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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Exp Eye Res. 2021 Oct 7;212:108787. doi: 10.1016/j.exer.2021.108787

β-cleavage of the prion protein in the human eye: Implications for the spread of infectious prions and human ocular disorders

Suman Chaudhary 1, Ajay Ashok 1, Aaron S Wise 1, Neil A Rana 1, Alexander E Kritikos 1, Ewald Lindner @, Neena Singh 1,*
PMCID: PMC8608713  NIHMSID: NIHMS1748316  PMID: 34624335

Abstract

Recently, we reported β-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly β-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from β-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigment epithelial cell line, also showed β-cleaved PrPC. Surprisingly, β-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that β-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, β-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for β1-integrin and amyloid-β, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models.

Keywords: Prion protein, β-cleavage, retina, aqueous humor, vitreous humor

1. Introduction

The prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-linked plasma membrane glycoprotein expressed ubiquitously. It is most abundant on neuronal cells and several cell types in the eye, including retinal ganglion cells (RGC) (Ashok et al., 2018; Orru et al., 2018; Sigurdson et al., 2019). PrPC is mostly known for its role in prion disorders, a group of neurodegenerative conditions of humans and animals where a conformational change in PrPC from a mainly α-helical to a β-sheet-rich PrP-scrapie (PrPSc) isoform renders it infectious and pathogenic (Singh, 2014; Singh et al., 2010, 2014). The infectious nature is due to the ability of exogenous PrPSc to transform host PrPC to its own, PrPSc conformation. The toxicity of PrPSc is believed to result from a combination of gain of toxic function and loss of physiological function of PrPC (Singh, 2014; Singh et al., 2010, 2014; Wulf et al., 2017). Several physiological functions have been described for PrPC, including iron and copper transport, neuroprotection, cell signaling, neuritogenesis, and other diverse functions, some ascribed to its post-translational processing (Ashok et al., 2018, 2019; Ashok and Singh, 2018; Bogdan et al., 2016; Castle and Gill, 2017; Linden, 2017; Lindner et al., 2020).

Three main post-translational cleavages have been described for PrPC, namely α-, β-, and ɣ-cleavage (Bremer et al., 2010; Lewis et al., 2016; Linsenmeier et al., 2017). In addition, significant amounts of full-length (FL), glycosylated PrPC lacking the GPI-anchor is shed from the plasma membrane. α-Cleavage of PrPC occurs at residues 111/112 of human PrPC in an endocytic compartment or at the plasma membrane. The C-terminal ~18 kDa (C1) fragment accumulates on the plasma membrane, and the N-terminal (N1) soluble fragment of 12 kDa is released in the extracellular milieu. Shedding and α-cleavage are considered physiological (Linsenmeier et al., 2018), and majority of PrPC (~50%) on neurons is cleaved at this site (Chen et al., 1995). An alternate cleavage at the β-site occurs under pathological conditions. Two main events are associated with β-cleavage; conformational conversion to PrPSc (Prusiner, 1998), and oxidative stress (Watt et al., 2005). This cleavage occurs on the cell surface near amino acid ~90, close to the octapeptide repeat region. The resulting C-terminal fragment of 20 kDa (C2) remains linked to the plasma membrane, while the soluble N-terminal fragment (N2) of ~9 kDa is released in the extracellular milieu. ɣ-Cleavage occurs close to the C-terminus between amino acids 170-200, resulting in a membrane bound C3 fragment of 6-7 kDa, and the corresponding N-terminal soluble fragment N3 (Lewis et al., 2016).

α-Cleavage is mediated by the A-disintegrin-and-metalloproteinase (ADAM) family of enzymes. ADAM10, and probably ADAM17 are implicated in this cleavage (Altmeppen et al., 2011, 2012; Pradines et al., 2009; Vincent et al., 2000; Vincent et al., 2001). β-cleavage is mediated by reactive oxygen species (ROS), and occupancy of Fe2+ and Cu1+ in the octapeptide repeat region facilitate this cleavage (McDonald et al., 2014). The soluble N-terminal fragments and membrane-bound C-terminal products of α-, β-, and ɣ-cleavage and shed PrPC perform diverse physiological functions, some of which are tissue and cell-specific (Ashok et al., 2018, 2019; Singh et al., 2020). For example, it is well-established that α-cleavage disrupts the amyloidogenic region of PrPC spanning amino acids 106-126 that precludes the conversion of C1 to PrPSc, a necessary event for the spread of prion disease (Chen et al., 1995). N1 is believed to protect the cells from amyloid-β (Aβ) mediated toxicity and other insults (Guillot-Sestier et al., 2009, 2012). C2, on the other hand, includes the amyloidogenic region, and readily undergoes conversion to PrPSc (Chen et al., 1995). Interestingly, C1 and not C2 is involved in regulating mRNA transcription of p53 and activating caspase-3, thus displaying pro-apoptotic function (Sunyach et al., 2007). Additionally, both C1 and C2 lack the octapeptide repeat region that contains binding sites for several ligands, some of which are implicated in ocular disorders (Lindner et al., 2020; Martins et al., 2010; Singh et al., 2013; Tripathi et al., 2015).

PrPC is known to protect photoreceptor cells from light-induced damage in mouse models, and PrPSc is known to mediate their destruction in addition to widespread damage to the retina (Frigg et al., 2006; Striebel et al., 2021). In human retinal pigment epithelial (ARPE-19) cells and trabecular meshwork (TM) cells important for regulating intra-ocular pressure, downregulation of PrPC creates iron deficiency and alters the extracellular matrix (Ashok et al., 2019; Asthana et al., 2017), suggesting a significant role in ocular pathophysiology. Surprisingly, PrPC is cleaved at the β-site in human ocular tissues (Ashok et al., 2019; Asthana et al., 2017), suggesting a response to light-induced oxidative stress or an alternate stimulus. Since C2, the C-terminal product of β-cleavage, is readily transformed to PrPSc, its presence increases the potential for infectious prion disease. It is therefore essential to identify the stimuli that lead to β-cleavage of PrPC in the human eye.

Here, we compared the cleavage of PrPC in the human eye with a select number of nocturnal and diurnal species with and without the tapetum lucidum, a layer of tissue that reflects light (Ollivier et al., 2004), and in response to a variety of stimuli. We report that β-cleavage of PrPC is limited to the human eye, and does not change significantly in reponse to a variety of insults. These observations are discussed in the context of human ocular disorders, and infection and dissemination of PrPSc in the human eye that is likely to differ from animal models conventionally used for such studies.

2. Methods

2.1. Human and animal eye tissue

Studies on human and animal eye globes were conducted in accordance with the tenets of the Declaration of Helsinki. Mice and rats used in this study were housed in the Association of Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved Animal Resource Center (ARC) at Case Western Reserve University (CWRU), School of Medicine. All animal experiments were approved by Institutional Animal Care and Use Committee at School of Medicine, CWRU, and were conducted in accordance with the guidelines of the Association for Research in Vision and Ophthalmology on the use of animals in research.

2.2. Tissue isolation from eye globes

Ocular tissues were isolated from eye globes as described in previous reports (Ashok et al., 2020b; Fernandez-Godino et al., 2016; Keller et al., 2018; MacNeil et al., 2007). The details of source, isolation and extraction of various ocular tissues are provided in the supplementary file.

2.3. Antibodies and chemicals

Details of chemicals and their sources as well as a list of antibodies used in the study are provided in the supplementary file and Table S1. Lipopolysaccharide (LPS) was obtained from Sigma Aldrich, USA (L2630). Mini-hepcidin PR73 was a kind gift of Elizabeth Nemeth (UCLA) and Thomas Ganz (UCLA). Replication-defective adenovirus expressing bioactive TGF-β2 (AdhuTGF-β2) or vector (AdEmpty) were obtained from University of Iowa. Sodium iodate was from Sigma Aldrich, USA (S4007-100G). ADAM10 (sc-41410) and ADAM17 (sc-36604) siRNA were obtained from Santa Cruz Biotechnology, Inc, USA.

2.4. Cell lines and experimental design

ARPE-19 (CRL-2303™) and BE(2)-M17 (CRL-2267™) cells were obtained from ATCC, and primary cultures of TM cells were established from human eye globes as described (Ashok et al., 2019, 2020a; Keller et al., 2018). Details of culture conditions and authentication of TM cells are provided in the supplementary file.

2.5. Western Blotting and RT-PCR

Tissue and cell lysates were fractionated by SDS PAGE and analyzed by Western Blotting as described (Ashok et al., 2019, 2020b; Ashok and Singh, 2018). The proteins of interest were visualized by probing with specific antibodies (Supplementary Table S1). (Full western blots and different exposures of blots are provided in supplementary Figure S4 and S5) Semi-quantitative RT-PCR was conducted as in previous reports (Ashok et al., 2020b) (List of primer sequences is provided in supplementary Table S2).

3. Results

3.1. PrPC is β- cleaved in the human eye

Human PrPC is an N-linked glycosylated protein that migrates between 27 and 37 kDa. α-Cleavage of PrPC disrupts the epitope for 3F4, and releases the 12 kDa (N1) N-terminal soluble fragment in the extracellular milieu. The 18 kDa (C1) C-terminal fragment remains attached to the plasma membrane, β-cleavage releases an N-terminal fragment of 9 kDa (N2), and the 20 kDa (C2) C-terminal fragment that includes the amyloidogenic region of PrPC and the epitope for 3F4 remains attached to the plasma membrane (C2) (Figure 1 A) (Chen et al., 1995; Wulf et al., 2017).

Figure 1. Processing of PrPc in the retina of human and animal species.

Figure 1.

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(A) Schematic representation of full length (FL, 27-37 kDa), C1 (α-cleaved, 18 kDa), and C2 (β-cleaved, 20 kDa) forms of PrPC. C-terminal antibodies 8H4 and G12 react with FL, C1 and C2. 3F4 reacts with FL and C2, not C1. (B) WB of glycosylated and deglycosylated retinal lysates from human, pig, cattle, goat, mouse (C57BL/6), and rat reveal mainly C2 in the human retina (lanes 1 & 2), and predominantly FL PrPC in animal species (lane 3-8 & 10-13). Human brain homogenates processed in parallel show mostly FL PrPC and C1 as expected (lanes 9, 14 & 15). (C) Quantification by densitometry shows the approximate ratio of FL vs C2 and C1 in 3 different estimations. (D) WB of glycosylated and deglycosylated lysates from human ocular tissues show mainly C2 in the retina, sclera, iris, corneal epithelium, and corneal endothelium (lanes 1-10). All membranes were re-probed for β-actin as a loading control. (E) Quantification by densitometry shows the approximate ratio of FL vs C2 in 3 different experiments.

To evaluate whether β-cleavage of PrPC is species-specific or associated with light-induced oxidative stress (Watt et al., 2005), we compared the cleavage pattern of human PrPC with common diurnal and nocturnal species including the human, pig, cattle, goat, mouse, and rat. Of these, cattle and rats have tapetum lucidum, while the others lack this membrane. Retinal lysates were deglycosylated, and analyzed by Western blotting (WB). Probing with 8H4 that binds FL, C1, and C2 fragments of PrPC (Figure 1 A) revealed mainly β-cleaved C2 fragment of 20 kDa in human retinal lysates (Figure 1 B, lanes 1 & 2). Retinal lysates from pig, cattle, goat, mouse, and rat showed mainly FL PrPC, and barely detectable C1 and C2 (Figure 1 B, lanes 3-8 & 10-13). Human brain lysates showed mainly FL and α-cleaved C1 as expected (Figure 1 B, lanes 9 & 14, 15). Quantitation of FL and C2 and C1 revealed significantly more C2 in the human retina, and mainly FL in the animal species. Human brain showed mainly FL and C1 as expected (Figure 1 C) (Walmsley et al., 2009). Quantitation by densitometry revealed a ratio of FL vs C2 and C1 of ~24:46:30 in the human retina, and almost 75-95% FL in animal species. Human brain showed a ratio of ~65:1:34 as expected (Figure 1 C). A similar evaluation of lysates from human retina, sclera, iris, corneal epithelium, and corneal endothelium showed minimal FL, and mainly 3F4-reactive C2, indicating that PrPC in human ocular tissues is cleaved at the β-site (Figure 1 D, lanes 1-10). Quantification revealed a ratio of FL to C2 of ~10-20:80-90 in the retina, sclera, and iris, and ~35:65 in the corneal epithelium and endothelium (Figure 1 E). The ratios of FL, C2 and C1 showed some variability between experiments, and are not absolute.

3.2. Soluble full length PrPC and N-terminal fragments are detected in the aqueous and vitreous humor

The presence of mainly β-cleavage PrPC in the human ocular tissues suggested the presence of N-terminal fragment N2 in the aqueous and vitreous humor (AH & VH). To explore this possibility, equal volume of AH and VH from post-mortem human eyes was evaluated by WB. Probing with the N-terminal antibody POM2 (Figure 2 A) revealed, in addition to N1 and N2, soluble FL glycosylated PrPC which was more abundant in the VH relative to the AH. The N2 fragment of 9 kDa and N1 at 12 kDa was more abundant in the AH than the VH (Figure 2 B, lanes 1-4). Higher levels of N2 in the AH was surprising since the AH is replenished in its entirety every 100 minutes, and the VH remains more or less unaltered from birth. Quantification revealed a ratio of FL to N1 and N2 of ~65:4:31 in the AH, and ~92:2:6 in the VH (Figure 2 C).

Figure 2. Soluble FL PrPC and N-terminal fragments are detected in the AH and VH.

Figure 2.

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(A) Schematic representation of FL PrPC (27-37kDa), N1 (12 kDa) and N2 (9 kDa) fragments of PrPC. (B) WB of human AH and VH shows significant amounts of FL PrPC and N2 fragment in the AH (lane 1-4). Levels of N2 are much higher in the AH relative to the VH (lanes 1 & 3 vs 2 & 4). Low levels of N1 are detected in the AH and VH (lanes 1-4). (C) Quantification by densitometry demonstrates the approximate ratio of soluble FL PrPC and N1 and N2 fragments in the AH and VH in 4 independent estimations.

3.3. β-cleavage of PrPC is not altered by a variety of insults

Oxidative stress is a known cause of β-cleavage of PrPC, and N2 is believed to provide protection from reactive oxygen species (ROS) (Haigh et al., 2015a, 2015b). To evaluate whether β-cleavage of PrPC is altered by oxidative stress or other insults, ARPE-19 cells were exposed to bacterial lipopolysaccharide (LPS), synthetic mini-hepcidin (PR73), and bioactive TGFβ2, stimuli known to upregulate cytokines and increase intracellular iron and iron-catalyzed ROS (Ashok et al., 2020a). After 48 hours, untreated and deglycosylated lysates were subjected to WB and probed with 8H4. No change in the ratio of FL:C2:C1 was observed by any of the treatments relative to untreated controls (Figure 3 A, lanes 1-12). Human brain homogenate fractionated in parallel showed α-cleavage as expected (Figure 3 A, lane 13 & 14). Quantification by densitometry showed a ratio of FL to C2 and C1 of ~5-10:90-95:0 by all insults (Figure 3 B). Likewise, exposure to sodium iodate (NaIO3), an oxidizing agent used to replicate age related macular degeneration (AMD) in mice, did not alter the levels of C2 (Figure 3 C, lanes 1-4). Quantification by densitometry showed a ratio of FL to C2 of ~16:84 in control and ~30:70 NaIO3 treated samples. (Figure 3 D). However, exposure to H2O2 increased the amount of β-cleaved PrPC in ARPE-19 cells marginally (Figure 3 E, lanes 1-4), as reported earlier (Watt et al., 2005), increasing the amount of C2 to almost 90% (Figure 3 F).

Figure 3. Various insults do not influence β-cleavage of PrPC.

Figure 3.

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(A) WB of glycosylated and deglycosylated lysates from ARPE-19 cells exposed to 1 μg/mL LPS, 50 μM of PR73 and virally encoded bioactive TGFβ2 or empty vector show minimal change in the amount of C2 generated relative to untreated control (lanes 3-6 vs 1-2 & 11-12 vs 7-10). Human brain homogenate fractionated in parallel shows FL and C1 as expected (lane 13 & 14). (B) Quantification by densitometry shows the approximate ratio of FL vs C2 in ARPE-19 cells in 3 different experiments, and human brain in 6 experiments. (C) Likewise, WB of glycosylated and deglycosylated ARPE-19 cells exposed to 10 mM of NaIO3 show no change in the amount of C2 relative to untreated control (lane 3 & 4 vs 1 & 2). (D) Quantification by densitometry shows the approximate ratio of FL vs C2 in 3 different estimations. (E) There is a marginal increase in C2 after exposure to 1 μM of H2O2 relative to untreated control (lane 3 & 4 vs 1 & 2). All membranes were re-probed with β-actin as a loading control. (F) Quantification by densitometry shows the approximate ratio of FL vs C2 in 5 independent estimations.

3.4. β-cleavage is reversed by DMSO, not by N-acetyl-L-carnosine

To explore whether β-cleavage is reduced by dimethyl sulfoxide (DMSO) as reported previously (Watt et al., 2005), ARPE-19 cells were exposed to 1% and 1.5% DMSO for 16 hours. Subsequently, untreated and deglycosylated lysates were analyzed by WB. Probing with 3F4 revealed reversal of β-cleavage by DMSO (Figure 4 A, lanes 3-6 vs 1 & 2). Quantification revealed a ratio of FL to C2 of ~45:55 with DMSO as opposed to ~25-75 in controls (Figure 4 B). Likewise, treatment of primary human TM cells with 1.5% DMSO for 16 hours followed by probing of deglycosylated lysates with the C-terminal antibody G-12 (Figure 4 C) that binds C1 and C2 showed a significant decrease in C2 and increase in FL PrPC (Figure 4 C, lanes 2 & 3 vs 1), with a decrease in the ratio of FL to C2 and C1 of ~51:47:2, and ~56:30:14 with 1.5% and 3% DMSO respectively relative to ~20:79:1 in controls (Figure 4 D). It is notable that DMSO reduced C2, and increased C1 in cells. Exposure of ARPE-19 cells to DMSO and the antioxidant N-acetyl-L-carnosine (NAC) followed by probing of deglycosylated lysates with 3F4 revealed reversal of β-cleavage by 1.5% DMSO as above, but not by NAC relative to untreated controls (Figure 4 E, lanes 2-4 vs 1). The ratio of FL to C2 remained unchanged in NAC treated cells relative to controls at ~35:65, and changed to ~70:30 in DMSO treated cells (Figure 4 F). Likewise, probing of deglycosylated lysates from primary human TM cells exposed to increasing concentrations of NAC did not alter the levels of C2 (Figure 4 G, lanes 1-5), which remained at a ratio of FL to C2 and C1 at approximately ~10:65-75:15-25 (Figure 4 H).

Figure 4. β -cleavage of PrPC is reversed by DMSO, but not NAC.

Figure 4.

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(A) WB of glycosylated and deglycosylated ARPE-19 cell lysates treated with 1 & 1.5% DMSO shows a dose dependent reduction in C2 relative to untreated control (lane 3-6 vs 1 & 2). (B) Quantification shows the approximate ratio of FL vs C2 in 4 independent evaluations. (C) Deglycosylated lysates of primary human TM cells exposed to 1.5% DMSO show an increase in FL, reduction in C2, and exposure to 3% DMSO indicate increase in FL, reduction in C2 and an increase in C1 relative to the control (lanes 2 & 3 vs 1). (D) Quantification of the approximate ratio of FL, C2, and C1 of 5 independent estimations. (E) WB of deglycosylated ARPE-19 treated with 5 and 10μM of NAC does not alter C2 levels (lanes 3 & 4 vs 1), though DMSO reduces C2 as in (A) above (lane 2 vs 1). (F) Quantification by densitometry shows the approximate ratio of FL vs C2 in DMSO and NAC treated lysates. (G) WB of deglycosylated lysates of primary human TM cells exposed to increasing concentration of NAC (0-1000 μM) does not show any change in C2 (lane 1-5). All membranes were re-probed with β-actin as a loading control. (H) Quantification of the ratio of FL vs C2 shows no change with increasing concentrations of NAC.

3.5. β-cleavage of the prion protein is not influenced by silencing of ADAM10/17.

Cleavage of brain PrPC at the α-site is mediated by ADAM10/17 (Liang and Kong, 2012). It is therefore likely that absence of ADAM10/17 in ocular tissue results in the alternate cleavage of PrPC at the β-site. To evaluate this possibility, mRNA extracted from primary human TM cells, cornea, CB and retina was subjected to RT-PCR with specific primers. Both ADAM10 and ADAM17 were amplified from ocular tissues (Figure 5 A, lanes 1-7). However, silencing of ADAM10 in primary human TM cells followed by WB of glycosylated and deglycosylated lysates with G-12 did not show any change in the amount of C1 or C2 despite efficient downregulation of ADAM10 (Figure 5 B, lanes 3 & 4 vs 1 & 2). A similar ratio of FL to C2 and C1 of ~10:80:10 was observed (Figure 5 C). Likewise, silencing of ADAM10 or ADAM17 in ARPE-19 cells, though effective in reducing their expression (Figure 5 D, lanes 2 & 3 vs 1), did not alter the amount of C1 or C2 when the same lysates were probed with 3F4 (upper panel), and re-probed with G-12 (lower panel) (Figure 5 E, lanes 3-6 vs 1 & 2). The ratio of FL to C2 and C1 remained at ~25:75 and ~35:55:10 despite downregulation of ADAM10/17 (Figure 5 F). Thus, ADAM10 and ADAM17 are probably not the principal enzymes responsible for α-cleavage of PrPC in the ocular tissue.

Figure 5. Cleavage of PrPC in ocular tissue is not influenced by ADAM10 or ADAM17.

Figure 5.

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(A) Amplification of ADAM10 and ADAM17 from primary human TM cells, cornea, ciliary body (CB), and retina by RT-PCR shows a band specific to both the genes, confirming their synthesis in ocular tissues (lanes 1-7). GAPDH was amplified from the same samples as a control. (B) Silencing of ADAM10 followed by WB of glycosylated and deglycosylated lysates from primary human TM cells shows downregulation of ADAM10 (lanes 3 & 4 vs 1 & 2, upper panel), but no change in C2 or C1 (lane 3 & 4 vs. 1 & 2). The membrane was re-probed for β-actin as a loading control. (C) Quantification of the ratio of FL vs C2 and C1 shows no change by downregulation of ADAM10 in 3 independent evaluations. (D) Silencing of ADAM10 or ADAM17 in ARPE-19 cells followed by WB confirms efficient downregulation (lanes 2 & 3 vs 1). (E) WB of the same lysates following deglycosylation shows no change in C2 or C1 relative to the control (lanes 3-6 vs. 1 & 2). The membranes were re-probed for β-actin as a loading control. (F) Quantification of the ratio between FL, C1 and C2 shows minimal change with downregulation of ADAM10 or 17 in three independent evaluations.

3.6. Interference with β-cleavage induces cell death

To visualize the site of PrPC cleavage and the fate of N1 and N2 fragments, a GFP tag was inserted between amino acids 38 and 39 of human PrPC (Gu et al., 2003), and PrPC-GFP was expressed in ARPE-19 and BE(2)-M17 cells, retinal and brain-derived cell lines, respectively (Figure 6 A). ARPE-19 cells were also transfected with untagged PrPC (Figure 6 B). Surprisingly, stable clones emerged from PrPC transfected ARPE-19 and PrPC-GFP transfected BE(2)M17 cells, but not from PrPC-GFP ARPE-19 cells. Lysates from transfected BE(2)-M17 cells were separated into detergent soluble and insoluble fractions (Gu et al., 2003, 2007), an established method where normally folded PrPC partitions in the detergent-soluble phase and aggregated forms are isolated in the detergent insoluble phase. Evaluation of lysates by WB revealed partitioning of PrPC from ARPE-19 cells and PrPC-GFP from BE(2)M17 cells in the soluble phase as expected (Figure 6 B & C). Probing of WB with 3F4 revealed soluble PrPC at the expected molecular mass in ARPE-19 cells, and PrPC-GFP migrating at 54 kDa in BE(2)M17 cells as expected. α-Cleaved PrPC-GFP co-migrated with brain-derived PrPC between 27-37 kDa (Figure 6 C, lanes 1 & 3). α-Cleavage of PrPC-GFP in M17 cells has been demonstrated earlier (Gu et al., 2003, 2007).

Figure 6. Interference with β cleavage of PrPC causes cytotoxicity in ARPE-19 cells.

Figure 6.

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(A) Schematic of GFP tag inserted between amino acids 38 and 39 of human PrPC (Gu et al., 2003; Haldar et al., 2015). (B) WB of lysates of ARPE-19 cells show partitioning of PrPC from non-transfected (NT), vector (vec) transfected, and PrPC transfected (PrPC) lysates in the detergent soluble phase (lanes 1-3), and none in the detergent insoluble fractions (lanes 4-6). (C) Transfection of BE(2)-M17 cells with PrPC-GFP followed by separation into detergent soluble and insoluble fractions, likewise, show partitioning of PrPC-GFP and glycosylated C1 in the detergent-soluble phase (lanes 1 & 2). Human brain homogenate was fractionated in parallel as a control (lane 3), and the membrane was re-probed for β-actin as a loading control. (D) Fluorescent and phase-contrast imaging of PrPC-GFP transfected ARPE-19 cells show a signal in the Golgi area on day 2 as expected (panel 1 & 1P). However, most of the transfected cells die after 4-5 days (panels 3 & 3P-5 & 5P), while non-transfected cells remain healthy and proliferate. Scale bar: 25 μm (E) WB of PrPC-GFP transfected ARPE-19 cell lysates separated into detergent soluble and insoluble fractions show partitioning of majority of PrPC-GFP in the detergent-insoluble phase, and a decline in the signal by day 6 (lanes 7-12 vs 1-6). Re-probing with anti-GFP antibody shows reactivity with FL, non-cleaved PrPC-GFP at 54kDa. The membrane was re-probed with β-actin as a loading control.

Since we failed to obtain stable clones of PrPC-GFP in ARPE-19 cells, expression of PrPC-GFP was examined in transiently transfected cells 2-6 days post-transfection (Figure 6 D). Top panels 1-5 are fluorescent images, and panels 1P-5P are superimposed phase contrast images (Figure 6 D). On day 2 and 3 post-transfection, a green fluorescent signal from PrPC-GFP was noted in the Golgi area, endosomes, and the plasma membrane. However, almost all transfected cells died by day 6, while non-transfected cells remained healthy and proliferated as noted in phase images of the same field (Figure 6 D, panels 1P-5P). ARPE-19 cells treated with vector-GFP did not show any cell death from day 2 to day 6 (data provided in supplementary Figure S3).

Subsequently, cell lysates were collected on post-transfection day 1-6, and separated into detergent soluble and insoluble fractions. Analysis by WB and probing with 8H4 showed the expected migration of PrPC-GFP at 54 kDa until day 3 in the detergent soluble fraction (Figure 6E, lanes 1-6). However, the majority of PrPC-GFP partitioned in the detergent-insoluble fraction thereafter, and the signal was lost by day 6 (Figure 6E, Lanes 7-12). Re-probing of the same membrane for GFP detected the signal until day 3, after which it was lost (Figure 6E, lanes 7-12).

Thus, in ARPE-19 cells, the GFP tag interferes with β-cleavage of PrPC-GFP, which leads to aggregation and cell death. In BE(2)-M17 cells, α-cleavage of PrPC-GFP occurs normally, and the cells remain healthy.

4. Discussion

We report cleavage of PrPC at the β-site in human ocular tissues and human ARPE-19 and TM cells. This contrasts with mainly α-cleavage in the human brain, and FL PrPC in the retina of common diurnal and nocturnal species. Significant levels of soluble FL and N2 were detected in the AH, suggesting dynamic processing of PrPC in cells surrounding the anterior chamber. The amount of C2 was not altered by a variety of insults. However, H2O2 increased C2 at the expense of FL PrPC, and was reversed by DMSO (Gironi et al., 2020; Watt et al., 2005). The relevance of these observations to common ocular disorders and dissemination of infectious PrPSc in ocular tissues is discussed.

Historically, α-cleavage of PrPC has received more attention because it protects cells from infection by PrPSc (Chen et al., 1995). β-Cleavage, on the other hand, is diagnostic of prion disease, and is associated with oxidative stress (Prusiner, 1998; Watt et al., 2005). The presence of mainly β-cleaved PrPC in the human eye is surprising. The retina, sclera, TM, corneal endothelium and epithelium, and ciliary body, regions with varying susceptibility to different wavelengths of light and oxidative stress all show β-cleavage. Considering that the retina of common diurnal and nocturnal species show mainly FL PrPC, it is unlikely that light-induced oxidative stress is the trigger. We did not observe any deletion or alternate splicing of PrPC mRNA in human retinal tissue in comparison to the human brain, ruling out this possibility (supplementary Figure S1). Surpisingly, insertion of the GFP tag in the N-terminus blocked β-cleavage and led to aggregation of PrPC-GFP, which resulted in cytotoxicity. In BE(2)-M17 cells α-cleavage was unhindered, and the cells remained healthy (data not shown). Expression of GFP alone did not have any effect, ruling out toxicity by GFP itself (supplementary Figure S3). An alternate explanation is that human ocular tissue and ARPE-19 cells lack the enzyme(s) responsible for α-cleavage, resulting in the alternate β-cleavage. However, downregulation of ADAM10 or ADAM17 did not alter α- or β-cleavage of PrPC in ARPE-19 or primary human TM cells, ruling out this possibility. Confluency of ARPE-19 cells, another factor known to influence PrPC cleavage pattern (Haigh et al., 2015b), likewise, had no effect on the amount of β-cleaved PrPC (supplementary Figure S2).

Several other toxic insults such as exposure to sodium iodate, an oxidizing agent that damages ARPE-19 cells selectively (Kannan and Hinton, 2014), hepcidin, that increases iron-mediated ROS (Nemeth et al., 2004), TGFβ2, that upregulates hepcidin (Ashok et al., 2020a) and LPS, that induces cytokine-mediated toxicity (Gao et al., 2020), did not alter the amount of C2 in ARPE-19 or primary human TM cells. Surprisingly, neither did the antioxidant NAC (Ashok et al., 2020a; Schimel et al., 2011). H2O2, which is known to induce β-cleavage of PrPC, increased C2 marginally (McMahon et al., 2001; Watt et al., 2005). However, an almost complete reversal of β-cleavage was observed with DMSO (Watt et al., 2005), and α-cleavage was restored to a small extent. Though encouraging, DMSO has several effects on membrane lipids, including depletion of cholesterol (Alam and Layman, 1982; Tuncer et al., 2018; Verheijen et al., 2019). However, depeletion of cholesterol with cyclodextrin did not alter the cleavage pattern, making it an unlikely cause (Misuri et al., 2017; Zidovetzki and Levitan, 2007). Further exploration is necessary to resolve this question.

It is known that PrPC modulates the production of amyloid-β (Aβ) (Jarosz-Griffiths et al., 2016; Parkin et al., 2007; Qin et al., 2019), a toxic proteolytic product of amyloid precursor protein (APP) implicated in Alzheimer’s disease (AD), and is a known receptor for Aβ-mediated toxicity (Ashok et al., 2020c; Chen et al., 2010; Da Costa Dias et al., 2011; Griffiths et al., 2011; Hooper and Turner, 2008; Salazar and Strittmatter, 2017). Two binding sites on PrPC have been identified for Aβ, including amino acids 23-27 and 95-110 (Ashok et al., 2020c; Chen et al., 2010; Da Costa Dias et al., 2011; Griffiths et al., 2011; Hernandez-Rapp et al., 2014; Hooper and Turner, 2008; Salazar and Strittmatter, 2017). Both sites are present in N1, which is believed to protect the cells by sequestering toxic Aβ in the extracellular milieu (Guillot-Sestier et al., 2012). N2 contains one binding site, and the other site is included in cell-associated C2 (Figure 7). It is unclear whether both binding sites are necessary for Aβ-mediated toxicity, or if one site on C2 is sufficient for mediating the toxic signal. It is notable that APP is expressed in ocular tissues, and manifestations of AD are obvious in the retina where deposits of Aβ are prominent in ‘drusen’ associated with AMD (Shah et al., 2017). It is likely that PrPC contributes to the pathogenesis of AMD through Aβ, a significant question given the abundant expression of C2 in the retinal pigment epithelial cells.

Figure 7: Schematic representation of full length (FL), α-cleaved (C1, 18 kDa), β-cleaved (C2, 20 kDa), and ɣ-cleaved (C3, 6 kDa) forms of PrPC.

Figure 7:

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Sites of different physiological cleavage sites of PrPC are shown. These cleavages also result in their respective N terminal fragments which are released in the extracellular milieu. These may contain one (β-cleaved) or two (α or ɣ-cleaved) Aβ binding sites, playing a protective role against Aβ toxicity. However, β-cleaved C terminal fragment (C2) is susceptible to PrPSc conversion, and contains the binding site for α-Synuclein (α-Syn), increasing the susceptibility of cells to infection by PrPSc, and toxicity by α-Syn. Therefore, the type of PrPC cleavage in different tissues can translate into the susceptibility of a particular tissue to toxicity by Aβ or α-Syn aggregates.

PrPC also binds β1-integrin at a site included in C2, and activates biochemical pathways implicated in cell adhesion and migration (Ashok et al., 2019; Filla et al., 2017; Gagen et al., 2014; Hajj et al., 2007; Loubet et al., 2012; Martins et al., 2010; Richardson et al., 2015). Downregulation of PrPC activates the RhoA-ROCK pathway via aggregation of β1-integrin, inducing endothelial to mesenchymal transition in a variety of cell types (Ashok et al., 2019; Kim et al., 2017; Loubet et al., 2012). In a recent gene ontology study, the membrane environment of PrPC was suggested as conducive to cross-talk between TGFβ and integrin signaling (Ghodrati et al., 2018). This is of significance in ARPE-19 cells, trabecular meshwork cells, and corneal endothelial cells, where such changes are associated with AMD, primary open angle glaucoma (POAG), and Fuch’s dystrophy, respectively (Ashok et al., 2018, 2019; Rao et al., 2017).

It was surprising to observe significant amounts of FL PrPC and N2 in the AH since it turns over in its entirety every 100 minutes. The significance of these observations is unclear. It is possible that N2 protects sensitive structures of the eye from light-induced damage, protects the lens, cornea, and trabecular meshwork from Aβ-induced toxicity, or performs other functions. On the other hand, FL soluble PrPC could serve as a substrate for PrPSc, and promote dissemination of prion infectivity. Moreover, C2 can be readily converted to PrPSc, and being membrane anchored, is toxic as well. The presence of C1 in ocular tissue of mice and sheep therefore suggests that the infectivity and spread of PrPSc in animal models of scrapie is likely to differ from the human eye, and studies in mouse models do not represent the human eye where infectivity and dissemination of PrPSc is concerned.

In conclusion, our data demonstrate a unique cleavage pattern of PrPC in the human eye that differs from the human brain and retinas of diurnal and nocturnal species, and has implications for several ocular disorders. Since this cleavage pattern is conducive to PrPSc infection and dissemination, re-evaluation of studies related to PrPSc infection and toxicity in animal models is suggested.

Supplementary Material

1

Highlights.

  1. The prion protein (PrPC) is mainly β-cleaved in the human ocular tissue.

  2. This contrasts with mainly α-cleaved PrPC in the human brain.

  3. β-Cleaved PrPC is relatively more susceptible to prion infectivity.

  4. This raises concerns of acquiring prion infection via the ocular route.

  5. PrPC in most animals is α-cleaved, requiring re-evaluation of infectivity studies.

Acknowledgements

Special thanks to Mellissa A. Pottinger (Recovery Technician) at the Lions Eye Bank. The authors alone are responsible for the content and writing of the manuscript. Supported by R01 NS 092145 (to NS).

Abbreviations

Aβ

amyloid-β

ADAM

A-disintegrin-and-metalloproteinase

AH

aqueous humor

ARPE-19

human retinal pigment epithelial cells

PrPC

prion protein

PrPSc

scrapie prion protein

RGC

retinal ganglion cells

ROS

reactive oxygen species

TM

trabecular meshwork

VH

vitreous humor

Footnotes

Disclosure

S. Chaudhary, None; A. Ashok, None; A.S. Wise, None; A.E. Kritikos, None; N.A. Rana, None; E. Lindner, None; N. Singh, None.

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