J or H mtDNA haplogroups in retinal pigment epithelial cells: Effects on cell physiology, cargo in extracellular vesicles, and differential uptake of such vesicles by naïve recipient cells

https://doi.org/10.1016/j.bbagen.2020.129798Get rights and content

Highlights

  • RPE cells with identical nuclear DNA but different mtDNA haplogroups (H, J) and differences in AMD risk (H<J) are studied.

  • Barrier function and energy metabolism are reduced in J- when compared to H-cybrids.

  • EVs from J-, but not H-cybrids, are taken up by naïve RPE cell monolayers and induce barrier function loss.

  • EV uptake involves ligands (fibronectin, annexin A2) previously shown to characterize EVs of oxidatively stressed RPE.

  • Characterization of EV bioactive molecules from diseased RPE cells might provide for eye disease biomarker discovery.

Abstract

Purpose

Extracellular vesicles (EVs) are predicted to represent the internal state of cells. In polarized RPE monolayers, EVs can mediate long-distance communication, requiring endocytosis via protein-protein interactions. EV uptake from oxidatively stressed donor cells triggers loss in transepithelial resistance (TER) in recipient monolayers mediated by HDAC6. Here, we examine EVs released from RPE cells with identical nuclear genes but different mitochondrial (mt)DNA haplogroups (H, J). J-cybrids produce less ATP, and the J-haplogroup is associated with a higher risk for age-related macular degeneration.

Methods

Cells were grown as mature monolayers to either collect EVs from apical surfaces or to serve as naïve recipient cells. Transfer assays, transferring EVs to a recipient monolayer were performed, monitoring TER and EV-uptake. The presence of known EV surface proteins was quantified by protein chemistry.

Results

H- and J-cybrids were confirmed to exhibit different levels of TER and energy metabolism. EVs from J-cybrids reduced TER in recipient ARPE-19 cells, whereas EVs from H-cybrids were ineffective. TER reduction was mediated by HDAC6 activity, and EV uptake required interaction between integrin and its ligands and surface proteoglycans. Protein quantifications confirmed elevated levels of fibronectin and annexin A2 on J-cybrid EVs.

Conclusions

We speculate that RPE EVs have a finite set of ligands (membrane proteoglycans and integrins and/or annexin A2) that are elevated in EVs from stressed cells; and that if EVs released by the RPE could be captured from serum, that they might provide a disease biomarker of RPE-dependent diseases.

Introduction

Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic abnormalities and environmental insults [1]. Inflammation, smoking [2] and polymorphisms in some complement proteins can increase the risk for AMD [3]. Hence, the concept has emerged that abnormalities in controlling oxidative stress and the complement system may lead to inflammation in retinal pigment epithelium (RPE)/Bruch's membrane, generating a pathological environment favorable for AMD development [4].

A feature of atrophic AMD is localized damage and ultimately loss of RPE cells, predominantly at the posterior pole. Mitochondria play an essential role in cell health and death. The majority of the RPE cell's energy is generated in the mitochondria by oxidative phosphorylation (OXPHOS) [5]. Mitochondria contain circular DNA, inherited through the maternal linage. The highly polymorphic mitochondrial DNA is critical for energy production, since 13/37 genes code for protein subunits used in OXPHOS. In OXPHOS, a proton gradient across the mitochondrial inner membrane is created, which is used to generate ATP. Under certain conditions however, partial uncoupling of the two processes occurs, increasing the proton leak and decreasing ATP production. Importantly, single nucleotide polymorphisms of the mitochondrial DNA (haplogroups) can change mitochondrial function [6]. These haplogroups are associated with geographic origins of different populations, allowing the establishment of a mtDNA phylogenetic tree, and providing a tool for medical genetics [7]. Interestingly, the J, T, and U haplogroups have been found to be associated with AMD [[8], [9], [10], [11]], while the H-haplogroup is not [12], yet all are of European origin. While it is unclear how mtDNA variants contribute to AMD risk and by which cellular mechanisms or pathways, Kenney and coworkers have speculated that the altered energy metabolism and modified expression of nuclear genes might play a role [13]. In other diseases such as Parkinson's disease and Alzheimer's disease, mitochondrial cybrids have provided important insights as to how mtDNA induces differences in mitochondrial biology, and how altered mitochondrial function influences cell physiology; however animal-based models to assess these mtDNA genotype–phenotype correlations should be developed and studied in addition [14]. We have created cybrids (cytoplasmic hybrids) which were generated by introducing mitochondria from individuals that are either haplogroup J or H into an immortalized human RPE cell (ARPE-19 cells) from which the mitochondria (U5 haplogroup) were removed, so the two cybrids carry the same nuclear genes [13]. Specifically, while cell viability is similar between the two APRE-19 cell haplogroups, J-cybrids generate significantly less reactive oxygen species and ATP when compared to H-cybrids. In addition, expression levels for nuclear genes involved in complement and metabolism are also altered in J- versus H-cybrids [13]. We have shown that mitochondrial ATP production is affected by age; human embryonic RPE cells have small, dynamic mitochondria that generate large amounts of ATP; cells derived from old donors have larger, less dynamic mitochondria that generate smaller amounts of ATP [15]. And age-related accumulation of damaged mtDNA has been reported in the RPE, which is also exacerbated by disease [16] and in subjects with AMD gene mutations [17].

RPE pathology in AMD has been shown to be initiated in multiple patches at the posterior pole, rather than to emanate from a single location. We have speculated that this pattern might be mediated by short-distance communication, such as gap-junction communication [18,19], and long-distance communication that might be mediated by extracellular vesicles (EVs) [20], between diseased and healthy parts of the tissue.

EVs are small vesicles with an approximate size of 40–150 nm. They are released from many different cells and consequently have been isolated from many body fluids. Our understanding of EVs has covered the spectrum from EVs as the cell's garbage bin, to intercellular communication devices, to promising therapeutic targets [21]. This change in sentiment occurred as we discovered that EVs not only contain protein, but also RNA, miRNA, lcRNA and play a crucial role in intercellular communication [22].

We have started characterizing EVs in intercellular communication in RPE monolayers. Previously, we have shown that EVs are released from both the apical and basal surfaces of ARPE-19, the same cells used here but with a U5 mitochondrial DNA haplogroup, as well as primary porcine RPE cells when grown as stable monolayers [20]. The ARPE-19 cell EVs were carefully characterized based on morphology, size and markers based on MISEC 2018 guidelines [23]. In short, nanoparticle tracking analysis confirmed that the average diameter of EVs was 127.5 ± 10.0 nm with an average zeta-potential of −25.70 ± 3.07 mV. Transmission electron microscopy (EM) demonstrated that some of the vesicles exhibited the central depression characteristic for exosomes, and immunogold labelling EM confirmed the presence of the exosome marker tetraspanin protein CD81, previously shown by Knickelbein and colleagues to be present on ARPE-19 cell EVs [24]. Additional experiments to confirm purity of the samples using ELISA measurements to quantify syntenin-1 (exosome marker) [25] and ApoB (modified lipoprotein particles known to be secreted by ARPE-19 cells) [26], revealed that the EV preparations contained syntenin-1, whereas no ApoB was detected [20]. Furthermore, two additional markers, annexin A2 and fibronectin were identified in these vesicles; two proteins that are within the list of top 100 proteins that are often identified in exosomes (exocarta.org). Follow-up experiments were performed on ARPE-19 cell EVs to characterize their potential function in cell-cell communication [20,27]. Using transfer assays, adding labeled or un-labeled EVs released from either control or H2O2-stimulated monolayers, we showed EV uptake by confocal microscopy and a functional readout [20]. Confocal microscopy demonstrated that EVs from oxidatively stressed cells added to a monolayer of naïve recipient cells were taken up within ~10–20 min, with very few EVs remaining on the cell surface. In contrast, EVs from control were internalized slowly (taking ~100 min) and incompletely, with ~80% of the control EVs remaining outside the cell [20]. As a functional readout, we demonstrated that stress EVs taken up by recipient cells resulted in a loss of barrier function (transepithelial resistance [TER]) that was mediated by HDAC6 activity present in the EVs [20]. TER loss was dependent upon the presence of EVs and not other secreted proteins, as ARPE-19 cell supernatant depleted of EVs, was ineffective [20]. Using these two readouts (imaging and TER), we showed that uptake is dependent on ligands on the EV surface [27]. Those include three ligand-receptor interactions, integrins, proteoglycans, and annexin A2. Elevated levels of these ligands were identified on stress EVs when compared to control EVs; and uptake could be inhibited by interfering with integrin signaling, stripping of proteoglycans from the recipient cells as well as knocking down gene expression for annexin A2 [20,27]. In contrast, elevating annexin A2 gene expression allowed for uptake of EVs from non-stressed cells [20]. These results suggest that EVs from stressed cells differ from their control counterparts in the level of ligands present on their surfaces. Not only does that affect uptake by recipient cells, but suggests that oxidative stress in general leads to increased targeting of ligands, such as fibronectin and annexin A2, to EVs.

Here, we ask the question whether oxidative stress mediated by mitochondrial haplogroups results in the production of EVs with similar characteristics as those generated by H2O2.

Section snippets

Cell culture

Parent ARPE-19 cells (ATCC® CRL-2302™; American Type Culture Collection, Manassas VA, obtained as passage 20) that carry the U5 mitochondrial DNA haplogroup between passages 20–35 and ARPE-19 cell lines that carry either the H- or J-haplogroup (called transmitochondrial cybrids) [28] between passages 10–18 were expanded in 100 mm dishes (Thermo Fisher) with Dulbecco's modified Eagle's medium (DMEM). ARPE-19 cells were provided with 10% fetal bovine serum (FBS), cybrids with 10% dialyzed fetal

Transmitochondrial J-cybrids have reduced energy metabolism

Cybrids (cytoplasmic hybrids) are cells with identical nuclear genes but different mitochondrial DNA, generated by introducing mitochondria of choice into rho0 cells from which mitochondria were removed [7]. The RPE cybrids chosen here are the H- and J-haplogroups. The H-haplogroup is associated with lower, the J-haplogroup with higher risk of developing AMD [[8], [9], [10], [11], [12]]. Cybrids with the H-haplogroup have been reported to grow and proliferate slower than those with the

Discussion

The main results of the current study are: 1) In comparison to H-cybrids, J-cybrid cells develop monolayers with reduced barrier function, and ATP synthesis is reduced in both aerobic phosphorylation and glycolysis; 2) Addition of EVs released from J- but not H-cybrids to naïve RPE monolayers resulted in TER reduction, and EV uptake was confirmed by imaging; 3) Two ligand-receptor interactions, integrins and proteoglycans known to be involved in stress EV uptake by naïve RPE cells were

Declaration of Competing Interest

The authors have no financial or non-financial competing interests to disclose.

Acknowledgements

In the Rohrer laboratory, the study was supported in part by the Department of Veterans Affairs (I01RX000444, I01BX003050 and IK6BX004858), the National Institutes of Health (R01EY024581), and the South Carolina SmartState Endowment; Cris Kenney is supported by NIH R01EY0127363 and Unrestricted grants to the Department of Ophthalmology from Research to Prevent Blindness, New York, NY and the Discovery Eye Foundation, Los Angeles, CA . The authors would like to thank James Chao for help with the

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