Mechanistical retinal drug targets and challenges

Rinnakkaistallenteet Terveystieteiden tiedekunta

2018

Mechanistical retinal drug targets and challenges

Kaarniranta

Elsevier BV

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Mechanistical retinal drug targets and challenges

Kai Kaarniranta, Heping Xu, Anu Kauppinen

PII: S0169-409X(18)30076-0

DOI: doi:10.1016/j.addr.2018.04.016

Reference: ADR 13297

To appear in: Advanced Drug Delivery Reviews Received date: 11 December 2017

Revised date: 27 March 2018

Accepted date: 20 April 2018

Please cite this article as: Kai Kaarniranta, Heping Xu, Anu Kauppinen , Mechanistical retinal drug targets and challenges. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Adr(2018), doi:10.1016/

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Advanced Drug Delivery Reviews 2018

Invited review

Mechanistical retinal drug targets and challenges

Kai Kaarniranta^1,2,3, Heping Xu^4,5, Anu Kauppinen⁶

1Department of Ophthalmology; Institute of Clinical Medicine; University of Eastern Finland; Kuopio, Finland; ²Department of Ophthalmology; Kuopio University Hospital; Kuopio, Finland; ³Department of Molecular Genetics, University of Lodz, Lodz, Poland⁴Centre for Experimental Medicine, The Wellcome-Wolfson Institute of Experimental Medicine, Queen’s University Belfast, Belfast, UK;

5Aier School of Ophthalmology, Central South University, China; ⁶School of Pharmacy, University of Eastern Finland, Kuopio, Finland

Funding support:

Kai Kaarniranta: European Commission: EC H2020 MSCA - ITN – 722717; Academy of Finland (296840), the Finnish Eye Foundation, the Päivikki and Sakari Sohlberg Foundation, the Business of Finland, the Kuopio University Hospital VTR (5503757) and University of Eastern Finland

Heping Xu: European Commission: EC H2020, MSCA - ITN – 722717; Fight for Sight: 1361/62, 1425/1426, 1574/1575, and 5057/5058; Diabetes UK: 13/0004729, 16/0005537).

Anu Kauppinen: Academy of Finland (297267, AK307341), the Emil Aaltonen Foundation, the Päivikki and Sakari Sohlberg Foundation, the Finnish Cultural Foundation, and the Finnish Eye Foundation.

Correspondence:

Professor Kai Kaarniranta, Department of Ophthalmology; Institute of Clinical Medicine; University of Eastern Finland; Kuopio; Tel: +358-17-172484; Fax: +358-17-172486; Email: kai.kaarniranta@uef.fi Professor Heping Xu, Centre for Experimental Medicine, The Wellcome-Wolfson Institute of

Experimental Medicine, Queen’s University Belfast, Belfast, UK, Tel: +44(0)2890976463; Email:

heping.xu@qub.ac.uk

Key words: aging, autophagy, phagocytosis, inflammation, oxidative stress

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Abstract

The retina is constantly exposed to light that increases reactive oxygen species in retina.

Oxidative stress, inflammation and neurodegeneration are the major contributors in the most common retinal diseases, such as age-related macular degeneration (AMD), glaucoma and diabetic retinopathy (DR). Emerging developments and research for novel therapy targets and drug delivery to the posterior segment offer a promising future for the treatment of retinal diseases including rare hereditary diseases.

In this review we discuss about promising mechanistical retinal drug targets. Vascular endothelial growth factor (VEGF) signaling and anti-VEGF treatments are excluded.

1. Introduction

The mature retina is organized into three nuclear layers; ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) that are interconnected by synapses [Figure 1].

Retinal ganglion cells (RGCs) form the most inner layer of retinal cells. Their axons form the optic nerve and transmit visual information into the visual cortex. RGCs undergo apoptotic cell death in glaucoma and other optic neuropathies [1]. The mechanisms of RGC death are still a matter of intense investigation, and several factors including growth factor deprivation and oxidative stress have been proposed to participate in RGC degeneration in glaucoma. Rod and cone photoreceptors of the ONL are the light sensing cells of the eye that are responsible for black/white and colour vision respectively. The photoreceptors initiate the phototransduction, which leads to the action potential in retinal ganglion cells. The retinal pigment epithelial (RPE) cells are situated between the photoreceptor cells and the choroid [Figure 1]. The RPE cells are vitally important for vision by maintaining the viability of photoreceptor cells [2-4]. Daily phagocytosis of photoreceptor outer segments (POS) and their degradation in RPE cell lysosomes (heterophagy) are critical for visual cycle and maintaining vision [4- 6]. RPE cells are phagocytically the most active cells in the whole body, uptaking and degrading up to 10% of the POS daily. Apical microvilli of the RPE extend around the POS and ingest shed rod and cone outer segment discs into the RPE as membrane-bound phagosomes [2]. These phagosomes fuse with lysosomes to form phagolysosomes. Lysosomal acid hydrolases degrade the outer segment material that is re-used in photoreceptors. The degradation process is critically controlled by acidification of lysosomes with vacuolar-type H+-ATPases (V-ATPase) [7]. Decreased lysosomal enzyme activity evokes an accumulation of lipofuscin, increases oxidative stress and protein aggregation and enhances RPE degeneration. We discuss about promising drug targets that coincide with normal cellular homeostasis in retina.

2. Targeting phagocytosis

Phagocytosis processes comprise recognition and binding, internalization, and finally digestion of POS discs in lysosomes [8; Figure 2]. [9,10]. Initially, the integrin ITGAV-ITGB5 (αVβ5) regulates the binding process [11,12]. Subsequently, MERTK (c-mer proto-oncogene tyrosine kinase) enhances the POS ingestion [13,14]. MERTK phosphorylates protein tyrosine kinase 2 during the binding process and links the signaling between integrin ITGAV-ITGB5 and MERTK [15,16].

Efficiency of phagocytosis varies according to circadian rhythm which is controlled by MFGE8 (milk fat globule-EGF factor 8 protein [17]. It is widely accepted that defects in phagocytosis are detrimental to photoreceptors and RPE [13,14, 18]. It has been documented that decrease of integrin ITGAV- ITGB5 associates with the accumulation of lipofuscin in RPE cell lysosomes. Lipofuscin is a heterogenous protein-lipid-carbohydrate aggregate which includes toxic fluorophores such as, N- retinylidene-N-retinylethanolamine (A2E) compound. Lipofuscin toxic compounds disturb lysosomal membrane stability, inhibits mitochondrial respiration and accelerate RPE degeneration [18]. Thus, regulatory proteins of POS phagocytosis are critical pharmaceutical targets to maintain retinal homeostasis. In addition, for improving the POS clearance, many modulators of visual cycle have been developed in order to treat various retinal diseases [Figure 2]. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting

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enzyme of the visual cycle, based on the idea that an attenuation of visual pigment regeneration could reduce the formation of toxic retinal conjugates [19,20].

3. Targeting autophagy

In addition to POS phagocytosis, lysosomal degradation of cytosolic material in a process called autophagy is a key therapy target in age-related retinal diseases [Figure 2]. Disturbed lysosomal clearance leads to RPE degeneration, which can clinically be observed by pigment mottling, increased autofluorescence and accumulation of extracellular drusen deposits. All those are hallmarks of increased cellular stress and RPE degeneration process that one day may lead to cell death [21].

Constant oxidative stress induces protein misfolding and aggregation if heat shock proteins (Hsps, molecular chaperones), proteasomes, or the protective capacity of autophagy have been exceeded [22]. Hsps working with the ubiquitin proteasome system and autophagy, are responsible for cellular quality control.Essential tasks of Hsps include assistance in the refolding of damaged proteins, facilitation their translocation to correct intracellular localizations, and mitigation of the harmful effects of protein misfolding and aggregation [23]. Hsp-related chaperone therapy has been discussed to be potentially preventive in the progression of retinal neurodegenerative diseases. Soluble damaged proteins are targeted to proteasomes for degradation, while larger proteins complexes undergo the autophagy clearance. Dysfunctional autophagy has been associated with several neuropathological conditions and a considerable number of studies have proved autophagy as a potential target for pharmacological modulation to achieve cellular protection in ganglion cells, photoreceptors, and in RPE cells [24-28].

Yoshinori Ohsumi was awarded with the Nobel Prize 2016 in Physiology or Medicine for his efforts on autophagy research [29]. Autophagy is an intracellular degradation process that removes long-lived or aggregated proteins, lipid droplets, and organelles from the cytoplasm in all eukaryotic cells [30]. Autophagy is host defence response to many environmental stresses observed in retinal diseases such as, nutrient deprivation, hypoxia, or oxidative stress. In autophagy cellular energy is reused by recycling free fatty acids, amino acids and nucleotides after their degradation process [22,31].

The autophagy process undergo the enlarging of autophagosome membrane from omegasome origin to the mature double-membrane autophagosome. The autophagosome itself is not active in degradation process. Once the autophagosome fuse with lysosomes, lysosomal enzymes are released into autophagosome cavity and start degrade its contents. There is strong evidence that pharmacological induction of autophagy improves cellular survival [24,32]. Adenosinemonophosphate-activated protein kinase (AMPK) is a key energy and stress sensor that regulates the cellular metabolism. AMPK stimulates autophagy by inhibiting mTOR complex 1 (mTORC1), a major negative regulator of autophagy [33]. mTOR belongs to the PI3K-related kinase family. It consists of the central regulatory catalytic core of two functionally distinct multiprotein complexes, mTORC1 and mTORC2. Rheb, Raptor, PRAS40-P, mLST8 and Deptor are regulatory units for mTORC1, while Sin1, Rictor, mLST8 and Protor are the corresponding units for mTORC2 [Figure 2]. mTORC1 regulates autophagy rather than mTORC2 which is involved in the control of cytoskeleton organization and cell survival. The modulation of mTORC1 function is a potential target for the development of therapeutics for neurodegenerative diseases including retinal diseases. Rapamycin, sirolimus, temsirolimus (CCI-779), everolimus (RAD-001), deferolimus (AP-23573) are examples of mTORC1 inhibitors. Metformin, resveratrol and adenosine analogue 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) activate AMPK and inhibit mTOR [24,33,34; Figure 2]. Autophagy is also required to support daily phagocytosis within RPE cells and keep retinoid levels for vision [4, Figure 1]. Decline in retinal clearance system may lead to increased oxidative and endoplasmic reticulum stress, mitohondrial damage, and inflammation [35,36].

4. Targeting the endoplasmic reticulum and mitochondria

Oxidative stress refers to the progressive cellular damage caused by reactive oxygen species, contributing to protein misfolding, and evoking functional abnormalities during the cellular senescence

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of the RPE cells [37]. Free radicals also damage the lysosomes of RPE cells, which lose their capacity to degrade photoreceptor outer segment material, resulting in the accumulation of lipofuscin—a recognized hallmark of ageing [38,39]. Lipofuscin contains vitamin A-derived fluorophores that has been shown to inhibit mitochondrial respiration and increase endoplasmic reticulum stress (ER) [40-42;

Figure 1]. To maintain proteostasis and cell function, the ER activates an adaptive quality control measure known as the unfolded protein response (UPR). The UPR is initiated by three independent transmembrane stress transducers; 1) inositol-requiring kinase-1 (IRE1), 2) double-stranded RNA- activated protein kinase-like ER kinase (PERK), and 3) activating transcription factor-6 (ATF6) [43].

There is evidence of autophagy-ER-mitochondrial crosstalk in RPE cells and the AMD development [22,44]. ER-mitochondria contact sites are involved in the autophagosome formation, and many proteins in the mitochondria-associated ER membrane (MAM) compartments are necessary for the autophagic vesicle formation [45; Figure 2]]. Humanin (HN) is a prominent member of a newly discovered family of mitochondrial-derived peptides (MDPs) expressed from an open reading frame of mitochondrial 16S rRNA [44]. The more potent variant humanin G (HNG) protects the RPE mitochondria and ER, and reduces apoptosis at both gene and protein levels [46,47; Figure 2]. The therapeutic use of HN could potentially prove to be a valuable approach for the treatment of AMD

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5. Inflammatory pathways as therapeutic targets 5.1. Retinal defence system

Inflammation is known to be involved in various retinal degenerative diseases, and properly controlled inflammation would be a novel approach for therapy. Retina is an immune privileged tissue due to (1) the blood retinal barriers (BRBs) that prevent the free migration of circulating immune cells and molecules into the retinal parenchyma, and (2) the immunosuppressive microenvironment of the retina. The “privilege” ensures that the retina is not affected by systemic immune system under normal physiological conditions. During systemic infection, a small number of circulating immune cells may infiltrate the retina [48], but they only induce inflammation when encounter specific antigens. When the retina suffers from exogenous or endogenous insults, the first line of defence is the retinal innate immune system rather than circulating immune cells. This may be a strategy to minimise retinal immune response, therefore protecting the neuroretina from inflammation-mediated damage.

As an immune privileged tissue, the retina has its own defence system, including resident immune cells and the complement system, although under normal physiological conditions their activity is suppressed by the microenvironment. Microglia are the main resident immune cells of the retina. Microglia are distributed in three layers of the neuronal retina, the ganglion cell layer (GCL), the inner plexiform layer (IPL), and the outer plexiform layer (OPL) [49]. They can quickly respond to exogenous pathogens and endogenous danger molecules, and become activated. Active microglia phagocytise invading pathogens, dead cells, or damaged molecules and play an important role in retinal homeostasis [50,51]. Microglial activation is tightly regulated by various membrane or soluble molecules released by surrounding neurons. Examples of the inhibitory pathways include the CX3CL1/CX3CR1, CD200/CD200R, SIRP/CD47, and the endocannabinoids/cannabinoids receptors (CB1, C2) pathway [49]. Active microglia are highly phagocytic and play an important role in the clearance of retinal dead cells and debris [50]. However, uncontrolled microglial activation is known to contribute to many retinal pathologies, such as photoreceptor degeneration, and retinal angiogenesis [50,51]. How this protective response becomes detrimental in retinal degenerative diseases remains poorly defined. Active microglia may release inflammatory molecules such as TNF-, IL-1, and CCL2 that may directly kill retinal neurons or may escalate inflammation by recruiting circulating immune cells.

When retinal diseases progress towards the advanced stages (e.g., neovascular AMD or proliferative diabetic retinopathy), retinal immune privilege may be compromised due to breakdown of the BRB. Circulating immune components including leukocytes and complement proteins may infiltrate the retina and participate in retinal immune response.

Extending treatment strategies to systemic immune regulation might be necessary.

5.2. Targeting microglial activation

When targeting microglial activation for therapy, it is important to ensure that the beneficial role of microglia is preserved when eliminating the detrimental roles (Figure 1). Microglia is activated upon engaging exogenous or endogenous pathogens/danger molecules using cell surface or cytosolic pathogen recognition receptors (PRRs) [52], including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs). After engaging with relevant ligands, the PRR-associated kinases, such as the interleukine-1 receptor-associated kinase (IRAKs) and the receptor-interacting protein (RIP), are activated (phosphorylated), leading to a cascade of intracellular signal transduction

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and inflammatory gene and protein expression. Corticosteroids are widely used to control retinal inflammation and they can certainly supress microglial activation. However, their application is limited, particularly in chronic retinal degenerative conditions, due to the well- known adverse effects (e.g., induce cataract and increase intraocular pressure). Target- specific and safe anti-inflammatory therapies are needed.

It would be a risk to block PRRs to control microglial activation as it would jeopardise the protective response of host defence. However, targeting relevant kinase of the PRRs could be a novel approach for the management of uncontrolled microglial activation.

IMO-8400 (Iderapharma), a selective antagonist of TLR7, 8, and 9 inhibiting the MyD88 signalling pathway, has been tested in conditions whereby lesions are related to the over- activation of TLRs [53], such as psoriasis, Waldenstrom's macroglobulinemia (NCT02092909), and dermatomyositis (NCT02612857). Their applications in uncontrolled microglial activation remains to be determined.

Microglia can be activated towards the pro-inflammatory phenotype (classical activated M1 phenotype) or anti-inflammatory/wound-healing phenotype (alternatively activated M2 phenotype) [54]. The former is related to the production of inflammatory cytokines such as TNF, IL-1, and nitric oxide, whereas the latter is related to the release of anti-inflammatory, tissue-repair and angiogenic growth factors, including IL-10, TGF, CCL22, and VEGF. Modulating microglial activation towards M2 phenotype is believed to be an effective approach to limit inflammatory damage in neuroinflammatory and neurodegenerative conditions. Despite intensive research in this area, clinical translation has not been materialized.

The toxic effect of active microglia is mediated through the release of inflammatory cytokines and chemokines such as TNF and IL-1. Therefore, targeting these cytotoxic mediators are also effective approaches of immune therapy. In fact, an anti-TNF

monoclonal antibody has been shown to be effective in the treatment of retinal and CNS autoimmune diseases [55]. It must be noted that inflammatory cytokines can be released by multiple cell types, particularly when the BRB is broken down during retinal inflammation.

Apart from active microglia, tissue cells and infiltrating immune cells can all produce inflammatory cytokines. Strictly speaking, anti-TNF is not a microglia-targeted therapy.

5.3. Targeting the complement system

The complement system is an important part of the innate immunity. The majority of complement proteins are produced in the liver and released as a latent form to circulation for systemic distribution. The complement system can be activated by at least three pathways, the classic pathway (CP), the alternative pathway (AP) and the mannose-binding pathway (MBP) [56]. The complement system may participate in immune response at multiple levels.

Firstly, complement fragments such as C3a and C5a are anaphylatoxins and may induce vasodilation and smooth muscle contraction. They can also induce immune cell migration and activation through relevant receptor (C3aR, C5aR and C5L2). Secondly, C3b can opsonize pathogens or apoptotic bodies and enhance their clearance by phagocytes. Finally, the end product of complement activation is the formation of C5b-9 (membrane attack complex, MAC), which can lyse target cells, although sublytic levels of MAC may participate in immune modulation [57,58].

The complement activation pathway can be targeted at the initiation, amplification, and effector stages (Figure 3). Indeed, various compounds have been developed to specifically target different steps of complement activation cascade. Figure 3

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illustrates the key steps of complement activation and the compounds that are currently on trials or approved for treating inflammatory conditions.

As the retina is separated from blood circulation by the BRB, circulating complement proteins cannot reach the retina under normal physiological conditions. We and others have shown that retinal cells, particularly RPE and microglia express various complement proteins [59,60]. Interestingly, the expression levels of complement regulators (CFH, CD46, CD59, C1INH) are generally higher than those of complement activators (e.g., C3, C5, C6, C7, C8, C9) [60]. We believe that the retina has a complement regulatory system [61] that protects the retina from uncontrolled complement activation under pathophysiological conditions. Under ageing and oxidative conditions, the regulatory system may become dysfunctional leading to uncontrolled complement activation and retinal pathology. For example, CFH, the key regulator of the AP, is highly expressed by RPE cells under physiological conditions [62]. The expression of CFH is down-regulated by inflammatory cytokines and under oxidative stress conditions. On the other hand, the expression of CFB, a key protein required for the AP activation, is increased during ageing [63]. When targeting the complement system for the management of retinal diseases, whether the drug should be administered systemically or locally is an important consideration. Due to the existence of the local complement regulatory system, we think that intraocular administration would be more appropriate even when the BRB is compromised.

Uncontrolled complement activation is known to be involved in a number of inflammatory and degeneration retinal diseases, including uveoretinits, AMD, and diabetic retinopathy. Targeting the complement system for the management of retinal inflammatory and degenerative diseases has been a hot area of research in recent years [61]. When targeting the complement system for therapy, it is important to understand the beneficial and detrimental elements of complement activation in retinal pathology under specific disease conditions. Although inappropriate levels of MAC and C3a/C5a may cause tissue damage, C3b can promote phagocytosis of dead cells and debris and is beneficial for resolution of inflammation and tissue remodelling. Ideally, the therapy should be sufficient to remove unwanted complement activation without shutting down necessary biology response pertinent to tissue repair and homeostasis. The chosen therapies should also consider the mechanism of complement activation, i.e., what triggers complement activation, which complement pathways are involved and at which stages of the disease. In autoimmune uveoretinitis, autoantibody-mediated immune complex may induce the CP complement activation, however the cascade is ultimately amplified by the AP pathway [64]. Therefore, inhibition of the AP can effectively control complement activation in uveoretinitis. If retinal pathology is caused by excessive amount of MAC, targeting the terminal pathway i.e., preventing the assembling of MAC using anti-C5, anti-C6 or over-expression of CD59 would be an ideal approach for therapy. If C3a- or C5a-mediated immune activation underlies retinal pathology, targeting the C3a/C3aR or C5a/C5aR pathway should be considered. Due to lack of mechanistic insights on how the complement system contributes to retinal pathology in aforementioned diseases, current approaches are restricted to the inhibition of complement activation (e.g., C3 or C5 inhibitors or CFD inhibitor, for more details please see details in our recent review article [61].

5.4. Targeting the inflammasome

Apart from microglia and the complement system, inflammation may also occur in retinal cells, i.e. as a tissue-cell autonomous inflammatory response. Typical example of tissue autonomous inflammation is inflammasome activation in RPE cells. Inflammasomes are

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multiprotein complex assembled in response to pathogens or danger molecules [65. The inflammasome complex typically contains a sensor protein, an adaptor protein and a procaspase-1. Inflammasome activation results in the cleavage of inactive procaspase-1 into an active cysteine-protease enzyme, caspase-1, which can activate proinflammatory cytokines IL-1 and IL-18. Inflammasome activation may also lead to pyroptosis, a pyrogenic inflammatory-type of cell death. Inflammasomes can be activated in immune cells (e.g.

macrophages) as well as tissue cells that express sensor proteins, such as members of the NLR (NLRP1, NLRP3, and NLRP4) or AIM2-like receptor (ALR) family [66]. Among these, NLRP3-mediated inflammasome activation in RPE cells is the most intensively investigated pathway [67,68], and activation of this pathway is known to contribute to the pathogenesis of AMD. NLPR3 can be activated by a wide array of microbe- and host-derived triggers. In the context of retinal inflammatory and degenerative diseases, NLRP3 is likely to be activated by endogenous ligands such as ATP and crystalline substances such as silica and alum [69]. Other events that can induce NLRP3 pathway activation include potassium efflux, the generation of mitochondrial reactive oxygen species (ROS) [70], cathepsin release from lysosome, and the release of mitochondrial DNA to cytosol [71]. In addition, complement MAC [72] and aluRNA [73] can also activate NLRP3. AluRNA-induced NLRP3 inflammasome activation results in increased IL-18 production, which is known to be involved in the development of geographic atrophy (GA) [73].

In addition to GA, inflammasome activation is also known to contribute to the pathogenesis of diabetic retinopathy [74]. Targeting the inflammasome pathway may benefit patients with GA and DR. As an innate immune response to endogenous danger stimuli, inflammasome in retinal cells or microglia/macrophages is initially a protective response.

Why this protective response becomes detrimental is poorly understood. Although excessive amounts of IL-1 and IL-18 are detrimental, appropriate levels of these cytokines may be required for retinal homeostasis, particularly under chronic stress conditions. RPE cells constantly produce high levels of IL-18 [75], and NLPR3 inflammasome activation-induced IL-18 has anti-permeability and anti-angiogenic functions [76] and can protect choroidal neovascularization in animal models of AMD [77,78]. Until we know the exact role of inflammasome activation in retinal homeostasis during aging and chronic oxidative stress conditions, and how uncontrolled inflammasome activation contributes to retinal pathologies, it will be difficult to safely and effectively target the inflammasome pathways for the management of retinal diseases.

A number of NLPR3-targeted therapies are being developed for the management of cancer and autoimmune diseases [Figure 4]. These are summarised in a recent review by Ozaki et al. [79] Some compounds indirectly target NLRP3 (Glyburide, 16673-34-0), whilst others target caspase-1 (Pralnacasan – VX-740, VX-765, Parthenolide) and ATP receptor P2X7 (AZD9056; CE-224535; GSK1482169). In addition, antibodies against IL-18 (GSK 1070806) and IL-1 (Canakinumab, Rilonacept) or IL-1 receptor (Anakinra) have also been developed and tested in various conditions related to inflammasome over-activation (e.g. rheumatoid arthritis, B cell non-Hodgkin’s lymphoma, inflammatory bowel disease). Once we know precisely the role of inflammasome activation in retinal inflammatory and degenerative conditions, we will be able to apply these compounds to ophthalmic conditions.

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6. Conclusions

Therapeutic strategies for the treatment of AMD coincide with the suppression of oxidative stress and inflammation as well as the maintenance of active clearance mechanisms of the RPE. Pharmacological research should focus on finding new molecular targets in the AMD pathogenetical pathway and on developing longer-lasting agents or new drug delivery systems. Since AMD is complex disease drug development will be challenging especially taking account of personalized medicine. However, possible breakthroughs in AMD treatments may open new therapy options in other retinal diseases.

7. References

[1] N. Rodríguez-Muela, P. Boya, Axonal damage, autophagy and neuronal survival, Autophagy 8 (2012) 286-288.

[2] O. Strauss, The retinal pigment epithelium in visual function, Physiol. Rev. 85 (2005) 845-881.

[3] K. Palczewski, Retinoids for treatment of retinal diseases, Trends Pharmacol. Sci., 31 (2010) 284-295.

[4] J.Y. Kim, H. Zhao, J. Martinez, T.A. Doggett, A.V. Kolesnikov, P.H.Tang, Z. Ablonczy, C.C. Chan, Z. Zhou, D.R. Green, T.A. Ferguson, Noncanonical autophagy promotes the visual cycle, Cell 154 (2013) 365-376.

[5] K. Kaarniranta, D. Sinha, J. Blasiak, A. Kauppinen, Z. Veréb , A. Salminen , M.E.

Boulton, G. Petrovski, Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration, Autophagy 9 (2013) 973-984.

[6] S. Guha, J. Liu, G. Baltazar, A.M. Laties, C.H. Mitchell, Rescue of compromised lysosomes enhances degradation of photoreceptor outer segments and reduces lipofuscin-like autofluorescence in retinal pigmented epithelial cells, Adv. Exp. Med. Biol. 801 (2014) 105- 111.

[7] M. Valapala, C. Wilson, S. Hose, I.A. Bhutto, R. Grebe, A. Dong, S. Greenbaum, L. Gu, S. Sengupta, M. Cano, S. Hackett, G. Xu, G.A. Lutty, L. Dong, Y. Sergeev, J.T. Handa, P.

Campochiaro, E. Wawrousek, J,S. Jr Zigler, D. Sinha, Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase- MTORC1 signaling, Autophagy 10 (2014) 480-496.

[8] D. Sinha, M. Valapala, P. Shang, S. Hose, R. Grebe, G.A. Lutty, J.S. Jr Zigler, K.

Kaarniranta, J.T. Handa, Lysosomes: Regulators of autophagy in the retinal pigmented epithelium, Exp. Eye Res. 144 (2016) 46-53.

[9] N.B. Caberoy, Y. Zhou, W. Li, Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis, EMBO J. 29 (2010) 3898-3910.

[10] B.M. Kevany, K. Palczewski, Phagocytosis of retinal rod and cone photoreceptors.

Physiology (Bethesda), 25 (2010) 8-15.

ACCEPTED MANUSCRIPT

[11] S.C.Finnemann, V.L. Bonilha, A.D. Marmorstein, E. Rodriguez-Boulan, Phagocytosis of rod outer segments by retinal pigment epithelial cells requires α(v)β5 integrin for binding but not for internalization, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 12932- 12937.

[12] E.F. Nandrot, Y. Kim, S.E. Brodie, X. Huang, D. Sheppard, S.C. Finnemann, Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin, J. Exp. Med. 200 (2004) 1539- 1545.

[13] P.M. D’Cruz, D. Yasumura, J. Weir, M.T. Matthes, H. Abderrahim, M.M. LaVail, D.

Vollrath, Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat, Hum. Mol. Genet. 9 (2000) 645-651.

[14] A. Gal, Y. Li, D.A. Thompson, J. Weir, U. Orth, S.G. Jacobson, E. Apfelstedt-Sylla, D.

Vollrath, Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa, Nat. Genet. 26 (2000), 270-271.

[15] W. Feng, D. Yasumura, M.T. Matthes, M.M. LaVail, D. Vollrath, Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells, J. Biol. Chem. 277 (2002) 17016-17022.

[16] S.C. Finnemann, Focal adhesion kinase signaling promotes phagocytosis of integrin- bound photoreceptors, EMBO J. 22 (2003) 4143-4154.

[17] E.F. Nandrot, M. Anand, D. Almeida, K. Atabai, D. Sheppard, S.C. Finnemann, Essential role for MFG-E8 as ligand for alphavbeta5 integrin in diurnal retinal phagocytosis, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12005- 12010.

[18] C. Vives-Bauza, M. Anand, A.K. Shiraz, J. Magrane, J. Gao, H.R. Vollmer-Snarr, G.

Manfredi, F.C. Finnemann, The age lipid A2E and mitochondrial dysfunction synergistically impair phagocytosis by retinal pigment epithelial cells. J. Biol. Chem. 283 (2008) 24770- 24780.

[19] C. Bavik, S.H. Henry, Y. Zhang, K. Mitts, T. McGinn, E. Budzynski, A. Pashko, K.L.

Lieu, S. Zhong, B. Blumberg, V. Kuksa, M. Orme, I. Scott, A. Fawzi, R. Kubota, Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity, PLoS One 10 (2015) e0124940.

[20] P.D. Kiser, J. Zhang, M. Badiee, J. Kinoshita, N.S. Peachey, G.P. Tochtrop, K.

Palczewski, Rational Tuning of Visual Cycle Modulator Pharmacodynamics, J. Pharmacol.

Exp. Ther. 362 (2017) 131-145.

[21] K. Kaarniranta, P. Tokarz, A. Koskela, J. Paterno, J. Blasiak J, Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration, Cell Biol Toxicol. 33 (2017) 113-128.

[22] D.A. Ferrington, D. Sinha, K. Kaarniranta, Defects in retinal pigment epithelial cell proteolysis and the pathology associated with age-related macular degeneration. Prog. Retin.

Eye Res. 51 (2016) 69-89.

ACCEPTED MANUSCRIPT

[23] A. Subrizi, E. Toropainen, E. Ramsay, A.J. Airaksinen, K. Kaarniranta, A. Urtti, Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration,

Pharm. Res. 32 (2015) 211-221.

[24] J. Viiri, M. Amadio, N. Marchesi, J.M. Hyttinen, N. Kivinen, R. Sironen, K. Rilla, S.

Akhtar, A. Provenzani, V.G. D'Agostino, S Govoni, A. Pascale, H. Agostini, G. Petrovski, A.

Salminen, K. Kaarniranta, Autophagy activation clears ELAVL1/HuR-mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human retinal pigment epithelial cells, PLoS One 8 (2013) e69563.

[25] I. Johansson, V.T. Monsen, K. Pettersen, J. Mildenberger, K. Misund, K. Kaarniranta, S.

Schønberg, G. Bjørkøy, The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells, Autophagy 11 (2015) 1636-1651.

[26] S.K. Mitter,C. Song, X. Qi, H. Mao, H. Rao, D. Akin, A. Lewin, M. Grant, W. Jr Dunn, J. Ding, C Bowes Rickman, M. Boulton, Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD, Autophagy 10 (2014) 1989-2005.

[27] R. Russo, C. Nucci, M.T. Corasaniti, G. Bagetta, L.A. Morrone, Autophagy dysregulation and the fate of retinal ganglion cells in glaucomatous optic neuropathy,

Prog. Brain. Res. 220 (2015) 87-105.

[28] J. Yao, L. Jia, K. Feathers, C. Lin, N.W. Khan, D.J. Klionsky, T.A. Ferguson, D,N.

Zacks, Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration, Autophagy, 12 (2016) 2439-2450.

[29] K. Kaarniranta, G. Petrovski, A. Kauppinen, The Nobel Prized cellular target autophagy in eye diseases, Acta Ophthalmol. 95 (2017) 335-336.

[30] L. Galluzzi, J.M. Bravo-San Pedro, B. Levine, D.R. Green, G. Kroemer,Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles, Nat. Rev. Drug Discov. 16 (2017) 487-511.

[31] P. Boya, L. Esteban-Martínez, A. Serrano-Puebla, R. Gómez-Sintes, B. Villarejo-Zori, Autophagy in the eye: Development, degeneration, and aging, Prog. Retin. Eye Res. 55 (2016) 206-245.

[32] Y. Kitaoka, Y. Munemasa, K. Kojima, A. Hirano, S. Ueno, H. Takagi, Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration, Cell Death Dis. 17 (2013) 4:e860.

[33] K. Kaarniranta, A. Kauppinen, J. Blasiak, A. Salminen, Autophagy regulating kinases as potential therapeutic targets for age-related macular degeneration, Future Med. Chem. 4 (2012) 2153-2161.

[34] Y. Cheng, X Ren, W.N. Hait, J-M Yang, Therapeutic targeting of autophagy in disease:

biology and pharmacology, Pharmacol. Rev. 65 (2013) 1162-1197.

ACCEPTED MANUSCRIPT

[35] N. Piippo, A. Korkmaz, M. Hytti, K. Kinnunen, A. Salminen, M. Atalay, K. Kaarniranta K, Kauppinen A, Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells, Biochim. Biophys. Acta 1843 (2014) 3038-3046.

[36] J. Liu, D.A. Copland, S. Theodoropoulou, H.A. Chiu, M.D. Barba, K.W. Mak, M. Mack, L.B. Nicholson, A.D. Dick, Impairing autophagy in retinal pigment epithelium leads to inflammasome activation and enhanced macrophage-mediated angiogenesis, Sci. Rep. 6 (2016) 20639.

[37] K. Kaarniranta, A. Salminen, E.L. Eskelinen, J. Kopitz J, Heat shock proteins as gatekeepers of proteolytic pathways-Implications for age-related macular degeneration (AMD), Ageing Res Rev. 8 (2009):128-139.

[38] M. Boulton, A. Dontsov, J. Jarvis-Evans, M. Ostrovsky, D. Svistunenko, Lipofuscin is a photoinducible free radical generator, J. Photochem. Photobiol. B. 19 (1993) 201–204.

[39] A. Terman, U.T. Brunk, Lipofuscin, Int. J. Biochem. Cell Biol. 36 (2004) 1400–1404.

[40] J.R. Sparrow, B. Cai, N. Fishkin, Y.P. Jang, S. Krane, H.R. Vollmer, J. Zhou, K.

Nakanishi, A2E, a fluorophore of RPE lipofuscin: Can it cause RPE degeneration? Adv. Exp.

Med. Biol, 533 (2003) 205-211.

[41] C.L. Nordgaard, P.P. Karunadharma, X. Feng, T.W. Olsen, D.A. Ferrington, Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age- related macular degeneration, Invest. Ophthalmol. Vis Sci. 49 (2008) 2848-2855.

[42] J. Feng , X. Chen , X. Sun, F. Wang, X. Sun, Expression of endoplasmic reticulum stress markers GRP78 and CHOP induced by oxidative stress in blue light-mediated damage of A2E-containing retinal pigment epithelium cells, Ophthalmic Res. 52 (2014) 224-233.

[43] A. Salminen, K. Kaarniranta, A. Kauppinen, Regulation of longevity by FGF21:

Interaction between energy metabolism and stress responses, Ageing Res Rev. 37 (2017) 79- 93.

[44] L. Minasyan, P.G. Sreekumar, D.R. Hinton, R. Kannan, Protective Mechanisms of the Mitochondrial-Derived Peptide Humanin in Oxidative and Endoplasmic Reticulum Stress in RPE Cells, Oxid. Med. Cell Longev. 2017;2017:1675230.

[45] M. Hamasaki, N. Furuta, M. Matsuda, A. Nezu, A. Yamamoto, N. Fujita, H. Oomori, T.

Noda, T. Haraguchi, Y.Hiraoka, A. Amano, T. Yoshimori, Autophagosomes form at ER- mitochondria contact sites, Nature 495 (2013) 389-393.

[46] D. Matsunaga, P.G. Sreekumar, K. Ishikawa, H. Terasaki, E. Barron, P. Cohen, R.

Kannan, D.R. Hinton DR. Humanin Protects RPE Cells from Endoplasmic Reticulum Stress- Induced Apoptosis by Upregulation of Mitochondrial Glutathione. PLoS One 11 (2016) e0165150.

[47] P.G. Sreekumar, K. Ishikawa, C. Spee, H.H. Mehta, J. Wan, K. Yen, P. Cohen, R.

Kannan, D.R. Hinton, The Mitochondrial-Derived Peptide Humanin Protects RPE Cells From

ACCEPTED MANUSCRIPT

Oxidative Stress, Senescence, and Mitochondrial Dysfunction, Invest. Ophthalmol. Vis. Sci.

57 (2016) 1238-1253.

[48] H. Xu, A. Manivannan, J. Liversidge, P.F. Sharp, J.V. Forrester, I.J. Crane, Requirements for passage of T lymphocytes across non-inflamed retinal microvessels, J.

Neuroimmunol. 142 (2003) 47-57.

[49] M. Chen, H. Xu, Parainflammation, chronic inflammation, and age-related macular degeneration. J. Leuk. Biol., 98, (2015) 713-725.

[50] M. Karlstetter, R. Scholz, M. Rutar, W.T. Wong, J.M. Provis, T. Langmann, Retinal microglia: Just bystander or target for therapy? Prog. Retin. Eye Res. 45 (2015) 2030- 2057.

[51] T. Langmann, Microglia activation in retinal degeneration, J. Leuk. Biol. 81 (2007), 1345-1351.

[52] C.S. Jack CS, N. Arbour, J. Manusow, V. Montgrain, M. Blain, E. McCrea, A. Shapiro, J.P. Antel, TLR signaling tailors innate immune responses in human microglia and astrocytes. J. Immunol. (Baltimore, Md.: 1950), 175 (2005) 4320-4330.

[53] D.M. Balak, M.B. van Doorn, R.D. Arbeit, R. Rijneveld, E. Klaassen, T. Sullivan, R.

Rissmann, IMO-8400, a toll-like receptor 7, 8, and 9 antagonist, demonstrates clinical activity in a phase 2a, randomized, placebo-controlled trial in patients with moderate-to- severe plaque psoriasis, Clin. Immunol. (Orlando, Fla.) 174 (2017) 63-72.

[54] J.D. Cherry, J.A. Olschowka, M.K. O'Banion, Neuroinflammation and M2 microglia:

The good, the bad, and the inflamed. J.Neuroinflamm. 11 (2014) 98.

[55] D.J. Shealy, S. Visvanathan, Anti-TNF antibodies: Lessons from the past, roadmap for the future. Handb. Exp. Pharmacol., 181 (2008), 101-129.

[56] P.F. Zipfel, C. Skerka, C, Complement regulators and inhibitory proteins. Nature Rev.

Immunol. 9 (2009) 729-740.

[57] A. Lakkaraju, K.A. Toops, J. Xu, Should I stay or should I go? trafficking of sub-lytic MAC in the retinal pigment epithelium, Adv. Exp. Med. Biol. 801 (2014) 267-274.

[58] K. Lueck, S. Wasmuth, J. Williams, T.R. Hughes, B.P. Morgan, A. Lommatzsch, J.

Greenwood, S.E. Moss, D. Pauleikhoff, D, Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration, Eye (London, England) 25 (2011) 1074-1082.

[59] D.H. Anderson, M.J. Radeke, N.B. Gallo, E.A. Chapin, P.T. Johnson, C.R. Curletti, L.S.

Hancox, J. Hu, J.N. Ebright, G. Malek, M.A. Hauser, C.B. Rickman, D. Bok, G..

Hageman, L.V. Johnson, The pivotal role of the complement system in aging and age- related macular degeneration: Hypothesis re-visited, Prog. Retin. Eye Res. 29 (2010) 95-112.

[60] C. Luo, M. Chen, M. H. Xu, Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid, Mol. Vis. 17 (2011) 1588-1597.

[61] H. Xu, M. Chen, Targeting the complement system for the management of retinal inflammatory and degenerative diseases. Eur. J. Pharmacol. 787 (2016) 94-104.

ACCEPTED MANUSCRIPT

[62] M. Chen, J.V. Forrester, H. Xu, Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments, Exp. Eye Res. 84 (2007) 635-645.

[63] M. Chen, E. Muckersie, M. Robertson, J.V. Forrester, H. Xu, Up-regulation of complement factor B in retinal pigment epithelial cells is accompanied by complement activation in the aged retina, Exp. Eye Res. 87 (2008), 543-550.

[64] M. Chen, E. Muckersie, C. Luo, J.V. Forrester, H. Xu, Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis, Eur. J. Immunol. 40 (2010), 2870-2881.

[65] S.K. Vanaja, V.A. Rathinam, K.A. Fitzgerald, Mechanisms of inflammasome activation:

Recent advances and novel insights, Trends Cell Biol. 25 (2015), 308-315.

[66] D. Sharma, T.D. Kanneganti, The cell biology of inflammasomes: Mechanisms of inflammasome activation and regulation, J. Cell Biol. 213 (2016), 617-629.

[67] A. Kauppinen, H. Niskanen, T. Suuronen, K. Kinnunen, A. Salminen, K. Kaarniranta, Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells-implications for age-related macular degeneration (AMD), Immunol. Lett., 147 (2012), 29-33.

[68] W.A. Tseng, T. Thein, K. Kinnunen, K. Lashkari, M.S. Gregory, P.A. D'Amore, B.R.

Ksander, NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: Implications for age-related macular degeneration, Invest.

Ophthalmol. Vis. Sci. 54 (2013) 110-120.

[69] S.L. Cassel, S. Joly, F.S. Sutterwala, The NLRP3 inflammasome: A sensor of immune danger signals, Sem. Immunol. 21 (2009) 194-198.

[70] K. Shimada, T.R. Crother, J. Karlin, J. Dagvadorj, N. Chiba, S. Chen, S., V. K.

Ramanujan A.J. Wolf, L. Vergnes, D.M. Ojcius, A. Rentsendorj, M. Vargas, C. Guerrero, Y. Wang, K.A. Fitzgerald, D.M. Underhill, T. Town, M. Arditi, Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis, Immunity 36 (2012), 401- 414.

[71] R. Zhou, A.S. Yazdi, P. Menu, J. Tschopp, A role for mitochondria in NLRP3 inflammasome activation, Nature 469 (2011), 221-225.

[72] K. Triantafilou, T.R. Hughes, M. Triantafilou, B.P. Morgan, The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation, J. Cell Sci. 126 (2013) 2903-2913.

[73] V. Tarallo, Y. Hirano, B.D. Gelfand, S. Dridi, N. Kerur, Y. Kim, W.G. Cho, H. Kaneko, B.J. Fowler, S. Bogdanovich, R.J. Albuquerque, W.W. Hauswirth, V.A. Chiodo, J.F.

Kugel, J.A. Goodrich, S.L. Ponicsan, G. Chaudhuri, M.P. Murphy, J.L. Dunaief, B.K.

Ambati, Y. Ogura, J.W. Yoo, D.K. Lee, D. Provost, D.R. Hinton, G. Núñez, J.Z. Baffi, M.E. Kleinman, J. Ambati, DICER1 loss and alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88, Cell 149 (2012), 847-859.

[74] W. Chen W, M. Zhao, S. Zhao, Q.Lu, L.Ni, C. Zou, L. Lu, X. Xu, H. Guan, Z. Zheng, Q,.Qiu, Activation of the TXNIP/NLRP3 inflammasome pathway contributes to inflammation in diabetic retinopathy: a novel inhibitory effect of minocycline, Inflamm.

Res. 66 (2017):157-166.

ACCEPTED MANUSCRIPT

[75] H.R. Jiang, X. Wei, W. Niedbala, L. Lumsden, F.Y. Liew, J.V. Forrester, IL-18 not required for IRBP peptide-induced EAU: Studies in gene- deficient mice, Invest.

Ophthalmol. Vis. Sci, 42 (2001) 177-182.

[76] J. Shen, D.F. Choy, T. Yoshida, T. Iwase, G. Hafiz, B. Xie, S.F. Hackett, J.R. Arron, P.A.. Campochiaro, Interleukin-18 has antipermeablity and antiangiogenic activities in the eye: Reciprocal suppression with VEGF, J. Cell Physiol. 229 (2014) 974-983.

[77] S.L. Doyle SL, M. Campbell, E. Ozaki , R.G. Salomon , A. Mori , P.F. Kenna , G.J.

Farrar , A.S. Kiang , M.M. Humphries, E.C. Lavelle, L.A. O'Neill, J.G. Hollyfield, P.

Humphries, NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components, Nat. Med., 18 (2012), 791-798.

[78] S.L. Doyle, E. Ozaki, K. Brennan, M.M. Humphries, K. Mulfaul, J. Keaney, P.F. Kenna, A. Maminishkis, A.S. Kiang, S.P. Saunders, E. Hams, E.C. Lavelle, C. Gardiner,P.G.

Fallon, P. Adamson, P. Humphries, M. Campbell, IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration, Sci. Transl. Med. 6 (2014), 230ra44.

[79] E. Ozaki, M. Campbell, S.L. Doyle, Targeting the NLRP3 inflammasome in chronic inflammatory diseases: Current perspectives. J. Inflamm. Res. 8 (2015) 15-27.

Figure legends

Graphical abstract.RPE cells in the macula are constantly exposed to the daily phagocytosis of photoreceptor outer segments (POS; heterophagy). In aged RPE cells, degradation of POS decreases and lipofuscin start to accumulate in lysosomes as a result of the coincident decline of lysosomal enzyme activity. The impaired lysosomal enzyme activity inhibits autophagic flux. Lipofuscin increases oxidative stress that lead to protein misfolding, cellular organelle damages and protein aggregation. Prior to aggregation, heat-shock proteins (Hsps) attempt to refold misfolded proteins. Once Hsp repair capacity is exceeded, individual polypeptides can be degraded by the ubiquitin (Ub) targeted proteasome, while aggregates are degraded by autophagy. p62/SQSTM1 sorts proteins between proteasomal and autophagic clearance pathways. It binds to Ub cargos and to LC3. Disturbed proteostasis and accumulated toxic compounds trigger the progression from para-inflammation to chronic inflammation and evoke the AMD-associated formation of extracellular drusen formation and complement activation. Similar intracellular mechanisms are obvious also in ganglion cells, photoreceptors and microglia, although lipofuscin accumulation is the most prominent in RPE cells and phagocytosis is characteristic in both RPE and microglia.

Figure 1. (A) Optical coherent tomography A-scan and (B) B-scan from macula.

Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer;

RPE, retinal pigment epithelium.

Figure 2. Schematic presentation of RPE drug targets for phagocytosis, AMPK, mTOR and autophagy. Abbreviations: AICAR, adenosine analogue 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, adenosinemonophosphate-activated protein kinase; ER, endoplasmic reticulum; mLST-8, target of rapamycin complex subunit LST8; mTOR, mechanistical target

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of rapamycin; mTORC1, mechanistical target of rapamycin complex 1; PRAS40-P, Phospho proline-rich Akt substrate of 40kDa; Rheb, Ras homolog enriched in brain;

Figure 3. Key steps of the complement activation cascades and available compounds targeting the complement system. Initiation: The complement system can be activated by at least three pathways, which all lead to the formation of C3 convertase. Amplification: when C3 is cleaved into C3a and C3b, C3b can form C3 convertase with the fragment of factor B (Bb) amplifying the complement activation cascade. C3b is essential for the formation of C5 convertase with C4b or Bb. The effectors of the complement system include C3a, C5a and C5b-9. Complement activation can be effectively controlled at each step.

Figure 4. Inflammasome-related drugs and their targets. Currently, there are already several compounds available at regulating either the priming or the activation phase of inflammasome signaling. In addition, the functionality of the cytokines IL-1 and IL-18 released from the cell by caspase-1-mediated maturation can be controlled. Abbreviations:

ASC, apoptosis-associated speck-like protein containing caspase-recruitment domain [CARD]; Cys-LT-RA, cysteinyl leukotriene receptor antagonist; IKK, inhibitor of nuclear factor kappa-B kinase subunit beta; IL-1R, IL-1 receptor; NF-B, nuclear factor kappa beta;

NLRP3, Nucleotide-binding domain and Leucine rich repeat Receptor containing a Pyrin domain 3; TLR, Toll-like receptor.