Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence

doi:10.1167/iovs.65.4.32

. 2024 Apr 1;65(4):32.

doi: 10.1167/iovs.65.4.32.

Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence

Saeed Shahhossein-Dastjerdi^{1

2}, Mark E Koina³, George Fatseas¹, Frank Arfuso⁴, Tailoi Chan-Ling^{1

5}

Affiliations

¹ Bosch Institute, The University of Sydney, Sydney, Australia.
² Now at Cell, Tissue & Organ Bank, Forensic Medicine Research Centre, Tehran, Iran.
³ Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, Canberra, Australia.
⁴ School of Human Sciences, The University of Western Australia, Crawley, Australia.
⁵ https://orcid.org/0000-0002-8225-3671.

PMID: 38648041
PMCID: PMC11044829
DOI: 10.1167/iovs.65.4.32

Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence

Saeed Shahhossein-Dastjerdi et al. Invest Ophthalmol Vis Sci. 2024.

. 2024 Apr 1;65(4):32.

doi: 10.1167/iovs.65.4.32.

Authors

Saeed Shahhossein-Dastjerdi^{1

2}, Mark E Koina³, George Fatseas¹, Frank Arfuso⁴, Tailoi Chan-Ling^{1

5}

Affiliations

¹ Bosch Institute, The University of Sydney, Sydney, Australia.
² Now at Cell, Tissue & Organ Bank, Forensic Medicine Research Centre, Tehran, Iran.
³ Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, Canberra, Australia.
⁴ School of Human Sciences, The University of Western Australia, Crawley, Australia.
⁵ https://orcid.org/0000-0002-8225-3671.

PMID: 38648041
PMCID: PMC11044829
DOI: 10.1167/iovs.65.4.32

Abstract

Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence.

Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation.

Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years.

Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

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Conflict of interest statement

Disclosure: S. Shahhossein-Dastjerdi, None; M.E. Koina, None; G. Fatseas, None; F. Arfuso None; T. Chan-Ling, None

Figures

**Figure 1.**
Initial RPE development in first trimester between eight to 12 WG. All data for this study represent observations made on one human specimen per age group. A minimum of 5 fields of view at three levels separated by 50 µm intervals were captured and analyzed for all specimens at each age and location. (A) Pseudostratified columnar RPE and primitive ChC evident at eight WG. (B) RPE with different levels of nuclei (N) containing evenly dispersed chromatin, and primitive endothelial linings (*EnL*) with red blood cell (*asterisk*) evident. *Arrow* is pointing to incipient RPE cell membrane. (C) High-magnified view showing primitive apical tight junctions (TJ, *circle*), partial attachment of lateral borders, and randomly distributed melanosomes (M) and mitochondria (m). (D) Primitive inner neuroblastic layer (*iNL*) loosely attached to RPE through outer limiting membrane (*OLM*) at 10 WG. (E) *Circle* showing development of apicolateral junctional complexes (JC), incipient RPE cell membrane (*arrow*), and ChC. (F) High-magnified view showing a possible apoptotic nucleus (*red asterisk*) with chromatin margination, surrounded by a group of melanosomes and randomly localized mitochondria. (G) Montaged view showing differentiation from pseudostratified epithelia to simple cuboidal layer at 12 WG. (H) RPE cell membrane (*vertical arrowhead*), incipient patchy elastic layer (*horizontal arrow*), primitive inner and outer collagenous layers (*red asterisks*) are evident. (I) High-magnified view showing apicolateral JC (*circle*). (J) Pseudostratified columnar epithelia in association with iNL through OLM at eight WG differentiates to cuboidal epithelium at 12 WG. This summary diagram is based on tracings of actual cells or cell clusters at each developmental stage and were developed to represent a summation of huge numbers of fields of views to show changes in RPE morphology at the whole cell/cell cluster level. Images have been drawn to scale and represent a higher magnification view of cell detail than would have been possible by photographing the TEM thin sections stained with toluidine blue.

**Figure 2.**
RPE interact with POS and choriocapillaris in second trimester between 16 to 24 WG. One specimen per age was examined throughout this study where five fields of view at three levels separated by 50 µm were examined per specimen. Cellular enlargement, apicolateral specialization, and incipient formation of underlying BrM are evident. (A) The nuclei (N) of primitive cells of the inner neuroblastic layer (*iNL*), outer limiting membrane (*OLM*), and underlying primitive POS are evident at 16 WG. (B) Developing iNL contains nuclei of outer nucleus layer (*ONL*), large number of mitochondria (m), and tight junctions between adjacent cells (*circle*). (C) Apical development of RPE with microvilli (*horizontal arrow*) and junctional complexes (JC) (*circles*) evident. (D) Melanosomes randomly located in the cytoplasm, patchy elastic layer (*red asterisk*), and endothelial cells evident at 20 WG. Representative regions in *boxes* are shown in E and F. (E) RPE cell membrane (*blue arrow*), RPE and choroidal basal lamina (*red arrowheads*), inner and outer collagenous fibers (*red asterisks*), elastic layer (*blue asterisk*), and fenestration toward RPE (*red arrow*) evident. Basal infoldings are not present at this early age. (F) Elastic layer (*asterisk*) and pericyte (P) are shown. (G) Montaged view showing development of iNL including ONL, outer plexiform layer, amacrine and horizontal cells (*circles*) in proximal and distal areas of the inner nucleus layer (*INL*) at 24 WG. Accumulation of mitochondria (*asterisk*) evident beyond the OLM in primitive inner segment of photoreceptors. (H) Primitive POS, JC (*circle*), and localization of organelles evident. (I) Schema showing no significant change in cell width from 16 to 24 WG.

**Figure 3.**
Organelle relocation at 26 WG, coincident with eye opening. *Circles* show junctional complexes (JC) between adjacent RPE cells. Apical-basal redistribution of RPE organelles occurs sooner in the region adjacent to the ONH than in the peripheral region (PR). (A) Shows human RPE at 26 WG in the region adjacent to the ONH. Apical microvilli (*blue arrow*) and phagosome (Ph) are evident. Melanosomes (M, *bracket*) migrate to the apical region, and mitochondria (m) are localized in the perinuclear region (*red arrow*). Elastic layer (EL), RPE basal lamina (*RBL*), choroidal basal lamina (*CBL*), and BES are evident. (B) In contrast, apical microvilli (*blue arrow*), melanosomes, and mitochondria are located randomly in RPE cells from the periphery of a 26 WG specimen indicative of a disc to peripheral topography of RPE maturation. Group of large spherical melanosomes (*bracket*) are in the basolateral region. (C) Semiquantitative ultrastructural analysis demonstrates the average area of phagosomes, complex granules, and mitochondria in the region adjacent to the ONH is significantly greater than in the PR at 26 WG (P < 0.05). Accompanied with these changes, melanosomes occupy a larger area in the peripheral region compared to the region adjacent to the ONH. (D) More than 65% ± 3% of melanosomes localized apically in the region adjacent to the ONH, whereas 46% ± 5% are apically distributed in the mid periphery region. Melanosome distribution in RPE cells at 26 WG is expressed as mean ± SEM, per RPE cell.

**Figure 4.**
Characterization of BES at 26 WG, coincident with eye opening. (A) *Circles* show BES between adjacent RPE cells with different levels of nuclei (N). Melanosomes (M) migrate to the apical region. (B) The high magnified view shows the BES as a triangular space along the lateral border of RPE cells (*arrows*) delineated by the two cell membranes of adjacent RPE cells and RPE basal lamina of BrM. Mitochondria (m) are localized in the perinuclear region.

**Figure 5.**
Autophagy and mitophagy evident from 32 WG. (A) RPE and BrM evident at 32 WG. Red blood cells and endothelial cells (*EnC*) are visible in the ChC. Representative regions in boxes are shown in B and C. (B) High-magnified montage showing mitophagosome (*mPh*) and autophagosome (*aPh*). A well-characterized BrM is evident at this age because all its layers are discernible; RPE basal lamina (1), inner collagenous (2), elastic (3) outer collagenous (4) and choroidal basal lamina (5). (C) Circle shows apicolateral junctional complexes (JC). Melanosomes (M) and mitophagosome evident in the RPE cytoplasm adjacent to the basolateral border of RPE cells. Triangle shows the BES between adjacent RPE cells. (D) The JC (*circle*) and BES (*triangles*) delineate the apicolateral and basolateral borders of adjacent RPE cells at 36 WG. Representative region in box is shown in E. (E) Basal infoldings of RPE cell membrane (*asterisk*), mitochondria (*bracket*), and mitolysosomes (*mLy*) are evident. (F) Low-magnified view showing outer segment discs (*asterisk*) surrounded by apical microvilli (*curve*), JC (*circle*), mitochondria (*bracket*), and adjacent BES (*triangle*). Representative region in box is shown in G. (G) Various structures showing autophagosomes (*circle*), autolysosome (*aLy*), late (*top-right*) and early (*bottom-left*) mitophagosomes. (H) Photoreceptor outer segment discs recognized for phagocytosis and phagosome (Ph) evident adjacent to the apicolateral JC.

**Figure 6.**
Lipofuscin formation and exocytosis to the BES evident at six years. (A) The apicolateral junctional complexes (*oval*) and the BES (*triangle*) and underlying BrM are evident at six years. Boxes are represented in B and C. (B) Montaged view showing apical melanosomes (M), mitochondria (m), and mitophagosomes (*mPh*) in the basolateral region. (C) Autophagosome (*aPh*), autolysosome (*aLy*), and mitophagosomes (*mPh*) (*bracket*) are evident. Lipofuscin (Lf) exocytosed into the BES. (D, E) The representative images delignated the number of fenestrae (*red solid triangles*) in the choriocapillaris towards the RPE adjacent to the optic nerve head (D) and peripheral region (E). Red blood cells (*RBC*, *stars*), endothelial cells (*EnC*) extension surrounding the lumen, and pericyte (P) are evident. (F) Junctional complexes (*circle*), melanosomes, and mitochondria (*bracket*) adjacent to the basolateral border of RPE, and BES containing autophagic byproducts (*asterisk*) are evident at nine years. Mitochondria and mitophagosomes localized in the basolateral region adjacent to BES (*triangle*). (G) The phagolysosome (*PhLy*), Golgi apparatus (GA), lysosome (Ly), and autophagic byproduct deposition into the BES are evident. Mitochondria and mitophagosomes localized in the basolateral region adjacent to BES. (H) Lipofuscin granule evident adjacent to the BES (*triangle*) at 17 years. *Box* is shown in I. (I) Phagosome (Ph), mitochondria, and mitophagosome are characterized in high magnification view. (J) Schematic in which the left panel (26 WG) shows a cell with distinct apical melanosomes and perinuclear mitochondria development with the evidence of phagosome and complex granules formation at 26 WG. There is no evidence of drusen formation at this age. The *right panel* (17 years) shows a fully functional RPE cell. Mitochondria distribute adjacent to basal infoldings and in the vicinity of the BES, the site of byproducts deposition.

**Figure 7.**
ChC maturation follows central to peripheral topography of development. A semi-quantitative analysis was performed by using iTEM software. The mean ± SEM of all measurements for each five different areas of 25 capillaries at six years in high-magnification images (20,000–60,000) were analyzed by using a one-way ANOVA with post-hoc tests for analysis, and P < 0.05 was considered significant. (A) Thickness of the endothelial cell basal lamina was observed to be greater toward RPE when compared to the scleral side in both region adjacent to the ONH and peripheral region and significantly more in the peripheral region when compared to the region adjacent to the ONH. (B) The number of fenestrae in the ChC are significantly greater toward the RPE cells when compared to the scleral side in both region adjacent to the ONH and periphery and significantly more in the region adjacent to the ONH when compared to the peripheral region. Data are expressed as mean ± SEM, per RPE cell.

**Figure 8.**
Nuclear to cytoplasm ratio changes during pigment epithelial development. (A) The column chart represents the increase in nucleus area relative to total cytoplasmic area (*N/C*) ratio between 10 to 20 WG, followed by a significant decrease by 28 WG, and plateau after birth. (B) RPE height exceeds width significantly (8–12 WG) coinciding with a significant increase in the N/C ratio (P < 0.05). From 16 WG, cell width exceeds height to the later stages of development, with the exception at 26 WG (coinciding with eye opening) and 32 WG (both width and height are approximately equal). This morphological change is coincident with the significant decrease in N/C ratio to a lower level than seen at 8 WG, suggesting increased emphasis on the number and distribution of various organelles during the later period of development. The nuclear to cytoplasm ratio is expressed as mean ± SEM at each age where 10 cells were measured at each age.

**Figure 9.**
Thickness increasing of BrM through aging. The thickness of BrM was not assessed before 32 WG because it was incomplete. The thickness of BrM increases from 36 WG to six years, with no obvious change up to 17 years. There is a substantial increase in the thickness of BrM from six years, and this increases further as a function of age. Although there was clearly a trend of increasing BrM thickness because of the small sample size, no statistical analyses were undertaken. The thickness of Bruch's membrane is expressed as mean ± SEM, at each age where 10 cells were measured at each age.

**Figure 10.**
(A) The schema details the 16 key ultrastructural morphological stages in the maturation of human retinal pigment epithelial cells from early fetus to adolescence. The inner neuroblastic layer (*iNL*), outer neuroblastic layer (*oNL*), RPE, BrM, and ChC development are evident. (1) The iNL is loosely attached to the pseudostratified columnar RPE through the outer limiting membrane (OLM). (2) RPE cell membrane and primitive vessels with a layer of endothelial cells in ChC is evident at eight WG. (3) Formation of junctional complexes (JC) evident from 10 WG. (4) Apoptotic nucleus evident from 10 WG. (5) Simple cuboidal RPE and development of apicolateral JC evident at 12 WG. (6) Inner, outer collagenous fibers and elastic layer of BrM evident from 12 WG. (7) RPE maturation evident by interaction of apical microvilli with primitive photoreceptors in the oNL and a mature apicolateral JC at 16 WG. (8) Formation of RPE, and endothelial cell basal lamina and pericyte evident at 20 WG. (9) Development of iNL including OLM, outer nucleus layer (*ONL*), outer plexiform layer (*OPL*), and inner nucleus layer (*INL*) evident at 24 WG. (10) Accumulation of mitochondria in primitive photoreceptor inner segments (IS) and random distribution of RPE organelles are evident at 24 WG. (11) Coincident with eye opening, distinct apical and perinuclear redistribution of melanosomes and mitochondria, phagocytosis, and BES are evident at 26 WG. (12) The oNL consisting of the presumptive POS with differentiated iNL (including amacrine, *horizontal*, and Müller cells) is evident by 26 WG. (13) Autophagy, mitophagy, and well-characterized BrM evident at 32 WG. (14) Basal infoldings of RPE cell membrane are evident at 36 WG. (15) Lipofuscin and mitochondrial basolateral re-distribution evident at six years. Adjacent RPE cells are sealed and attached together through apicolateral JC including tight junctions, adherens junctions, desmosome, and gap junctions along the lateral borders. (16) Exocytosis of heterophagic and autophagic byproducts into the BES evident at 17 Y. (B) The schema represents the contribution of heterophagy, autophagy and mitophagy in ensuring a viable and functional RPE-photoreceptor outer segment complex as evidenced by extensive ultrastructural observations throughout human fetal and early adolescent development. As the RPE develops it forms apical and basal infoldings. The apical microvilli accommodate rod and cone OS whereas the basal infoldings optimize cell surface area for exchange of metabolites with the choroidal circulation. Tight junctions, gap junctions, and desmosomes are formed to increase physical strength between the cells. Because of the dynamics of this structure, we see formation of the BES (*blue*). The photoreceptors are renewed daily by shedding their OS that are phagocytosed (heterophagosome) and digested through a process known as heterophagy. When single-membrane phagosomes fuse with single-membrane lysosomes, they form double-membrane heterophagosomes followed by single-membrane heterolysosomes where the process of digestion occurs. This process results in both digested materials able to be recycled by the RPE and undigested materials eventually forming single-membrane lipofuscin and deposition into the BES and drusen located below the RPE and in the inner collagenous layer thereafter. Double-membrane autophagosomes/mitophagosomes are also formed by fusion of misfolded proteins and damaged organelles including dysfunctional mitochondria with lysosomes where the digestive process occurs through a process known as autophagy/mitophagy by formation of single-membrane autolysosomes/mitolysosomes. This also results in both recyclable materials and undigested components forming lipofuscin and eventually drusen. These metabolic demands on the RPE, in the basal region of the cell, encourage mitochondria to locate to this proximity. The recycling process occurs within the cytosol of the RPE cells. This schema represents the temporal region adjacent to the ONH in adolescence.

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References

1. Wong WL, Su X, Li X, et al. .. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Global Health. 2014; 2: e106–e116. - PubMed
1. Beatty S, Koh H-H, Phil M, Henson D, Boulton M. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000; 45: 115–134. - PubMed
1. Hall MO, Bok D, Bacharach A.. Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J Mol Biol. 1969; 45: 397–406. - PubMed
1. Bok D, Hall MO.. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol. 1971; 49: 664–682. - PMC - PubMed
1. Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci. 1993; 1993: 189–195. - PubMed

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[1] Wong WL, Su X, Li X, et al. .. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Global Health. 2014; 2: e106–e116. - PubMed

[2] Wong WL, Su X, Li X, et al. .. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Global Health. 2014; 2: e106–e116. - PubMed

[3] Beatty S, Koh H-H, Phil M, Henson D, Boulton M. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000; 45: 115–134. - PubMed

[4] Beatty S, Koh H-H, Phil M, Henson D, Boulton M. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000; 45: 115–134. - PubMed

[5] Hall MO, Bok D, Bacharach A.. Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J Mol Biol. 1969; 45: 397–406. - PubMed

[6] Hall MO, Bok D, Bacharach A.. Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J Mol Biol. 1969; 45: 397–406. - PubMed

[7] Bok D, Hall MO.. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol. 1971; 49: 664–682. - PMC - PubMed

[8] Bok D, Hall MO.. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol. 1971; 49: 664–682. - PMC - PubMed

[9] Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci. 1993; 1993: 189–195. - PubMed

[10] Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci. 1993; 1993: 189–195. - PubMed

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Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence

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Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence

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