Differential mitochondrial and cellular responses between H vs. J mtDNA haplogroup-containing human RPE transmitochondrial cybrid cells.

Mitochondrial dysfunction is associated with several retinal degenerative diseases including Age-related Macular Degeneration (AMD). Human mitochondrial DNA (mtDNA) haplogroups are inherited from a common ancestral clan and are defined by specific sets of genetic differences. The purpose of this study was to determine and compare the effects of mtDNA haplogroups H and J on transcriptome regulation and cellular resilience to oxidative stress in human RPE cytoplasmic hybrid (cybrid) cell lines in vitro. ARPE-19 cybrid cell lines containing mtDNA haplogroups H and J were created by fusing platelets obtained from normal individuals containing H and J haplogroups with mitochondria-deficient (Rho0) ARPE-19 cell lines. These cybrids were exposed to oxidative stress using 300 µM hydrogen peroxide (H2O2), following which mitochondrial structural dynamics was studied at varying time points using the mitochondrial markers - TOMM20 (Translocase of Outer Mitochondrial Membrane 20) and Mitotracker. To evaluate mitochondrial function, levels of ROS, ΔΨm and [Ca2+]m were measured using flow cytometry, and ATP levels were measured using luminescence. The H and J cybrid cell transcriptomes were compared using RNAseq to determine how changes in mtDNA regulate gene expression. Inflammatory and angiogenic markers were measured using Luminex assay to understand how these mtDNAs influenced cellular response to oxidative stress. Actin filaments' morphology was examined using confocal microscopy. Following exposure to H2O2 stress, the J cybrids showed increased mitochondrial swelling and perinuclear localization, disturbed fission and fusion, increased calcium uptake (p < 0.05), and higher secreted levels of TNF-α and VEGF (p < 0.001), compared to the H cybrids. Calcium uptake by J cybrids was reduced using an IP3R inhibitor. Thirteen genes involved in mitochondrial complex I and V function, fusion/fission events, cellular energy homeostasis, antioxidant defenses, and inflammatory responses, were significantly downregulated with log2 fold changes ranging between -1.5 and -5.1. Actin levels were also significantly reduced in stressed J cybrids (p ≤ 0.001) and disruption in actin filaments was observed. Thirty-eight genes involved in mitochondrial and cellular support functions, were upregulated with log2 fold changes of +1.5 to +5.9 in J cybrids compared to H cybrids. Our results demonstrate significant structural and functional differences between mtDNA haplogroups H vs. J -containing cybrid cells. Our study suggests that the J mtDNA haplogroup can alter the transcriptome to increase cellular susceptibility to stress and retinal degenerations.

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells.

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 µg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1ß, TGF-α, TGF-ß1 and TGF-ß2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 µg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 µg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 µg/ml in all cultures and 60 µg/ml after 72 h. The CPFX 120 µg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells.

Altered Retrograde Signaling Patterns in Breast Cancer Cells Cybrids with H and J Mitochondrial DNA Haplogroups.

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.

Differential effects of risuteganib and bevacizumab on AMD cybrid cells.

PURPOSE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). METHODS: Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 µl (25,000 µg/ml) of Bmab or 1.0 mg/50 µl (20,000 µg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1ß, IFN-ß1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. RESULTS: The untreated AMD cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal cybrids. The Bmab-treated AMD cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD cybrids. The Bmab-treated cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated cybrids had higher expression levels of inflammatory (IL-6, IL1-ß), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. CONCLUSIONS: These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.

Mitochondria: The Retina's Achilles' Heel in AMD.

Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.

A two-step method for identifying photopigment opsin and rhodopsin gene sequences underlying human color vision phenotypes.

Purpose: To present a detailed, reliable long range-PCR and sequencing (LR-PCR-Seq) procedure to identify human opsin gene sequences for variations in the long wavelength-sensitive (OPN1LW), medium wavelength-sensitive (OPN1MW), short wavelength-sensitive (OPN1SW), and rhodopsin (RHO) genes. Methods: Color vision was assessed for nine subjects using the Farnsworth-Munsell 100 hue test, Ishihara pseudoisochromatic plates, and the Rabin cone-contrast threshold procedure (ColorDX, Konan Medical). The color vision phenotypes were normal trichromacy (n = 3), potential tetrachromacy (n = 3), dichromacy (n = 2), and unexplained low color vision (n = 1). DNA was isolated from blood or saliva and LR-PCR amplified into individual products: OPN1LW (4,045 bp), OPN1MW (4,045 bp), OPN1SW (3,326 bp), and RHO (6,715 bp). Each product was sequenced using specific internal primer sets. Analysis was performed with Mutation Surveyor software. Results: The LR-PCR-Seq technique identified known single nucleotide polymorphisms (SNPs) in OPN1LW and OPN1MW gene codons (180, 230, 233, 277, and 285), as well as those for lesser studied codons (174, 178, 236, 274, 279, 298 and 309) in the OPN1LW and OPN1MW genes. Additionally, six SNP variants in the OPN1MW and OPN1LW genes not previously reported in the NCBI dbSNP database were identified. An unreported poly-T region within intron 5(c.36+126) of the rhodopsin gene was also found, and analysis showed it to be highly conserved in mammalian species. Conclusions: This LR-PCR-Seq procedure (single PCR reaction per gene followed by sequencing) can identify exonic and intronic SNP variants in OPN1LW, OPN1MW, OPN1SW, and rhodopsin genes. There is no need for restriction enzyme digestion or multiple PCR steps that can introduce errors. Future studies will combine the LR-PCR-Seq with perceptual behavior measures, allowing for accurate correlations between opsin genotypes, retinal photopigment phenotypes, and color perception behaviors.

Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes.

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

Increased expression of ApoE and protection from amyloid-beta toxicity in transmitochondrial cybrids with haplogroup K mtDNA.

Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNP) patterns, represent populations of diverse geographic origins and have been associated with increased risk or protection of many diseases. The H haplogroup is the most common European haplogroup while the K haplogroup is highly associated with the Ashkenazi Jewish population. Transmitochondrial cybrids (cell lines with identical nuclei, but mtDNA from either H (n=8) or K (n=8) subjects) were analyzed by the Seahorse flux analyzer, quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC). Cybrids were treated with amyloid-ß peptides and cell viabilities were measured. Other cybrids were demethylated with 5-aza-2'-deoxycytidine (5-aza-dC) and expression levels for APOE and NFkB2 were measured. Results show K cybrids have (a) significantly lower mtDNA copy numbers, (b) higher expression levels for MT-DNA encoded genes critical for oxidative phosphorylation, (c) lower Spare Respiratory Capacity, (d) increased expression of inhibitors of the complement pathway and important inflammasome-related genes; and (e) significantly higher levels of APOE transcription that were independent of methylation status. After exposure to amyloid-ß1-42 peptides (active form), H haplogroup cybrids demonstrated decreased cell viability compared to those treated with amyloid-ß42-1 (inactive form) (p<0.0001), while this was not observed in the K cybrids (p=0.2). K cybrids had significantly higher total global methylation levels and differences in expression levels for two acetylation genes and four methylation genes. Demethylation with 5-aza-dC altered expression levels for NFkB2, while APOE transcription patterns were unchanged. Our findings support the hypothesis that mtDNA-nuclear retrograde signaling may mediate expression levels of APOE, a key factor in many age-related diseases. Future studies will focus on identification of the mitochondrial-nuclear retrograde signaling mechanism(s) contributing to these mtDNA-mediated differences.

Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial-nuclear interactions.

Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other 'modifiers' may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt-nuclear interactions.

Increased retinal mtDNA damage in the CFH variant associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness among the elderly in the developed world. Genetic analysis of AMD has identified 34 high-risk loci associated with AMD. The genes at these high risk loci belong to diverse biological pathways, suggesting different mechanisms leading to AMD pathogenesis. Thus, therapies targeting a single pathway for all AMD patients will likely not be universally effective. Recent evidence suggests defects in mitochondria (mt) of the retinal pigment epithelium (RPE) may constitute a key pathogenic event in some AMD patients. The purpose of this study is to determine if individuals with a specific genetic background have a greater propensity for mtDNA damage. We used human eyebank tissues from 76 donors with AMD and 42 age-matched controls to determine the extent of mtDNA damage in the RPE that was harvested from the macula using a long extension polymerase chain reaction assay. Genotype analyses were performed for ten common AMD-associated nuclear risk alleles (ARMS2, TNFRSF10A, CFH, C2, C3, APOE, CETP, LIPC, VEGF and COL10A1) and mtDNA haplogroups. Sufficient samples were available for genotype association with mtDNA damage for TNFRSF10A, CFH, CETP, VEGFA, and COL10A1. Our results show that AMD donors carrying the high risk allele for CFH (C) had significantly more mtDNA damage compared with donors having the wild-type genetic profile. The data from an additional 39 donors (12 controls and 27 AMD) genotyped for CFH alleles further supported these findings. Taken together, these studies provide the rationale for a more personalized approach for treating AMD by uncovering a significant correlation between the CFH high risk allele and accelerated mtDNA damage. Patients harboring this genetic risk factor may benefit from therapies that stabilize and protect the mt in the RPE.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Mitochondrial DNA haplogroups confer differences in risk for age-related macular degeneration: a case control study.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170). METHODS: Total DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene. RESULTS: The JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background. CONCLUSIONS: There is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.

The role of mitochondrial genes on nuclear gene expression in neovascular age related macular degeneration: analysis of nuclear VEGF gene expression after ranibizumab treatment in cytoplasmic hybrid retinal pigment epithelial cell lines correlated with clinical evolution.

PURPOSE: The present study tests the hypothesis that mitochondrial genes have retrograde signaling capacity that influences the expression of nuclear genes related to angiogenesis pathways. Cytoplasmic hybrid (cybrid) in vitro cell lines with patient specific mitochondria inserted into an immortalized retinal pigment epithelial cell line (ARPE-19) were used to test this hypothesis. This type of analysis can provide important information to identify the optimal regimen of anti-VEGF treatment, personalizing age-related macular degeneration (AMD) therapies. METHODS: Mitochondria deficient ARPE-19 cells (Rho0) were fused with AMD donor's platelets to create individual cybrid cell lines containing mitochondria from patients with phenotypic AMD disease and nuclear DNA from the immortalized RPE cell line. The cybrids were treated with Ranibizumab (Lucentis, Genentech, San Francisco, CA), at 4 different concentrations for 24 h, and subsequently the levels of reactive oxygen species (ROS), gene expression for VEGF-A, hypoxia-inducible factor 1-alpha (HIF1-a) and manganese superoxide dismutase (SOD2) were measured. The clinical evolution of the two AMD-donors were correlated with the molecular findings found in their 'personalized' cybrids. RESULTS: Cybrids from Patient-01 showed down-regulation of gene expression of VEGF-A and HIF-1a at both 1X and 4X Ranibizumab concentrations. Patient-01 AMD cybrid cultures had an increase in the ROS levels at 1X (P = 0.0317), no changes at 2X (P = 0.8350) and a decrease at 4X (P = 0.0015) and 10X (P = 0.0011) of Ranibizumab. Clinically, Patient-01 responded to anti-VEGF therapy but eventually developed geographic atrophy. Patient-02 cybrids demonstrated up-regulation of gene expression of VEGF-A and HIF-1a at Ranibizumab 1X and 4X concentrations. There was decreased ROS levels with Ranibizumab 1X (P = 0.1606), 2X (P = 0.0388), 4X (P = 0.0010) and 10X (P = < 0.0001). Clinically, Patient-02 presented with a neovascular lesion associated with a prominent production of intraretinal fluid in clinical follow-up requiring regular and repeated intravitreal injections of Ranibizumab with recurrent subretinal fluid. CONCLUSIONS: Our cybrid model has the potential to help personalize the treatment regimen with anti-VEGF drugs in patients with neovascular AMD. Further investigation is needed to better understand the role that the mitochondria play in the cellular response to anti-VEGF drugs. Future studies that focus on this model have the potential to help personalize anti-VEGF treatment.

The Transcriptome Profile of Retinal Pigment Epithelium and Müller Cell Lines Protected by Risuteganib Against Hydrogen Peroxide Stress.

Purpose: Oxidative stress contributes to the pathogenesis of vision-impairing diseases. In the retina, retinal pigment epithelium (RPE) and Müller cells support neuronal homeostasis, but also contribute to pathological development under stressed conditions. Recent studies found that the investigational drug risuteganib (RSG) has a good safety profile, provided protection in experimental models, and improved visual acuity in patients. The present in vitro study evaluated the effects of RSG in RPE and Müller cell lines stressed with the oxidant hydrogen peroxide (H2O2). Methods: Human RPE (ARPE-19) and Müller (MIO-M1) cell lines were treated with various combinations of RSG and H2O2. Trypan blue assay was used to investigate the effect of compounds on cell viability. Gene expression was measured using RNA sequencing to identify regulated genes and the biological processes and pathways involved. Results: Trypan blue assay found RSG pre-treatment significantly protected against H2O2-induced cell death in ARPE-19 and MIO-M1 cells. Transcriptome analysis found H2O2 regulated genes in several disease-relevant biological processes, including cell adhesion, migration, death, and proliferation; ECM organization; angiogenesis; metabolism; and immune system processes. RSG pre-treatment modulated these gene expression profiles in the opposite direction of H2O2. Pathway analysis found genes in integrin, AP-1, and syndecan signaling pathways were regulated. Expression of selected RSG-regulated genes was validated using qRT-PCR. Conclusions: RSG protected cultured human RPE and Müller cell lines against H2O2-induced cell death and mitigated the associated transcriptome changes in biological processes and pathways relevant to the pathogenesis of retinal diseases. These results demonstrate RSG reduced oxidative stress-induced toxicity in two retinal cell lines with potential relevance to the treatment of human diseases.

Impacts of Bacteriostatic and Bactericidal Antibiotics on the Mitochondria of the Age-Related Macular Degeneration Cybrid Cell Lines.

We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-ß1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro.

Differential modulation of cancer-related genes by mitochondrial DNA haplogroups and the STING DNA sensing system.

Activation of the Simulator of Interferon Genes (STING) system by mitochondrial (mt) DNA can upregulate type 1 interferon genes and enhance immune responses to combat bacterial and viral infections. In cancers, the tumor-derived DNA activates STING leading to upregulation of IFN-beta and induction of antitumor T cells. The entire mtDNA from the cell lines was sequenced using next-generation sequencing (NGS) technology with independent sequencing of both strands in both directions, allowing identification of low-frequency heteroplasmy SNPs. There were 15 heteroplasmy SNPs showing a range from 3.4% to 40.5% occurrence in the K cybrid cell lines. Three H haplogroup cybrids possessed SNP heteroplasmy that ranged from 4.39% to 30.7%. The present study used qRT-PCR to determine if cybrids of H and K haplogroups differentially regulate expression levels of five cancer genes (BRAC1, ALK, PD1, EGFR, and HER2) and seven STING subunits genes (CGAS, TBK1, IRF3, IκBa, NFκB, TRAF2, and TNFRSF19). Some cybrids underwent siRNA knockdown of STING followed by qRT-PCR in order to determine the impact of STING on gene expression. Rho0 (lacking mtDNA) ARPE-19 cells were used to determine if mtDNA is required for the expression of the cancer genes studied. Our results showed that (a) K cybrids have lower expression levels for BRAC1, ALK, PD1, EGFR, IRF3, and TNFRSF19 genes but increased transcription for IκBa and NFκB compared to H cybrids; (b) STING KD decreases expression of EGFR in both H and K cybrids, and (c) PD1 expression is negligible in Rho0 cells. Our findings suggest that the STING DNA sensing pathway may be a previously unrecognized pathway to target modulation of cancer-related genes and the PD1 expression requires the presence of mtDNA.

Differential Epigenetic Status and Responses to Stressors between Retinal Cybrids Cells with African versus European Mitochondrial DNA: Insights into Disease Susceptibilities.

Mitochondrial (mt) DNA can be classified into haplogroups, which represent populations with different geographic origins. Individuals of maternal African backgrounds (L haplogroup) are more prone to develop specific diseases compared those with maternal European-H haplogroups. Using a cybrid model, effects of amyloid-ß (Amyß), sub-lethal ultraviolet (UV) radiation, and 5-Aza-2'-deoxycytidine (5-aza-dC), a methylation inhibitor, were investigated. Amyß treatment decreased cell metabolism and increased levels of reactive oxygen species in European-H and African-L cybrids, but lower mitochondrial membrane potential (ΔΨM) was found only in African-L cybrids. Sub-lethal UV radiation induced higher expression levels of CFH, EFEMP1, BBC3, and BCL2L13 in European-H cybrids compared to African-L cybrids. With respect to epigenetic status, the African-L cybrids had (a) 4.7-fold higher total global methylation levels (p = 0.005); (b) lower expression patterns for DNMT3B; and (c) elevated levels for HIST1H3F. The European-H and African-L cybrids showed different transcription levels for CFH, EFEMP1, CXCL1, CXCL8, USP25, and VEGF after treatment with 5-aza-dC. In conclusion, compared to European-H haplogroup cybrids, the African-L cybrids have different (i) responses to exogenous stressors (Amyß and UV radiation), (ii) epigenetic status, and (iii) modulation profiles of methylation-mediated downstream complement, inflammation, and angiogenesis genes, commonly associated with various human diseases.

Mitochondrial DNA polymorphisms and biogenesis genes in primary and metastatic uveal melanoma cell lines.

PURPOSE: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into metastatic progression. METHOD: The entire mtDNA genomes were sequenced using Sanger sequencing from two primary UM cell lines (92.1 and MEL270) and two cell lines (OMM2.3 and OMM2.5) derived from liver metastases of the MEL270 patient. The mtDNA copy numbers determined by the ratio of nDNA versus mtDNA. qRT-PCR was used to evaluate expression levels of mitochondrial biogenesis genes. RESULTS: Sequencing showed that cell line MEL270 and metastases-derived OMM2.3 and OMM2.5 cell lines had homoplasmic single nucleotide polymorphisms (SNPs) representing J1c7a haplogroup, whereas 92.1 cells had mtDNA H31a haplogroup. mtDNA copy numbers were significantly higher in primary cell lines. The metastatic UM cells showed down-regulation of POLG, TFAM, NRF-1 and SIRT1 compared to their primary MEL270 cells. PGC-1α was downregulated in 92.1 and upregulated in MEL270, OMM2.3 and OMM2.5. CONCLUSIONS: Our finding suggests that within metastatic cells, the heteroplasmic SNPs, copy numbers and mitochondrial biogenesis genes are modulated differentially compared to their primary UM cells. Therefore, investigating pathogenic mtDNA variants associated with cancer metabolic susceptibility may provide future therapeutic strategies in metastatic UM.

Low frequency mitochondrial DNA heteroplasmy SNPs in blood, retina, and [RPE+choroid] of age-related macular degeneration subjects.

PURPOSE: Mitochondrial (mt) DNA damage is associated with age-related macular degeneration (AMD) and other human aging diseases. This study was designed to quantify and characterize mtDNA low-frequency heteroplasmy single nucleotide polymorphisms (SNPs) of three different tissues isolated from AMD subjects using Next Generation Sequencing (NGS) technology. METHODS: DNA was extracted from neural retina, [RPE+choroid] and blood from three deceased age-related macular degeneration (AMD) subjects. Entire mitochondrial genomes were analyzed for low-frequency heteroplasmy SNPs using NGS technology that independently sequenced both mtDNA strands. This deep sequencing method (average sequencing depth of 30,000; range 1,000-100,000) can accurately differentiate low-frequency heteroplasmy SNPs from DNA modification artifacts. Twenty-three 'hot-spot' heteroplasmy mtDNA SNPs were analyzed in 222 additional blood samples. RESULTS: Germline homoplasmy SNPs that defined mtDNA haplogroups were consistent in the three tissues of each subject. Analyses of SNPs with <40% heteroplasmy revealed the blood had significantly greater numbers of heteroplasmy SNPs than retina alone (p≤0.05) or retina+choroid combined (p = 0.008). Twenty-three 'hot-spot' mtDNA heteroplasmy SNPs were present, with three being non-synonymous (amino acid change). Four 'hot-spot' heteroplasmy SNPs (m.1120C>T, m.1284T>C, m.1556C>T, m.7256C>T) were found in additional samples (n = 222). Five heteroplasmy SNPs (m.4104A>G, m.5320C>T, m.5471G>A, m.5474A>G, m.5498A>G) declined with age. Two heteroplasmy SNPs (m.13095T>C, m.13105A>G) increased in AMD compared to Normal samples. In the heteroplasmy SNPs, very few transversion mutations (purine to pyrimidine or vice versa, associated with oxidative damage) were found and the majority were transition changes (purine to purine or pyrimidine to pyrimidine, associated with replication errors). CONCLUSION: Within an individual, the blood, retina and [RPE+choroid] contained identical homoplasmy SNPs representing inherited germline mtDNA haplogroup. NGS methodology showed significantly more mtDNA heteroplasmy SNPs in blood compared to retina and [RPE+choroid], suggesting the latter tissues have substantial protection. Significantly higher heteroplasmy levels of m.13095T>C and m.13105A>G may represent potential AMD biomarkers. Finally, high levels of transition mutations suggest that accumulation of heteroplasmic SNPs may occur through replication errors rather than oxidative damage.