms a stable complex with AMPK α and β subunits in the absence of γ subunit. 27 However, later it was reported that AMPK activation by CamKK2 occurs without the formation of the stable complex. 28 Another serine/threonine kinase, TAK1 (transforming growth factor-activated kinase 1), from the mitogen-activated protein kinase kinase kinase family also activates AMPK. TAK1 forms a heterotrimeric complex with TAK1-binding proteins -TAB1/2/3. Activation of TAK1 is mediated by interleukin-1, TGFβ, TNF-α, TLR and BCR. TAK1 regulates cell viability and survival via activation of MAPKs -Erk, p38/MAPK and JNK. Under starvation and autophagy, TAK1 activates AMPK and IκB kinase complex (IKK). TAK1 activates NF-κB via phosphorylation and stimulation of IKK complex and subsequent degradation of IκB. 29,30 Activated NF-κB and MAPKs then elevates the levels of inflammatory cytokines as well as anti-apoptotic proteins and represses caspase cascade. TAK1-AMPK signalling is activated in response to extracellular signal such as TRAIL leading to either autophagy or apoptosis depending on the cell types. 31 TAK1 regulates VEGF-, TNFα-and IL-1β-induced activation of AMPK and kinase cascade (p38, JNK, Erk1/2, Akt) by upregulating SOD2 in endothelial cells during angiogenesis. 32 Three phosphatasesprotein phosphatase 2A, protein phosphatase 2C and Mg 2+ /Mn 2+ -dependent protein phosphatase 1Ealso mediate regulation of Thr172 phosphorylation. 18 Under energy replete conditions, GSK3mediated (negative regulation of AMPK) inhibitory phosphorylation of Thr479 and subsequent inhibition of AMPK activity is exclusively due to increased phosphatase sensitivity towards Thr172. GSK3-induced AMPK inhibition is regulated by insulin/PI3K/Akt pathway, 23 and under serumstarvation, inhibition of GSK3β triggers autophagy via the LKB1-AMPK pathway. 33 Downstream targets of AMPK Downstream targets of AMPK include those implicated in protein metabolism, fatty acid biosynthesis, β-oxidation and carbohydrate metabolism (Figure 1), thereby controlling processes such as autophagy, proliferation, migration, apoptosis and transcription. Activated AMPK increases glucose uptake by inducing membrane translocation of GLUT4. AMPK inhibits glycogen synthase through phosphorylation (Ser7) and decreases the rate of glycogen synthesis. The increased glycolytic flux activates phosphofructo-2-kinase (Ser466), which stimulates glycolysis. [34][35][36] AMPK stimulates fatty acid oxidation via phosphorylation of Acetyl-CoA carboxylase 1 (ACC) and 2 (Ser79 and Ser212, respectively). Activation of ACC reduces fatty acid flux towards lipid synthesis and more towards mitochondrial β-oxidation. 37 AMPK controls cell growth and protein synthesis mainly by negative regulation of mTORC1. Under energy replete state, mTORC1 impedes autophagy by suppressing ULK1 and ULK2. AMPK inhibits mTOR/S6K1 (S6 kinase1) via phosphorylation of TSC2 and Raptor. 24,38 Through inhibition of mTORC1, AMPK inhibits anabolic reactions and promotes autophagy by phosphorylation (Ser317 Ser777) and activation of ULK1. 39 AMPK signalling is also integrated with the PI3K/ Akt/GSK3 pathways. Activation of AMPK elevates Akt phosphorylation (S473) by regulating PI3K and increases levels of PIP3, thereby increasing insulin sensitivity. 40 AMPK regulates transcriptional expression through stimulation of HIF-1α nuclear translocation and interaction with transcription factors as well as histone deacetylases (SIRT1, HDAC5). 41 AMPK activates many transcription factors and co-activators such as PGC1α, CREB, and FOXO in phosphorylationdependent manner. AMPK stimulates G1 arrest and controls cell cycle through direct phosphorylation and stabilization of p53 (Ser15). Under conditions of nutrient stress, AMPKdependent phosphorylation of p53 at Ser46 leads to apoptosis. 42 AMPK regulates cell proliferation and differentiation by inducing the phosphorylation of retinoblastoma protein (Ser804). 43 Under glucose depletion, AMPK activation reduces RNA pol I activity through phosphorylation of Pol Iassociated transcription initiation factor (TIF-1A) at Ser635. 44 Role of AMPK-associated pathways

Regulation of mitochondrial biogenesis
The alteration in mitochondrial morphology, function or mitochondrial DNA in neural retina, retinal ganglion cells (RGCs) or retina pigment epithelial (RPE) cells influence mitochondrial bioenergetics and has been linked to the pathophysiology of many ocular diseases including diabetic retinopathy (DR), age-related macular degeneration (ARMD) and glaucoma. 45,46 Therefore, regulation of mitochondrial biogenesis is critical to maintaining a normal mitochondrial network. In vivo stimulation of AMPKα2 protects photoreceptors and RPE cells against acute photoinjury and inherited retinal degeneration by increasing mitochondrial biogenesis. 47 AMPK regulates mitochondrial biogenesis via peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α) activation. PGC1α and PGC1β are both expressed in different compartments of the murine retina, highest in photoreceptors and the former being a negative regulator of oxidative stress-induced senescence in RPE cells and subsequent retinal degeneration. 48,49 PGC1α knockout mice show abnormalities in RPE cells which is linked to elevated senescence and differential expression of genes related to cell death and damage repair. Overexpression of PGC1α elevates expression of electron transport chain, fatty acid β-oxidation and antioxidant genes. 50 PGC1α function as a co-activator of nuclear receptors and nuclear respiratory factors (NRF1 and NRF2) which induce the expression of mitochondrial transcriptional factors (TFAM, TFBM1, TFBM2). A study has revealed the correlation of diminished mitochondrial DNA copy number in the diabetic retina owing to diminished mitochondrial transport of TFAM despite its increased expression in the nucleus as a primary mechanism of pathogenesis of DR. 51 Activation of PGC1α requires direct phosphorylation by AMPK and deacetylation by SIRT1. AMPK enhances SIRT1-mediated deacetylation of PGC1α and forkhead transcription factors (FOXO1 and FOXO3) by increasing NAD + /NADH ratio. 49,52 Therefore, during energy stress or mitochondrial insult, increased AMP/ATP ratio will activate AMPK, which stimulates PGC1α activity to increase mitochondrial gene expression. SIRT1 overexpression deacetylates inhibitory lysine residue on LKB1 and increases its activity, simultaneously activating downstream targets of LKB1 (AMPK and MARK1) in retinal endothelial cells. 53,54 The strict regulation of AMPK/ SIRT1/PGC1α pathway controls mitochondrial activity, and its repression in RPE cells can lead to generation of oxidative stress and subsequent retinal degeneration. 55 p38/ MAPK, Akt and GSK3β can also regulate PGC1α under different physiologic conditions. 56,57 GSK3β, a repressor of NRF2, phosphorylates and inhibits its nuclear translocation. Activated Akt inhibits GSK3β, thus facilitating the translocation of NRF2 and its binding to NRF1 promoter. This binding activates TFAM involved in mitochondrial DNA replication. 58 Intense physiological light-induced lysosomal degradation of photoreceptor outer segments in ARPE-19 cells is mediated by PGC1α overactivation. Overactivation of PGC1α up-regulates VEGF secretion and enhances choroid neovascularization through PGC1α/ERRα pathway in vivo. 59 Silencing of PGC1α in mammalian RPE triggers loss of epithelial integrity, induces epithelial-mesenchyme transition (EMT) and causes disorganization of outer retina complex and retinal degeneration. 60

Regulation of autophagy
Light-induced autophagic response in RPE cells protects against oxidative damage and subsequent retinal degeneration. 61 Autophagy slows down senescence, and inhibition of mTORC1 could delay the age-associated changes in RPE cells. 62 Differential expression of autophagy-associated genes and decreased autophagic reactivity accompanies the development of ARMD-like retinopathy in ageingaccelerated rat model. 63 These differentially expressed genes include those involved in immune response, kinase activity, mTOR, MAPK, PI3K-Akt, AMPK and FOXO signalling. mTORC1 and AMPK activate autophagy under nutrition starvation and energy stress, respectively. Under low glucose conditions, AMPK triggres autophagy by stimulating autophosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) (Ser317 Ser777). ULK1 is upstream kinase in autophagy initiation and direct substrate of mTORC1. mTORC1 phosphorylates inhibitory Ser757 residue on ULK1 and inhibits this process under energyefficient conditions. AMPK-mediated phosphorylationdependent inhibition of mTORC1 via phosphorylation of TSC2 (Thr1269 Ser1387) and RAPTOR (Ser722 Ser792) augments the reaction between ULK1 and AMPK. 64,24 During ER stress, cytosolic Ca 2+ flux activates AMPK by CAMKK2, triggering activation of autophagy through inhibition of mTORC1 and phosphorylation of ULK1, VPS34 and Beclin1. 65,39 AMPK phosphorylates Beclin1 and VPS34, which is class III phosphatidylinositol-3 kinase (PI3K) together with other autophagy-specific proteins to activate pro-autophagy complex. 66 ULK1-mediated phosphorylation of Beclin1 elevates the activity of this complex and induces autophagy. 67 Beclin deficiency causes lightinduced RPE degeneration in vivo due to oxidative stress-mediated damage of endoplasmic reticulum and mitochondria. 61 AMPK regulates autophagy at the transcriptional level by increasing co-activator associated arginine methyltransferase (CARM)-dependent H3 Arg17 dimethylation on autophagy-associated genes through transcription factor EB. 68 MEK/Erk and PI3K/Akt signalling have also been reported to regulate autophagy. 69,70 In vitro hypoxic conditions activate autophagy with the upregulation of autophagy-associated proteins Atg3, Atg5, LC3B-II and Beclin1 in murine retinal microvascular endothelial cells. This hypoxia-induced autophagic response is regulated by AMPK/mTOR and PI3K/Akt/mTOR pathways. 71 Hypoxia-induced autophagy is a crucial process in the progression of ischemic retinopathies, where retinal endothelial cells (RECs) are susceptible to hypoxiainduced oxidative stress. However, autophagy seems to have different roles in response to different ocular conditions. In both the glaucomatous and diabetic retina, an autophagic response is activated in RGCs. In diabetic retina, expression of LC3B-II increases via AMPK signalling leading to cell survival. In glaucomatous retina, Beclin1 and LC3B-II expression increases in response to IOP elevation resulting in apoptosis. 72

Regulation of inflammatory response
Inflammation is often present in different parts of the eye, including ocular surface, cornea, conjunctiva, uvea, sclera, retina and optic nerve. Inflammation is a critical process in the aetiopathogenesis of the ocular disorders such as DR, retinal vein occlusion, ARMD and uveitic glaucoma. During retina inflammation, AMPK activity decreases and inflammatory transcription factor NF-κB expression increases in vivo. 73 AMPK attenuates the inflammatory response through various signalling cascades. AMPK exerts anti-inflammatory effects through activation of SIRT1. Knock out of SIRT1 leads to enhanced STAT3/Erk phosphorylation and inflammation in mice. 74 SIRT1 reduces acetylation of c-Jun/c-fos transcription factors and inhibit phosphorylation of JNK, p38 and Erk1/2. 75 SIRT1 also inhibits NF-κB p65 through inhibitory deacetylation of Lys310 residue and reduces nuclear translocation of NF-κB p65 in activated macrophages. It also decreases IL-1β expression in mice model of inflammatory pain. 76 AMPK increases inhibitory phosphorylation of its substrate IKK (Ser177 Ser181) followed by inhibition of IκB (Ser32 Ser36) and p65 (Ser536) phosphorylation in endothelial cells. Phosphorylation of IκB is required for its degradation, which induces activation and nuclear translocation of NF-κB. 77,78 PI3K/Akt/GSK3β and JAK/STAT3 signalling mediates IL-10 dependent anti-inflammatory action of AMPK. To limit inflammatory response, IL-10 rapidly activates AMPK which then activates PI3K p55, Akt and JAK1 in macrophages. Akt increases mTORC1 activity in activated macrophages which mediates phosphorylation/ activation of STAT3 (Tyr705 Ser727). 79,80 In vitro activation of AMPK decreases STAT1 activity and inhibits vascular inflammation through up-regulation of MAPK phosphatase 1. Inhibition of inflammation is thought to occur due to diminished levels of pro-inflammatory cytokines. 81 Complement factor-B is a component of an alternative pathway having pivotal role in inflammation and pathogenesis of ARMD. Pharmacological AMPK activation attenuates pro-inflammatory cytokine-induced complement factor-B levels in human RPE cells. 82 AMPK and ocular diseases AMPK and its associated signalling pathways present a central role in the aetiopathogenesis of ocular diseases by affecting various ocular tissues such as retina, lens, cornea and uvea. AMPK activation or inhibition impacts pathological processes such as oxidative stress, apoptosis, inflammation, degeneration and neovascularization. Variety of phytochemicals and pharmacologic agents (Table 1) interferes with AMPK signalling and impedes these pathological processes.

Glaucoma
Glaucoma is an optic neuropathy disease, occurring due to the gradual death of RGCs through apoptosis. Although slow progressive, if left untreated, it damages the optic nerve and leads to irreversible vision loss. Degeneration of optic nerve involves cupping of optic disc and loss of axon and retinal ganglion cell body. With the increasing global prevalence, it has been estimated that the number of affected people will be approximately 111.8 million in 2040. 83 Multiple factors, including increased intraocular pressure, vascular abnormalities, oxidative damage, ischaemia, excitotoxicity and inflammation, contribute to the pathogenesis of glaucoma. 84 Phosphorylated AMPK levels increase in optic nerves of patients with glaucoma. Increased activation of AMPK leads to synaptic elimination and dendrite retraction in RGCs. 85,86 High deposition of extracellular matrix (ECM) components (fibronectin, collagen, matrix metalloproteinases) and TGFβ2 has also been observed in mammalian glaucomatous trabecular meshwork (TM) cells and aqueous humour of open-angle glaucoma patients, respectively. 87,88 AMPK activation with AICAR has known to down-regulate cytoskeletal and ECM proteins (F-actin, collagen I/IV, laminin) through RhoA phosphorylation (Ser188) in primary human TM cells. RhoA is a downstream target of AMPK that when unphosphorylated induces ECM deposition through RhoA/ROCK pathway. 89 Ocular hypertension also increases AMPK activation and lead to loss of RGCs. 85 It has been reported that with increased intraocular pressure (IOP), energy levels often get depleted in the optic nerve of mice model. 90 During energy stress, AMPK activation induces an inflammatory response in optic nerve and retina in mice model of glaucoma. AMPK induces NF-κB p65, facilitating the expression of proinflammatory cytokines. 91 A study has reported that with increasing IOP spatio-temporal expression of MAPKs (p38, p42/44, SAPK/JNK) in retina and optic nerve contributes to pathogenesis of glaucoma. 92 During oxidative stress, Akt and Erk phosphorylation increases in TM cells in vitro. 93 Elevated IOP and intravitreal inactivation of PI3K/Akt and JAK/STAT pathways decrease the survival (increases the loss) of RGCs in the mammalian retina. 94 PI3K and Akt activation inhibit the apoptosis of glaucomatous human TM cells. 95 Silibinin protects RGCs against photo-induced apoptosis in vitro. It down-regulates inflammatory cytokines through the activation of the MEK/Erk/CREB pathway. 96 Reduced activated Akt levels in glaucomatous retina and optic nerve leads to an elevation in MMP1/2/3 production in rat model. Phosphorylation-dependent activation of Akt protects RGCs through inhibition of TNF-α mediated MMP production. 97 Metformin has been reported to protect against vision loss and promote RGC survival via AMPK activation. Metformin reduces the odds of developing open-angle glaucoma in diabetic patients by 25%. 98

Diabetic retinopathy
Diabetic retinopathy (DR) has known to occur due to retinal vascular dysregulation as a complication of diabetes. The primary step in the pathophysiology of DR is microvascular damage due to hyperglycaemia followed by endothelial proliferation, neovascularization, vitreous haemorrhage and diabetic macular oedema leading to visual impairment. 99 A meta-analysis study has reported the prevalence of DR to be 42% in type 2 diabetes patients from the Indian population. Also, the prevalence of proliferative DR is higher in the Indian population among other populations of Asia. 100 During the progression of DR, energy levels get diminished in the hypoxic retinal environment, which stimulates AMPK activation. Activation of AMPK and ACC increases in the retina of DR rat model. 101 AMPK activation prolongs survival of photoreceptor via regulation of autophagy and mitochondrial function in vivo. 102,103 Hyperglycaemic conditions enhance O-linked N-acetylglucosamine-mediated modification of NF-κB p65 in DR. The activated NF-κB increases apoptosis of RGCs. α-lipoic acid, a natural antioxidant, rescues apoptosis in RGCs by attenuation of interaction between AMPK and O-linked β-N-acetylglucosamine transferase. This is accomplished by increased PPARγ and SIRT3 activation through AMPK signalling. 104,105 Microvascular damage in retina induces disruption of blood-retina barrier leading to diabetic macular oedema. Cytokine such as IL-1β causes imbalance of junctional proteins (claudin-1) via AMPK activation, which contributes to increased permeability of ARPE-19 cells. Fenofibrate, a PPARα agonist, attenuates imbalance in junction proteins and cytokine-induced hyperpermeability in ARPE-  106,107 Fenofibrate maintains RPE permeability through down-regulation of AMPK expression as well as NF-κB p65 translocation and also down-regulates inflammatory cytokine levels. 108 In contrast, sodium tanshinone IIA silate (STS, a diterpenoid derivative) attenuates decreased phosphorylation levels of AMPK and decreases paracellular permeability under high glucose conditions in ARPE-19 cells. This STS-mediated activation of AMPK increases phosphorylation of p300 histone acetyltransferase (Ser89). Thus, acetylation activity of p300 and subsequent activation of NF-κB p65 (Lys221) decrease, inhibiting its nuclear translocation. 109 Under hyperglycaemic and hypoxic conditions, p38 MAPK activation induces RPE barrier disruption. It contributes to the alteration of junction proteins expression. An ECM proteoglycan, Decorin down-regulates VEGF and HIF-1α expression in ARPE-19 cells and suppresses angiogenesis in RECs. It prevents the disruption of RPE monolayer by down-regulating the activation of p38. 110,111 AMPK activity decreases in the retina with the hyperglycaemia-induced caspase-3 activity. Overexpression of AMPK with AICAR decreases apoptosis of muller cells. 112 Metfrmin improves muller cell dysfunction via regulation of the components of retinal clock in AMPK-dependent manner in diabetic mice model. 113 miR-29 has been known to regulate AMPK/mTOR signalling, and increased apoptosis in DR. AMPK inactivation up-regulates mTOR phosphorylation accompanied by reduced miR-29 expression and decreased apoptosis in the affected retina of mice model. 114 Up-regulation of VEGF contributes significantly to angiogenesis and development of both proliferative and nonproliferative DR. Upon knockdown of AMPK, mouse embryonic fibroblasts show increased expression of VEGF, PDGF, and MMP-9 and increased migratory capacity in higher glucose conditions. Resveratrol and AICAR increase SIRT1/AMPK activation and subsequent NF-κB phosphorylation. Oral administration of resveratrol reduces leukocyte adhesion in the diabetic retina via downregulation of VEGF and ICAM, thereby suppressing inflammatory response. 115 The thickness of retinal layers decreases in the diabetic retina with the up-regulation of VEGF expression and apoptosis of RGCs. α-lipoic acid inhibits thinning of the retina through its antioxidant properties. Long-term administration of α-Lipoic acid decreases levels of 8-OHdG in the retina by suppressing VEGF expression and activation of NF-κB. 116,117 Metformin down-regulates inflammatory cytokines (ICAM-1 and MCP-1) through AMPK activation and inhibits angiogenesis in RECs. Pre-treatment with metformin suppresses intraretinal neovascularization and leukocyte adhesion in retinal vessels of diabetic mice model of retinal angiogenesis. 118 Akt and Erk signalling pathways have been linked to the apoptosis of retinal neuron cells. 119 During high-fat diet-induced DR phosphorylation levels of cellular kinases, Akt and AMPK decrease while pErk levels decrease in vivo. Administration of metformin reverses this diminished activation of kinases. It also downregulates cytokines (IL-6, IL-12, MCP-1 and VEGF) through depletion of p65 activation and suppresses retinal inflammation. However, administration of metformin during the progression of DR does not protect mice from the decreased retinal light response and neovascularization. 120 AMPK activation has also been correlated with the antiinflammatory effect of cartonectin in DR progression. Adipokine cartonectinin (CTRP3) has been reported as a novel screening biomarker for DR. CTRP3 levels decreases in the serum of DR patients with increasing VCAM1 (vascular cell adhesion molecule 1) levels which induces inflammation. 121 Artesunate shows protective effects against increase in retinal permeability and inflammation in diabetic rat model which is mediated via AMPK/SIRT1 signalling. 122 Lipids are known to elevate oxidative stress and apoptosis in retinal microvascular pericytes. Among fatty acids, palmitate but not oleate increases ceramide and diacylglycerol accumulation in retinal pericytes. Elevated levels of ceramide contribute to the induction of apoptosis by increasing NF-κB and Bax activity. AICAR-mediated activation of AMPK prevents palmitate-induced apoptosis by down-regulation of serine palmitoyltransferase and lowering ceramide and diacylglycerol levels, but it does not inhibit oxidative stress. 123 However, AMPK activation with resveratrol inhibits oxidative stress-induced apoptosis via AMPK/SIRT1/PGC1α signalling in hyperglycaemic conditions. 124 Hyperglycaemia increases apoptosis in RECs via generation of oxidative stress and decreases AMPK, SIRT1 and PGC1α expression while resveratrol and AICAR attenuate the modulation in their expression. AMPK activation has also been implicated in cytokinesand growth factor-induced (TGFβ, PDGF, VEGF, TNF-α) EMT and aggregate formation ARPE-19 cells via modulation of cellular signalling pathways. AMPK activation with AICAR attenuates altered expression of E-cadherin, fibronectin and α-smooth muscle actin. It also reduces levels of MMP2/9, IL-6 and VEGF through modulation of mTOR and MAPK (Erk, JNK, p38) pathways and suppresses EMT as well as aggregate formation in vitro. 125 Resveratrol and lycopene diminish ARPE-19 cell migration by reducing phosphorylation of PI3K, Akt, Erk and p38 and inhibition of PDGF receptor β in response to PDGF. 126,127 Crocetin also decreases phosphorylation of MAPKs (Erk, p38 and JNK) and decreases PDGF-induced ARPE-19 cell migration and proliferation. Crocetin inhibits ARPE-19 cell proliferation by G1 arrest via up-regulation of p21 in a p53-dependent manner. It inhibits TGFβ2-induced EMT by up-regulation of E-cadherin and ZO-1 and down-regulation of vimentin and α-SMA. It does so by decreasing phosphorylation of p38. 128,129 Lutein suppresses PDGF-induced RPE cell migration via inhibition of mitochondrial translocation of phospho-Akt. 130 Lutein also improves mitochondrial biogenesis in RPE cells via AMPK-mediated up-regulation of PGC1α, TFAM and NRF-1 in vitro and in vivo high glucose conditions. 131 Studies have reported impaired retinal blood flow due to microvascular damage in DR. 132,133 Pioglitazone instigates dilation of retinal vessels via PPAR-γ dependent production of nitric oxide (NO) in endothelial cells. Pioglitazone induces phosphorylation of endothelial nitric oxide synthase through AMPK, guanyl cyclase/cGMP and PI3K/Akt/ eNOS pathways in RECs. 134 Fenofibrate and adiponectin have shown vasodilatory properties through a similar mechanism. 135,136 Cilostazol also induced dilation of retinal arteriole via endothelium-dependent NO release and endothelium-independent K channel activation by cAMP/ PKA and AMPK pathways. 137 High glucose promotes angiogenesis by inducing the proliferation of endothelial cells via up-regulation of proangiogenic factors from muller cells. 138 Berberine protects muller cells against apoptosis via activation of the AMPK/ mTOR signalling in response to hyperglycaemia. It ensures cell survival via autophagy through up-regulation of Beclin-1 and LC3II. It also dampens the levels of the pro-apoptotic factor (Bax) and elevates that of anti-apoptotic factor (Bcl-2) via AMPK activation. 139 Berberine protects muller cells from oxidized low density lipoprotein (LDL)-induced apoptosis and autophagy through AMPK activation. It down-regulates oxidized LDL-induced expression of glial fibrillary acidic protein and also inhibits angiogenesis and inflammation by down-regulation of inflammatory cytokines. 140

Age-related macular generation
Pathological changes during the progression of ARMD involve degeneration of rods and cones owing to degeneration of RPE cells. Gradual deterioration of RPE pigmentation followed by drusen accumulation, geographic atrophy and choroidal neovascularization increases the degeneration of RPE and photoreceptors leading to vision loss. 141 According to a meta-analysis study, approximately 196 million people will be affected by ARMD in 2020 and will reach up to 288 million by 2040. 142 Increased production of inflammatory cytokines causes retinal inflammation leading to drusen formation. Accumulation of drusen increases the risk of developing ARMD. During retina inflammation rhodopsin levels and length of photoreceptor outer segments decrease contributing to impaired visual functions. pAMPK levels decrease in the inflamed neural retina with the decreased visual functions by photoreceptors and inner retina. Lower AMPK activity along with perturbations in other pathways in ARMD RPE compared to normal RPE contribute the pathogenesis of ARMD. 143 AMPK activation with AICAR decreases NF-κB p65 activation and suppresses rod photoreceptor damage. AMPK attenuates inflammatory response and impaired visual functions by increasing rhodopsin levels and length of outer segments. 73 During retinal inflammation-mediated cone photoreceptors dysfunction, PGC1α and pAMPK levels get depleted in vivo. AMPK activation with AICAR decreases TNF-α level and increases PGC1α in cone cells. AICAR also decreases glial fibrillary acidic protein expression in inflamed retina. 144 Increased activation of PI3K/Akt/mTOR pathway stimulates dedifferentiation of RPE cells which leads to photoreceptor degeneration. Rapamycin dependent inhibition of this pathway suppresses these changes in vivo. 145 Loss of PGC1α and NRF-2 both shows disorganization of Bruch's membrane, drusen-like deposits, damaged mitochondria, impaired autophagy in RPE and increased size of RPE cells in mice. This double knockout mice also shows ARMD-like phenotypes such as decreased rod cell function as well as thinning of outer nuclear layer and photoreceptor degeneration. 146 Metformin has shown good potential for the treatment of ARMD by targeting multiple pathological process such as inflammation, cell death and drusen formation. 147 In vivo activation of AMPK with metformin has shown to safeguard photoreceptors against light-induced degeneration. Intravitreal administration of metformin attenuates oxidative DNA damage and induces mitochondrial biogenesis via elevation in expression of PGC1α, TFAM, NRF1 and COXII in the retina. It also prevents the degeneration of rods and cones in the mice model of inherited retinal degeneration. 47 A retrospective study conducted by the researchers based in Florida has reported a reduced risk of ARMD development in patients who use metformin. 148 Oxidative stress and inflammation have a profound effect in degeneration of RPE. 149 Malondialdehyde (MDA) levels increase in the eye of ARMD patients. High dietary intake of linoleic acids increases MDA levels in mice's eyes. Dietary intake, as well as intravitreal administration of MDA, increases autophagy and choroidal neovascularization volumes in mice eyes. MDA impairs VEGF expression, disrupts cell junctions and causes autophagy dysfunction in RPE of ARMD patients. 150 Resveratrol decreases choroidal neovascularization volumes in mice model of ARMD by attenuation of decreased AMPK activation and increased NF-κB in RPE/choroid. It upregulates the levels of antioxidant enzymes and reduces the levels of inflammatory cytokines including VEGF, MCP-1 and ICAM1. 151 In vivo administration of curcumin suppresses TNF-α, MCP-1 and ICAM1 expression in RPE via inhibition of NF-κB and HIF-1α. 152 Astaxanthin also brings about similar changes via down-regulation of VEGFR1 and VEGFR2 and NF-κB activation in vivo. 153 Genipin, delphinidin, astaxanthin, piceatannol and a triterpenoid RTA 408 protect RPE cells from oxidative damage-mediated apoptosis through activation of NRF2 and its downstream targets including phase II metabolic enzymes. [154][155][156][157][158] Berberine inhibits apoptosis in D407, RPE cells in vitro by activation of AMPK and inhibition of caspase activities. 159 Artemisinin reduces apoptosis in D407 cells by restoring the loss of mitochondrial membrane potential and increased phosphorylation of AMPK, Erk (Thr202 Tyr204) and CREB (Ser133) in response to oxidative stress. 160,161 Artemisinin also inhibits apoptosis in RGC-5 cells via activation of p38 and Erk1/2, thereby protecting against photoinduced retinal damage in rats. 162 α-melanocyte-stimulating hormone (MSH) increases phosphorylation-dependent activation of Erk (Thr202 Tyr204), Akt (Ser473) and mTOR (Thr389) through MSH receptor in ARPE-19 cells. It protects cells against reactive oxygen species-induced apoptosis and increases cell survival through Akt/mTOR pathway. 163 Escin (triterpene of saponins) protects RPE cells from oxidative damage and apoptosis through Akt phosphorylation (Ser473 Thr308). Activated Akt then phosphorylates NRF2 (Ser40) facilitating its nuclear translocation and expression of phase II enzymes. 164 Cigarette smoke has known to increase oxidative stress and lipid peroxidation. It also induces lipid accumulation, ER stress as well as expression of VEGF in RPE cells. Morin hydrate attenuates these smoke-induced changes in RPE through activation of AMPK-NRF2 signalling. 165,166 UV-and H 2 O 2 -induced AMPK activation has been associated with RPE apoptosis. H 2 O 2 stimulates ceramide production in ARPE-19 cells leading to ER stress (phosphorylation of PERK and eIF2α), phosphorylationdependent activation of AMPK as well as ACC (Ser79) and inhibition of mTOR (S6). It also phosphorylates and activates MAPKs (p38, JNK, Erk) and induces apoptosis. Inhibitors of ceramide synthesis, AMPK activation and ER stress attenuate the apoptosis of ARPE-19 cells. 167 Another AMPK activator PF-06409577 inhibits UV-induced oxidative stress and apoptosis in ARPE-19 cells by stimulation of AMPK and ACC phosphorylation. 168 Besides, it also activates NRF2-dependent phase II genes in primary murine RPE cells.
Expression of genes related to mitochondrial fission and fusion (Mfn2, Oma1 and Fis1) gets imbalanced in RGCs of the ageing zebrafish retina. Levels of PINK1 and LC3B-II/ LC3B-I decreases in the ageing retina, indicating decreased mitophagy and autophagy compared to young retina. This response is mediated by increased phosphorylation and activation of Akt (Thr308 and Ser473), and mTOR. Resveratrol improves the expression of these genes and mitochondrial integrity in zebrafish retina but not mitochondrial DNA copy number. Resveratrol activates AMPK, SIRT1 and PGC1α, thereby decreasing mTOR phosphorylation in the ageing retina. 169 EMT of RPE cells is also associated with the pathogenesis of ARMD. In vitro knock out of PGC1α in RPE cells leads to mitochondrial dysfunction, down-regulation of autophagy-related genes and oxidative stress. These cellular changes lead to depletion of AMPK levels and upregulation of EMT markers. Knockout of PGC1α in vivo also causes loss of epithelial phenotype, EMT of RPE and retinal degeneration. 60 Increase in RPE of lipofuscin or its component di-retinal conjugate N-retinylidene-N-retinylethanolamine (known as A2E) induces apoptosis and modulates cell junctions and paracellular permeability in RPE cells. A2E, along with blue light damages fundus, causes thinning of the retina and impairs retinal neuron transduction in rats. 170 In vivo accumulation of A2E in RPE induces choroidal neovascularization via retinoic acid receptor interaction and VEGF up-regulation. 171 Upon blue light stimulation, A2E also elevates ER stress-mediated apoptosis of ARPE-19 cells via up-regulation of glucose-related protein 78(GRP78). GRP78 enhances autophagy activation by AMPK/mTOR signalling. GRP78 increases mTOR activation and inactivation of AMPK in A2E-burdened blue light stimulated ARPE-19 cells. 172 A2E is a precursor of alltrans-retinal. Excessive all-trans-retinal in RPE also induces loss of mitochondrial membrane potential and ERstress mediated apoptosis of RPE cells and may lead to retinal degeneration. 173 Paeoniflorin (monoterpene) inhibits all-trans-retinal induced mitochondrial dysfunction, ER stress and apoptosis by increasing phosphorylation of CAMKK2 and AMPK activation. 174 LB-100, an inhibitor of protein phosphatase 2A, suppresses oxidative damage by activation of NRF2 and AMPK-ACC phosphorylation in RPE cells. 175 Keratinocyte growth factor also counteracts UV-induced RPE cell damage through activation of Akt-mTOR-NRF2 signalling via KGF receptor interaction. 176 3H-1,2-dithiole-3-thione (dithiolethione from vegetables) shows similar effects in RPE cells through activation of Akt-mTORC1 pathway. 177

Corneal diseases
During the pathogenesis of corneal endothelial dystrophies and corneal oedema, cellular metabolism and ATP levels often get depleted in corneal endothelial cells. Reduced energy and metabolism lead to reduction in mitochondrial membrane potential and mitochondrial density. Subsequent loss of Na + -HCO 3 À transporter activates cell survival and mitophagy increases activation of AMPK and p53 (Ser15). 178 LKB1 deletion in mice epidermis causes hyperkeratinization of corneal epithelium and corneal opacity. 179 Corneal limbal epithelial cells are dependent on glycolysis due to less number of mitochondria. Their energy demands are met by the expression of GLUT1, whose expression increases during corneal wound healing. 180 During energy stress, GLUT1 expression requires AMPK activation (in hepatocytes). 181 Increased oxidative stress, thinning and loosening of the corneal epithelium occur in the diabetic dry eye mice model. SIRT1 and FOXO3 expression decrease in dry eye and increases in diabetic dry eye. 182 Desiccation stress induces autophagy through upregulation of LC3, LAMP1 and Akt/mTOR pathway. Chloroquine and trehalose deplete production of inflammatory cytokines as well as MMP-9 and NF-κB p65 in vitro dry eye disease model. Both of these compounds also attenuate increased phosphorylation of ERK (Thr180/ Thy204) and p38 (Thr180/Thy182), whereas only chloroquine increased phosphorylation of Akt (Ser473) and p70S6 kinase (Thy389), suggesting mTOR/Akt dependent activation of autophagy by chloroquine. 183,184 Hyperosmotic conditions in dry eye disease induce apoptosis of corneal epithelial cells. Substance P suppresses apoptosis through phosphorylation of Akt and increasing the antioxidant capacity of cells. 185 Elevated autophagy and mitophagy during corneal endothelial dystrophy have been known to occur due to phosphorylation-dependent activation of AMPK. 186 Differential expression of autophagy-related genes (ATG5, ATG7, p62, LC3B, LAMP1) was observed in cone-specific and peripheral areas of corneal epithelium from different grades of keratoconus patients. Oxidative stress conditions further impair autophagy in corneal epithelial cells as well as in keratoconus epithelium by decreasing phosphorylation of Akt (Ser473) and p70S6 kinase (Thr389). 187 Both of these are upstream and downstream kinase of AMPK and mTOR, respectively, suggesting the implication of Akt/mTOR signalling in oxidative stress-mediated autophagy deregulation. Raptor-mediated mTOR inhibition also induces autophagy in type 2 granular corneal dystrophy fibroblasts. 188 Mitochondrial structure and number get affected with the alteration in mitochondrial membrane potential accompanied by reduced expression of PGC1α, pAKT (Ser473 and Thr308) and mTOR (Ser2481) in type 2 granular corneal dystrophy fibroblasts. 189 p53 and p21 expression also decreases, indicating cell cycle dysregulation. 190 Rapamycin suppresses corneal neovascularization in chemically burned mice model through down-regulation of inflammatory cytokines -IL-1β and TNF-α. It also reduces VEGF expression through down-regulation of VEGFR1 and HIF-1α in corneal tissue. 191 Inflammatory cytokine such as IL-6 stimulates TGFβ signalling during corneal neovascularization in chemical injury and induces transdifferentiation of stromal cells into myofibroblasts. Elevated TGFβ1 secretion up-regulates α-SMA in stromal cells. Rapamycin suppresses TGFβ1-induced α-SMA expression and trans-differentiation of stromal cells through Erk1/2 activation. 192 Hyperglycaemia inhibits corneal epithelial wound healing through increased acetylation of p53 and decreased phosphorylation of Akt. IGFBP3, a downstream target of p53, negatively regulates IGF-1R/Akt dependent cell survival. Overexpression of SIRT1 in these cells increases wound healing capacity via reduced acetylation of p53 and activation of p53/IGFBP3/IGF-1R/Akt signalling. 193 SIRT1 promotes corneal epithelial wound healing and nerve regeneration through its downstream target miR-182 in diabetic mice. 194 Metformin delivery through the PLGA-PGE drug carrier also reduces corneal neovascularization. The underlying mechanism is not reported but might involve AMPK/mTOR signalling. 195 Rapamycin also alleviates replicative senescence in primary corneal epithelial cells through the reduction of cell proliferation and inflammatory cytokines. 196 Hyperglycaemia diminishes nerve innervations in corneal epithelium. VEGF-B expression increases in corneal epithelium following nerve injury and decreases under hyperglycaemic conditions. Subconjunctival administration of VEGF-B induces corneal nerve regeneration from diabetic trigeminal ganglia through VEGFR1 and phosphorylation of Akt, GSK3β and S6K implying activation of PI3K/Akt/GSK3β/mTOR signalling. 197 SIRT6 deletion in mice diminishes corneal epithelial wound healing and causes keratinization. SIRT6 deletion also up-regulates the expression of inflammatory cytokines. 198 SOX2 activation increases proliferation and wound healing capacity of corneal endothelial cells. It inhibits wnt/β-catenin through GSK3β-mediated degradation of β-catenin and activates Akt/FKHRL1 signalling. SOX2 activation increases mitochondrial energy production through the up-regulation of SIRT1 and down-regulation of AMPK. These changes help to reduce corneal oedema through decreased inflammatory cell infiltration and to maintain cell shape and function. 199 Reduced AMPK phosphorylation and increased NF-κB activation have been reported in conjunctival tissues in dry eye disease. AMPK exerts anti-inflammatory response in the conjunctiva of the mice model of dry eye. AMPK activation with AICAR maintains conjunctival goblet cell density and decreases levels of inflammatory cytokines and chemokines. 200 AMPK activation decreases the chances of immunemediated corneal graft rejection through its downstream anti-inflammatory signalling. AICAR decreases stromal opacity, corneal oedema and neovascularization in vivo after corneal transplantation. AICAR-mediated AMPK activation and mTOR inhibition decrease levels of inflammatory cytokines and increase levels of those cytokines required for immune tolerance (IL-4, IL-5, IL-13). AMPK activation reduces hemangiogenesis and increases graft survival. 201 Topical IGF-1 administration inhibits gradual loss of corneal nerves in diabetic mice through AMPKinduced mitochondrial biogenesis and energy production. IGF-1 activates ACC, Akt, p70S6K and PGC1α. 202

Uveal melanoma
Uveal melanoma (UM) is a malignancy of choroidal melanocytes from uveal tract including iris, choroid and ciliary body. Elevated activation of mTOR, MEK/MAPK and PI3K/Akt signalling have been reported during the pathogenesis of UM. 203,204 Inhibitors of these signalling molecules in combination (MEK, PI3K, PKC) induces cell death and regresses tumour growth in mice model of UM. 205,206 Everolimus, in combination with PI3K inhibitor GDC0941, reduces tumour growth in vivo. 207 Increased AMPK signalling is known to induce the survival of UM cells in BAP1 mutant UM progression. 208 The reduced proliferation of UM cells by Erk and Akt inhibitors accompanies AMPKmediated autophagy induction. Activated AMPK upregulates Beclin1 and LC3, whereas siRNA-mediated inhibition of AMPK-induced apoptosis in primary choroidal UM cells. 209 AICAR causes s phase arrest through p53 up-regulation and reduces proliferation of UM cells partially through AMPK activation and ACC phosphorylation. 210 Expression of PGC1α increases during metastatic melanoma which ensures cell survival through increased mitochondrial activity. Silencing PGC1α in these cells elevates cell migration and metastasis. 211 Increased expression of miR-21 has been reported in UM cells in vitro. miR-21 inhibits p53 expression and increases proliferation and invasion capacity of cells. 212 Expression of SIRT2 and other histone deacetylases increases in melanoma cells from UM patients compared to normal melanocytes. 213,214 SIRT2 is known to deacetylate and decrease the transcriptional expression of p53, which explains the increased migration capacity of miR-21 overexpressed uveal melanocytes. 215 Inhibitor of histone deacetylase (trichostatin, valproic acid) decreases proliferation of UM cells through G1 arrest and tumour growth in mice model. Valproic acid also induces the differentiation of UM cells into quiescent cells. 216 Other histone deacetylase inhibitors JSL-1 and tenovin-6 upregulates expression of p53 and its downstream targets (p21, p27, Bax and Puma) in UM cells and decreases tumour growth in xenografted mice model. They induce UM cell apoptosis through loss of mitochondrial membrane potential. Both of these also inhibits cell proliferation, migration and decreases cancer stem cell population. 214,217 TNF-α induced NF-κB activity increases in UM cells. Pristimerin (triterpenoid) inhibits NF-κB activation through p65 nuclear translocation. Pristimerin stimulates G1 arrest and intrinsic apoptosis in UM cell through transcriptional down-regulation of survivin. It also reduces cancer stemlike cell properties and cell invasion by decreasing expression of MMP2/9 at translational levels. 218 Level of VEGF secretion increases in UM cells as it stimulates angiogenesis. Increased plasma concentration of VEGF is also an important marker for metastatic UM. 219 Apigenin decreases VEGF expression through inhibitory phosphorylation of Akt and Erk1/2 in vitro UM cells. 220 Resveratrol decreases tumour growth in the mice model of UM through mitochondria-mediated apoptosis. 221 Epigallocatechin gallate decreases UM cell migration via Erk1/ 2 suppression. 222 Withaferin A (steroidal lactone) induces apoptosis and tumour regression through Akt inactivation in mice model. 223

Uveitis
Uveitis is the inflammation of uvea and associated structuresiris, ciliary body, choroid, retina and retinal vessels. Sometimes, it may also involve cornea and sclera. 224 The causes of uveitis include infections, trauma or autoimmunity. Phosphorylation of AMPK decreases in mice retina during the progression of endotoxin-induced uveitis. 225 AMPKα exerts anti-inflammatory activity in macrophages and dendritic cells by stimulating the production of IL-10 and decreasing IL-6 and TNF-α upon exposure to bacterial lipopolysaccharides. Deletion of AMPK in these cells elevates the expression of IFN-γ and IL-17 (proinflammatory) from T cells. In AMPK deficient cells, elevated phosphorylation of p65, Erk1/2 and p38 mediates inflammatory cytokine production while decreased phosphorylation of Akt, CREB and GSK3β inhibits IL-10 production. 226 AICAR pretreatment counteracts LPSinduced uveitis in rats. AICAR decreases cell infiltration into aqueous humour and leukocyte recruitment to retinal vasculature by reducing NF-κB p65 activity. AICAR also inhibits secretion of inflammatory cytokines and chemokines (MCP-1, ICAM-1, TNF-α) in the retina and aqueous humour. 227 KS23 (adiponectin derived peptide) activates AMPK and suppresses LPS-induced uveitis through attenuation of reduced translational expression of SIRT1 and PPAR-γ in mice. SIRT1 deacetylates p65 and inhibits proinflammatory cytokines expression in the retina through down-regulation of cytokine and chemokine receptors as well as TH17 cell populations. 225 Metformin reduces cell infiltrates in aqueous humour during uveitis progression. It also decreases levels of inflammatory cytokines, chemokines and VEGF in aqueous humour. Metformin brings about these changes through AMPK-induced NF-κB inactivation. 228 During uveitis, Muller glial cells become reactive and extend their processes up to the inner nuclear layer of the retina, leading to the breakdown of the blood-brain barrier and leukocyte adhesion to retinal vessels. Theissenolactone (fungal derivative) inhibits endotoxin-induced iris inflammation, inhibits glial cell activation and protects photoreceptor functions. It inhibits levels of cytokines (MCP-1, TNF-α) and MMP2/9 in aqueous humour in a rat model of uveitis. It also down-regulates endotoxin-induced p65 and Hsp90 which is required for TAK1 activity and inhibits leukocyte adhesion to retinal vessels. Theissenolactone suppresses inflammation through inhibition of NF-κB/ TAK1/IKK axis as it also inhibits endotoxin-induced TAK1 phosphorylation. 229 Glucosamine inhibits cellular infiltrates in the anterior segment of the eye in the rat model of uveitis. It attenuates increased expression of NF-κB and ICAM-1 in iris and ciliary body. 230 Glucosamine might show these protective effects through activation of AMPK-mTOR signalling-induced autophagy as it does in RPE cells. 231 SIRT1 expression decreases during uveitis. Resveratrol inhibits inflammatory response in RPE-choroid complex via up-regulation of SIRT1 and down-regulation of NF-κB P65 translocation. 232 A meta-analysis study has shown the potential therapeutic effect of rapamycin in uveitis. 233 A phase 3 study involving intravitreal administration of rapamycin for 5 months showed complete regression of ocular inflammation. 234 Chrysin reduces macrophage infiltration in vitreous and retina and improves increased permeability of retinal vessels in vivo model of uveitis. Chrysin inhibits pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, IFN-γ, IL-17A) and up-regulates TGFβ (antiinflammatory) expression in the uveitic retina through down-regulation of NF-κB p65 translocation. 235 Oxidative stress-mediated activation of p38 MAPK induces TNF-α secretion, microglial activation and apoptosis in the retina following endotoxin exposure. Cannabidiol suppresses microglia activation and inflammation in the uveitic retina of the rat. It suppresses the activation of p38 MAPK and apoptosis in RGCs. 236 Matairesinol inhibits autoimmune uveitis via regulation of Th17 cell differentiation by suppressing MAPK/ROR-γt signalling in vivo. 237 Microbial lipopolysaccharide elevates levels of pJNK in uveal melanocytes. JNK inhibitor XG-102 (peptide) reduces cell infiltration and expression of inflammatory chemokines in the uveitic retina. 238 Lutein and zeaxanthin inhibit the secretion of IL-8 by attenuation of endotoxin-induced pJNK and NF-κB in uveal melanocytes. 239

Retinitis pigmentosa
Pathology of retinitis pigmentosa involves retinal degeneration, abnormal deposition of retinal pigment, epiretinal membrane formation, night blindness and peripheral vision loss. Misfolded rhodopsin accumulates in outer rod segments and leads to photoreceptor death. AMPK activation with metformin improves synthesis and misfolding of mutant rhodopsin as well as its translocation to the plasma membrane in rod opsin cells. 240 Depletion of insulin levels leads to degeneration of cone cells following photoreceptor outer segment shortening and major rod death phase in mice model of retinitis pigmentosa. Constitutive activation of mTORC1 mutant or by insulin administration prolongs cone survival in mice model of retinitis pigmentosa via upregulation of genes involved in glucose metabolism (uptake, retention, utilization). 241,242 AICAR and rapamycin decrease ER stress and prolongs survival of mutant photoreceptor cells through AMPK/mTORC1 signalling activation. 243 Ciliary neurotrophic factor increases the survival of retinal Muller cells via JAK/STAT and MAPK/ Erk signalling activation. 244 Abnormal expression of SIRT1 has also been associated with retinal degeneration as studied in a mice model of retinitis pigmentosa. 245 Luteolin a plant flavone delays degeneration and functional deterioration of photoreceptor by regulating retinal oxidative stress and inflammation via inhibition of JNK signalling in vivo model of retinitis pigmentosa. 246 Administration of zeaxanthin also shows similar effects in retinal degeneration via p38, Erk, STAT3 and MCP1 signalling in vivo. 247

Retinoblastoma
Studies have reported that AMPK signalling inhibits cell proliferation via apoptotic cell death in different cancers including breast cancer, prostate cancer, and pancreatic cancer. [248][249][250] In vitro AICAR-induced activation of AMPK and ACC leads to s phase arrest and obstructs cell proliferation. AICAR inhibits retinoblastoma cell growth and retinoblastoma xenografts in mice by targeting neovascularization through AMPK/mTORC1/p21 signalling. 251,252 Metformin arrests the cell cycle at s phase and reduces proliferation through activation of AMPK and suppression of mTOR. Involvement of autophagy in the invasion and metastasis of retinoblastoma has already been reported. Metformin prolongs cell survival via up-regulation of autophagy marker LC3B. However, metformin did not inhibit the growth of human retinoblastoma xenografts in mice. 253,254 Salinomycin targets invasive retinoblastoma cells and induce apoptosis by disrupting their mitochondrial membrane potential via activation of AMPK signalling in vivo. 255 Rapamycin inhibits proliferation of retinoblastoma cells in vitro, arrests cell cycle by reducing the phosphorylation and activation of mTOR. 256 Isoflavones arrests retinoblastoma cell cycle at G1 decreases mTOR phosphorylation and retards xenografted human retinoblastoma growth in mice. 257 Gingerol inhibits retinoblastoma cell growth and causes G2/M phase arrest in vitro. It induces apoptosis by activation of PI3K and Akt phosphorylation. 258 Methyl eugenol decreases viability and induces G2/M arrest through PI3K/Akt/mTOR signalling. It induces autophagy in these cells via induction of LC3-II and suppression of p62 expression via inactivation of PI3K/Akt/ mTOR signalling. 259 Cataract A cataract is characterized by opacification of lens affecting the sharpness of the image on the retina, which causes visual impairment. A cataract is the common cause of blindness involving the interplay of several signalling pathways such as oxidative stress, NF-κB, MAPK, PKC, protein modification, inflammation and apoptosis. 260 Studies have shown that age-related inactivation of AMPK and autophagy leads to senescence of lens epithelial cells (LECs) and age-related cataract. Metformin-mediated activation of AMPK attenuates LEC senescence and impaired autophagy in vivo. AMPK activation by metformin also improves lysosomal dysfunction in LECs of age-related cataract patients. [261][262][263] EMT of LECs following cataract surgery causes posterior capsular opacification. As reported by a retrospective study the use of metformin in diabetic patients reduces the risk of development of posterior capsular opacification and its removal by laser capsulotomy compared to nondiabetic patients or cataract patients not using metformin. This protective effects of metformin against PCO formation is mediated via inhibition of EMT mainly by inactivation of Erk. 264 A natural compound, 3H-1,2-dithiole-3-thione inhibits EMT of LECs in vitro diabetic cataract via AMPK activation. AMPK activation by this compound mainly targets Snail/Slug signalling during its protective effects against EMT and oxidative stress. 265 Expression of SIRT1 increases and acetylation of p53 decreases in LECs from age-related cataract patients. With this differential expression, apoptosis also increases in LECs. 266,53 Silencing SIRT1 induces apoptosis in cataract mice. miR-211, which is highly expressed in LECs of cataract mice, binds SIRT1 and decrease its expression. 267 Activation of p38, JNK and Erk decreases in capsular bags from elderly cataract patients (>60 years) compared to young patients. 268 Thus, therapeutic agents that could modulate multiple signalling pathways should be tested to inhibit the growth of LECs. Mutations leading to disruption in mTOR signalling also contribute to cataract development. 269 Rapamycin inhibits LEC proliferation via induction of apoptosis by attenuation of growth factor-induced phosphorylation of Akt/ mTORC1, Erk and JAK/STAT pathways. 270 Dual inhibitor of mTORC1/2, PP242, inhibits proliferation and migration of LECs which might be of therapeutic use in the management of posterior capsular opacification. PP242 induces G0/G1 arrest and autophagy-mediated cells death by up-regulation of p53 and LC3-II/LC3-I. 271 Quercetin inhibits EMT of LECs in diabetic cataract via regulation of TGFβ2/PI3K/Akt signalling. 272 Andrographolide decreases growth factors-induced proliferation and migration of LECs by depleting phosphorylation of Akt. 273 L-carnitine and pcoumaric acid protect LECs from oxidative damage via decreasing phosphorylation and activation of p38, JNK and Erk. 274,275 Oxidative stress induces apoptosis in LECs, which contributes to cataract formation. miR-124 downregulates NF-κB p65 translocation in LECs and suppresses oxidative stress and apoptosis. 276 Rutin (vitamin P) also rescues LECs from oxidative stress-induced apoptosis by inhibition of NF-κB p65 translation. 277 Epigallocatechin attenuates oxidative stress-induced intrinsic and extrinsic apoptosis in LECs via down-regulation of p38 MAPK, Akt and Erk activation in vitro. 278 Honokiol also shows similar effects in LECs by down-regulation of p38 MAPK, Akt, Erk and JNK activation and NF-κB p65 translocation. 279 Parthenolide inhibits apoptosis of LECs by attenuation of oxidative stress-induced activation of Erk, Akt and NF-κB. 280 Magnolol shows anti-apoptotic effects in LECs burdened by oxidative stress through attenuation of reduced Erk phosphorylation and increased p38 and JNK phosphorylation. 281

Conclusion
As the role of AMPK is generally recognized in ocular diseases, there is a rising inclination in the therapeutic exploitation of the AMPK pathway for ocular disease treatment. However, emerging evidence indicates that RPE cells, RGCs and LECs might also use AMPK activation as a survival strategy to undergo metabolic adaptation in terms of energy regain and pathogenesis of the disease. These discoveries accentuate the intricacy of the cellular function of AMPK under pathological and physiological conditions. In spite of the known association between AMPK signalling under various pathological conditions and treatment, the functional evaluation of AMPK signalling axis in regulation of these processes using AMPK deficient genetic models is warranted. Furthermore, AMPK pathway blocking might constitute a more pertinent approach, then the AMPK pathway awaking for ocular disease treatment. Such documentation will work as a lighthouse for the development of more effectual AMPK modulators for ocular disease.
Aiming AMPK/ERK pathway may prove to be a hallmark for treatment in combination with MAPK signalling modifications which is due to the fact that ERK inhibitors prey the downstream of signalling cascade independent of the mutational status of the upstream component. Effective targeting of the pathway intermedial can be a streamlined expedient for integrally energized signalling cascades, such as the role of the MAPK pathway in cancer. Successful blocking of even a single step in the signalling cascade diminishes the downstream progression as well as the overall anomalous function of the pathway. It is a need of an hour that various studies are required to be carried out which investigates the combined effect of inhibitors and/or multitargeted molecules as these might provide potential information for methodical manipulation of signalling pathways which can be further utilized for treatment regime of the ocular diseases.

Author contributions
Dhaval Shukal and Pooja Malaviya designed the study, prepared the original draft and performed literature survey. Tusha sharma did the conceptualization of the study, writingreviewing and editing of the original draft.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.