Complement modulation reverses pathology in Y402H‐retinal pigment epithelium cell model of age‐related macular degeneration by restoring lysosomal function

Abstract Age‐related macular degeneration (AMD) is a multifactorial disease, which is characterized by loss of central vision, affecting one in three people by the age of 75. The Y402H polymorphism in the complement factor H (CFH) gene significantly increases the risk of AMD. We show that Y402H‐AMD‐patient‐specific retinal pigment epithelium (RPE) cells are characterized by a significant reduction in the number of melanosomes, an increased number of swollen lysosome‐like‐vesicles with fragile membranes, Cathepsin D leakage into drusen‐like deposits and reduced lysosomal function. The turnover of C3 is increased significantly in high‐risk RPE cells, resulting in higher internalization and deposition of the terminal complement complex C5b‐9 at the lysosomes. Inhibition of C3 processing via the compstatin analogue Cp40 reverses the disease phenotypes by relieving the lysosomes of their overburden and restoring their function. These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation.


| INTRODUCTION
Maintenance of cellular homeostasis is critical for cellular survival and is achieved through degradation of damaged cellular proteins/organelles and continuous recycling of metabolites. The autophagylysosome pathway plays an important role in maintaining this cellular homeostasis by sequestering unnecessary and damaged cellular components into autophagosomes, which ultimately fuse with lysosomes where their content is degraded. 1 A block in this system leads to the build-up of waste material, which has the potential to cause damage and even cell death. The oxygen-rich environment of the outer retina results in high levels of oxidized waste that needs to be removed to preserve vision. Retinal photoreceptors are highly metabolic cells subjected to light-induced oxidative stress, which increases the burden of damaged organelles and macromolecules. 2,3 The homeostasis of photoreceptors is critically dependent on an active autophagy response, that follows the circadian cycle and ensures that damaged macromolecules and metabolites are removed efficiently and cellular organelles (mitochondria, ribosomes, Golgi complex, and outer segments) are repaired. 4 Retinal pigment epithelial (RPE) cells are exposed to oxidative stress due to their constant exposure to light, high levels of lipid peroxidation products from photoreceptors, and high oxygen utilization. RPE cells phagocytose photoreceptor outer segments (POS) daily and both phagocytosis and autophagy converge into the lysosomes for degradation of their substrates. As such, RPE cells are highly susceptible to lysosomal dysfunction, which in turn has been shown to result in accumulation of large degradation resistant macromolecules, such as lipofuscin, and release of partially digested or undigested material. 2 Lipofuscin accumulation can increase lysosomal pH and reduce lysosomal degradation, which in turn impairs the functions of RPE leading to RPE and/or photoreceptor loss. 3,5 For these reasons, any impairment in autophagy could be detrimental to cellular functions of RPE and retinal cells.
Dysregulation of the autophagy-lysosome pathway has been implicated in a number of inflammatory, neurodegenerative, and agerelated diseases, 6 including age-related macular degeneration (AMD).
AMD is one of the most common causes of blindness, affecting one in three people by the age of 75. It accounts for 50% of blind and partially sighted registration with an estimated prevalence of 600 000 significantly visually impaired people in the United Kingdom and over 8 million worldwide. 7-10 AMD is a multifactorial progressive disease with a complex interaction between environmental, metabolic, and hereditary factors as well as chronic innate immune activation. 11 Vision loss in AMD is caused either by apoptosis of the RPE or overlying photoreceptors or both. AMD occurs in two advanced forms: "dry" AMD where cellular debris called drusen accumulates between the choroid and the retina with RPE and photoreceptor cell atrophy, and the "wet" form where neovascularization from the choroid occurs underneath the retina. Treatments are in widespread use for the choroidal neovascularization associated with wet AMD including anti-VEGF agents [12][13][14] ; however, no treatment exists for dry AMD and there is a huge unmet need for investigations into therapies for this disease. The James Lind Alliance priority setting exercise in AMD research identified the creation of treatments to stop the progress of dry AMD as the number one priority in AMD research. 15 Several lines of evidence suggest an association between dysfunctional autophagy-lysosome pathway and AMD. For example, increased autophagic flux and higher expression of autophagic key proteins have been observed in retinae of early AMD patients, which may suggest an increased demand for clearance of accumulated damaged organelles and macromolecules at the early stages of the disease. 16,17 However, the opposite was observed in late stages of AMD, which may reflect an overload or dysfunction of the autophagic system that is unable to cope with the increased demand for clearance of damaged organelles. This is corroborated by findings that key pathogenic features of AMD, namely increased mitochondrial damage, lipid peroxidation and accumulation of N-retinylidene-N-retinylethanolamine (A2E, a key component of lipofuscin) can result in decreased lysosomal activity in RPE cells and impairment of autophagosome-lysosome fusion. 17,18 Autophagic and exosomal proteins are found in drusen, leading to the hypothesis that increased autophagic activity together with lysosomal dysfunction may result in the release of intracellular proteins via exosomes. 17 Mice deficient in key components of autophagy (Beclin 1 and Atg7) develop severe retinal degeneration upon light exposure 19 and bi-allelic mutations in the autophagy regulator DRAM2 result in development of retinal degeneration with early macular cone photoreceptor involvement, suggesting an important role for autophagy in retinal homeostasis and function. 20 Despite these associations, it remains unclear whether changes in autophagic flux and function are a cause or a consequence of disease.
The paucity of information on the role of autophagy in the pathophysiology of AMD in previous studies was due to the lack of an adequate human in vitro AMD disease model that recapitulates many aspects of this multifactorial disease and can be used as a reliable source to study the role of autophagy in AMD. We have been able to overcome this limitation by developing a physiologically relevant RPE human disease model of AMD caused by the most common risk factor (complement factor H [CFH] polymorphism Y402H) that recapitulates key

Significance statement
Currently, there is no treatment for dry age-related macular degeneration (AMD), which comprises the majority of AMD pathology. In a collaborative effort, this study describes a novel link between uncontrolled complement activation and autophagy-lysosome axis, which is caused by increased deposition of the terminal attack complex C5b-9 at the lysosomes, leading to their overburdening and malfunction.
Using an inhibitor of C3 processing, Cp40, this study shows that all the disease phenotypes are reversed, relieving the lysosomes of their overburden and restoring their function.
These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation. features of AMD and displays an impaired autophagic response. 21 Recent data suggest that complement activation can contribute to regulation of autophagy-lysosome pathway in infective and inflammatory diseases. 22 For example, sublytic C5b-9 membrane attack triggers lysosomal membrane permeabilization and cathepsin leakage from lysosomes resulting in podocyte injury in idiopathic membranous nephropathy. 23 C3 protein has also been shown to be expressed intracellularly in pancreatic β cells, where it binds autophagy-related protein ATG16L1, regulating autophagy and protecting β cells from dying, highlighting a novel intracellular protective role for this important complement regulator. 24 Interestingly, complement activation of C3 in other cell types (eg, human CD4+ T cells) has been shown to occur via cathepsin L-mediated cleavage, resulting in C3a mediated stimulation of intracellular C3aR signaling in lysosomes, which sustains homeostatic T-cell survival. 25 Collectively this evidence suggests complex links between intracellular complement-mediated signaling, autophagy, and fluid-phase complement activation, which is cell typedependent.
In this article, we have used the induced pluripotent stem cell derived RPE (iPSC-RPE) disease model to investigate which steps of autophagy-lysosome pathway are affected in AMD patients with the Y402H-CFH polymorphism and the interplay between complement activation and the autophagy-lysosome pathway. Our data suggest that Y402H patient-specific RPE cells are characterized by increased C3 turnover, which results in increased C5b-9 deposition within lysosomes, resulting in their swelling and reduced membrane integrity.
Inhibition of C3 turnover with the compstatin analogue Cp40 results in the reversal of the RPE cellular phenotypes, the restoration of the lysosomal number, size and function, and a significant reduction in deposition of drusen-like deposits.

| High-risk RPE cells are characterized by an expanded and less functional lysosomal compartment and disrupted melanogenesis
In our previous study, we reported a significant increase in LC3 puncta and p62/SQSTM1 aggregates in RPE cells derived from high risk (homozygous for the Y402H-CFH polymorphism) and affected AMD patients compared to those derived from low-risk (homozygous for the wild type CFH) nonaffected subjects, which suggests autophagosome accumulation. 21 To investigate in detail which step of autophagy is affected in high-risk RPE cells, we analyzed markers of autophagosome initiation (ATG12-ATG5) and formation (LC3-I/II) as well as lysosomal (CTSD, LAMP1, and p62) and late endosomal/exosomal (CD63) markers. In accordance with our previous study, we observed a significant increase in the expression of LC3-II, p62, and ATG12-ATG5 ( Figure S1A) and LC3 and p62 puncta in RPE cells derived from high-risk AMD patients ( Figure S1B). We also performed autophagy flux experiments, by combining application of a known allosteric mammalian Target of Rapamycin Complex I (mTORC1) inhibitor and autophagy inducer, Rapamycin for 24 hours with Bafilomycin A1 (late stage autophagy inhibitor) treatment for the last 4 hours of treatment. These flux experiments showed a decrease in pS6 expression in both low-and high-risk RPE cells upon application of Rapamycin ( Figure S1C). As expected, low-risk RPE cells showed an increase in LC3-II expression upon application of Rapamycin, which was further augmented when late stages of autophagy were blocked by Bafilomycin A1 expression; however, this was not the case for high-risk RPE cells, where combined application of Rapamycin and Bafilomycin did not augment LC3-II expression compared to Rapamycin treatment alone ( Figure S1C). Together, these data suggest reduced autophagic flux in the high-risk RPE cells.
LC3 is a constitutive component of autophagosomal membranes, while p62 is a target of autophagy degradation. Increased expression of both markers in high-risk RPE cells suggests a block in the late stages of the autophagy-lysosome pathway; hence, we proceeded with analysis of markers involved in the degradation process. We did not see a difference in expression of early endosome marker RAB5 between low-and high-risk RPE cells ( Figure 1A), however, LAMP1 and CD63 expression was significantly increased in high-risk RPE cells ( Figure 1A), suggesting an expansion of the late endosomal/lysosomal compartment and/or increased exocytosis. These findings were further corroborated by increased Lysotracker staining in high-risk RPE cells ( Figure 1B). Interestingly, LAMP2 expression was significantly reduced in high-risk RPE cells, suggesting an impaired lysosomal maturation in high-risk RPE cells ( Figure 1A). The expression of the key lysosomal enzyme, Cathepsin D, and its activity were significantly reduced ( Figure 1A,C) in high-risk RPE cells. Together these data suggest that high-risk RPE cells are characterized by an expanded, albeit less functional lysosomal compartment.
We performed transmission electron microscopy (TEM) analysis to quantify the number of melanosomes and lysosomes. We observed the presence of melanosomes type 3 (where the lamellae are not tightly compacted) and mature melanosomes type 4 (where the melanin deposits are thick preventing visibility of fibrils in the internal F I G U R E 1 Assessment of lysosomal marker expression in low-and high-risk RPE cells. A, Western blotting quantification shows no changes in RAB5 expression, a significant increase of LAMP1 and CD63 and a significant decrease of CTSD, pro-CTSD, and LAMP2 expression. All quantification data were normalized to the total protein blots and shown as fold change against low-risk RPE cells. Data shown as mean ± SEM (data from at least six replicates); B, Fluorescence microscopy analysis shows a significant increase of acidic vesicles detected through LysoTracker Green DND-26 in high-risk RPE cells. Data shown as mean ± SEM (data from at least six replicates); C, Cathepsin D activity assay shows a significant decrease of activity in high-risk compared to low-risk RPE cells. The activity assays data were normalized against low-risk RPE cells and shown as fold change. Data shown as mean ± SEM (data from three replicates). Statistical significance was assessed using an unpaired t test. HR, high-risk retinal pigment epithelium cells; LR, low-risk retinal pigment epithelium cells; RPE, retinal pigment epithelium Collectively these data suggest the most affected step of autophagy-lysosome pathway in high-risk RPE cells is at the lysosomes, which despite an increase in number are unable to mature and process the last stage of autophagy due to a reduction in Cathepsin D expression and activity, preventing autophagosome maturation and acidification.

| Inhibition of mTORC1 by Rapamycin application has no beneficial impact on high-risk RPE cells
Published studies have shown that activation of lysosomal function can be achieved by suppressing mTORC1 activity. 28 Seven day Rapamycin treatments were carried out in high-risk RPE cells to assess the impact on lysosomal number and ultrastructure. There were no significant changes in Cathepsin D activity assays in high-risk RPE cells in response to Rapamycin treatment ( Figure S2A). The number of lysosome-like vesicles was unchanged in Rapamycin treated high-risk RPE cells ( Figure S2B,C); however, a significant increase in the number of stress vacuoles was observed ( Figure S2D). Together these data suggest that application of Rapamycin has no beneficial impact on the high-risk RPE cells, thus corroborating the clinical trials of mTORC1 inhibitor 29 (sirolimus), which have shown no evidence of anatomical or functional benefit in treated eyes of patients with geographic atrophy. Furthermore, one of the trials suggested that application of sirolimus might potentially be associated with effects detrimental to visual acuity, which could be due to increased cell stress observed in this study in high-risk RPE cells in response to Rapamycin treatment. terminal complement complexes in association with the Apo-E rich sub-RPE deposits is a key feature of AMD. Recent published work suggests that RPE cells are able to mitigate the effect of complement attack by endocytosis of C5b-9 and processing through the lysosomes. 38 Given the higher C3 turnover in high-risk RPE cells, we hypothesized that more C5b-9 deposition would occur on the surface of high-risk RPE cell leading to higher internalization and localization within lysosomes. Immunofluorescence data showed a significantly increased colocalization of C5b-9 with ZO-1 ( Figure 4B) and a significantly higher intracellular C5b-9 accumulation in the high-risk RPE when compared to low-risk control cells ( Figure 5A), suggesting possible endocytosis of excess C5b-9 from the cell membranes. We used the colocalization of C5b-9 with LAMP2 as an indicator of internalized complement proteins within the more mature lysosomes. This analysis showed a significantly increased C5b-9 at the lysosomes in the highrisk RPE cells ( Figure 5B). Together these data suggest increased C3 turnover in high-risk RPE cells, which leads to increased deposition of C5b-9 associated with RPE cells, followed by enhanced internalization and overloading of the lysosomes.

| Inhibition of C3 with compstatin analogue
Cp40 results in reversal of cellular phenotypes, restoration of lysosomal function, and reduction of drusen-like deposits in high-risk RPE cells  Figure 6A) and C5b-9 associated with Cp40 treated high-risk RPE cells ( Figure 6B). In addition, the intracellular and lysosomal C5b-9 expression was significantly reduced ( Figure 7A,B) in the highrisk RPE cells in response to Cp40 treatment. This was associated with increased CTSD expression and Cathepsin D activity ( Figure S5A,B) and decreased lysosome size in Cp40 treated high-risk RPE cells ( Figure S5C). TEM analysis showed significant improvements in RPE ultrastructure with Cp40 treatment increasing the number of type 4 melanosomes and reducing the number of lysosome-like vesicles and stress vacuoles ( Figure 8A,B). Most importantly, Cp40 treatment reduced the fragility of lysosomal membranes ( Figure 8C) and the volume of drusen-like deposits in high-risk RPE cells ( Figure 8D).

| DISCUSSION
Our group 21 and others have successfully applied the iPSC approach to generate disease models for AMD. 42 These have helped to identify and/or validate the disease risk alleles 43 generated by genome-wide association studies 42 and to highlight key cellular phenotypes in AMD-RPE related to reduced defense against oxidative damage, 44 mitochondrial DNA damage and disintegration, 45 higher expression of complement and inflammatory markers, 46 dysregulated autophagy, 16,21,47 and activation of inflammasome signaling. 48  inhibition. 55 In accordance with these studies, we have found an increased number of lysosomes with fragile membranes and enlarged size in RPE cells generated from Y402H-AMD as well as an increased C3b and C5b-9 associated with RPE cells, C5b-9 internalization, and overloading of the lysosomes, which we hypothesize leads to the lysosomal damage and formation of drusen-like deposits. All these phenotypes are restored upon Cp40 application, indicating that the complement-associated-lysosomal damage in high-risk RPE cells is directly linked to increased C3 turnover in the high-risk RPE cells.
Both C3 activation and depletion have been associated with retinal degeneration, 56,57 indicating that careful control of C3 expression and its cleavage fragments needs to be considered for AMD therapies.
In view of these studies, we generated iPSC lines with stably integrated shRNAs to C3, which resulted in 85% reduction in C3 mRNA levels and C3 secretion into the supernatant of high-risk RPE cells (data now shown). TEM analysis indicated that C3 knockdown did not restore the number of melanosomes and lysosomes, but most importantly had a detrimental effect in the cells as it increased the number of stress vacuoles, which can lead to cell bursting and death ( Figure S6). In contrast to this, inhibition of C3 via the compstatin analogue Cp40 restored the number of lysosomes and melanosomes and reduced lysosomal membrane damage, deposition of drusen-like deposits and C5b-9 internalization. Cp40 prevents the conversion of C3 to C3b, which is essential for the initiation, amplification, and for-
The media was then partially replaced every 2 to 3 days until pigmented cell patches appeared. The pigmented patches were surgically excised, disassociated using TrypLE Select Enzyme (10X) (ThermoFisher) and seeded on Matrigel-coated plates in RPE media.

| Lysotracker green DND-26 assay
Fully confluent RPE cells were grown on Nunc 4-well cell culture treated dishes with Nunclon Delta surface (ThermoFisher). The lysotracker probe stock solution was warmed up to room temperature and diluted to 1 μM final working concentration in warm RPE cell culture medium. Growth medium was removed from cell culture dishes and the diluted lysotracker probe added to cells and incubated for 1 hour at optimal growth conditions. Following the incubation, the probe was removed and replaced with warm growth medium (wash) and a final volume of growth medium. Cells were imaged immediately using Nikon A1R confocal microscope at the exact same laser power in series with a 4x line average method.
Average image intensity was quantified from a maximum intensity projection analysis of 6 x20 images using Image J program (NIH). were stained with uranyl acetate and lead citrate before being imaged on a 120 kV HT7800 TEM (Hitachi). At least 10 cells were imaged per sample, selected randomly along the length of the insert, provided that the cell was intact with a clearly demarcated cell membrane.

| Transmission electron microscopy
Objects identified in the images were measured using ImageJ with tools calibrated to the burned-in scale bar.  Table S2.