bims-nenemi 2024-09-08 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2024‒09‒08
twelve papers selected by
Marco Tigano, Thomas Jefferson University

Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression.
Mitochondrial permeability transition mediated by MTCH2 and F-ATP synthase contributes to ferroptosis defense.
Nucleoid-phagy: a novel safeguard against mitochondrial DNA-Induced inflammation.
Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.
Structure-destabilizing mutations unleash an intrinsic perforation activity of antiapoptotic Bcl-2 in the mitochondrial membrane enabling apoptotic cell death.
Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways.
Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus.
Mitochondrial dsRNA from B-ALL cells stimulates mesenchymal stromal cells to become cancer associated fibroblasts.
Mitochondrial DNA is a sensitive surrogate and oxidative stress target in oral cancer cells.
Defective post-transcriptional modification of tRNA disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.
Robustness and reliability of single-cell regulatory multi-omics with deep mitochondrial mutation profiling.
Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures.

Life Sci Alliance. 2024 Nov;pii: e202402764. [Epub ahead of print]7(11):

Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression.

Vanessa Xavier, Silvia Martinelli, Ryan Corbyn, Rachel Pennie, Kai Rakovic, Ian R Powley, Leah Officer-Jones, Vincenzo Ruscica, Alison Galloway, Leo M Carlin, Victoria H Cowling, John Le Quesne, Jean-Claude Martinou, Thomas MacVicar.

Mitochondrial gene expression is a compartmentalised process essential for metabolic function. The replication and transcription of mitochondrial DNA (mtDNA) take place at nucleoids, whereas the subsequent processing and maturation of mitochondrial RNA (mtRNA) and mitoribosome assembly are localised to mitochondrial RNA granules. The bidirectional transcription of circular mtDNA can lead to the hybridisation of polycistronic transcripts and the formation of immunogenic mitochondrial double-stranded RNA (mt-dsRNA). However, the mechanisms that regulate mt-dsRNA localisation and homeostasis are largely unknown. With super-resolution microscopy, we show that mt-dsRNA overlaps with the RNA core and associated proteins of mitochondrial RNA granules but not nucleoids. Mt-dsRNA foci accumulate upon the stimulation of cell proliferation and their abundance depends on mitochondrial ribonucleotide supply by the nucleoside diphosphate kinase, NME6. Consequently, mt-dsRNA foci are profuse in cultured cancer cells and malignant cells of human tumour biopsies. Our results establish a new link between cell proliferation and mitochondrial nucleic acid homeostasis.

DOI: https://doi.org/10.26508/lsa.202402764
FEBS Lett. 2024 Sep 03.

Mitochondrial permeability transition mediated by MTCH2 and F-ATP synthase contributes to ferroptosis defense.

Lishu Guo.

  The opening of the mitochondrial permeability transition pore (PTP), a Ca2+-dependent pore located in the inner mitochondrial membrane, triggers mitochondrial outer membrane permeabilization (MOMP) and induces organelle rupture. However, the underlying mechanism of PTP-induced MOMP remains unclear. Mitochondrial carrier homolog 2 (MTCH2) mediates MOMP process by facilitating the recruitment of tBID to mitochondria. Here, we show that MTCH2 binds to cyclophilin D (CyPD) and promotes the dimerization of F-ATP synthase via interaction with subunit j. The interplay between MTCH2 and subunit j coordinates MOMP and PTP to mediate the occurrence of mitochondrial permeability transition. Knockdown of CyPD, MTCH2 and subunit j markedly sensitizes cells to RSL3-induced ferroptosis, which is prevented by MitoTEMPO, suggesting that mitochondrial permeability transition mediates ferroptosis defense.

Keywords:  F‐ATP synthase; cyclophilin D; ferroptosis; mitochondrial carrier homolog 2; mitochondrial permeability transition

DOI:  https://doi.org/10.1002/1873-3468.15008
Autophagy. 2024 Sep 03. 1-3

Nucleoid-phagy: a novel safeguard against mitochondrial DNA-Induced inflammation.

Hao Liu, Jianming Xie, Cien Zhen, Lin Zeng, Hualin Fan, Haixia Zhuang, Du Feng.

  Mitochondria, the powerhouses of the cell, play pivotal roles in cellular processes ranging from energy production to innate immunity. Their unique double-membrane structure typically sequesters mitochondrial DNA (mtDNA) from the rest of the cell. However, under oxidative or immune stress, mtDNA can escape into the cytoplasm, posing a threat as a potential danger signal. The accumulation of cytoplasmic mtDNA can disrupt cellular immune balance and trigger cell death. Our research unveils a novel quality control mechanism, which we term "nucleoid-phagy", that safeguards cellular homeostasis by clearing mislocalized mtDNA. We demonstrate that TFAM, a key protein involved in mtDNA folding and wrapping, accompanies mtDNA into the cytoplasm under stress conditions. Remarkably, TFAM acts as an autophagy receptor, interacting with LC3B to facilitate the autophagic clearance of cytoplasmic mtDNA, thereby preventing the activation of the pro-inflammatory CGAS-STING1 pathway. This study provides unprecedented insights into cytoplasmic mtDNA quality control and offers new perspectives on mitigating inflammatory responses in mitochondrial-related diseases.

Keywords:  Autophagy; CGAS-STING1; LIR; TFAM; mitochondria DNA

DOI:  https://doi.org/10.1080/15548627.2024.2395145
Aging Cell. 2024 Aug 29. e14282

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

John Sturgis, Rupesh Singh, Quinn R Caron, Ivy S Samuels, Thomas Micheal Shiju, Aditi Mukkara, Paul Freedman, Vera L Bonilha.

  Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the PolgD257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed in vivo and ex vivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration.

Keywords:  D257A; mitochondria; mitochondrial DNA (mtDNA); polymerase gamma (POLG); retina; retinal degeneration

DOI:  https://doi.org/10.1111/acel.14282
Mitochondrial Commun. 2023 ;1 48-61

Structure-destabilizing mutations unleash an intrinsic perforation activity of antiapoptotic Bcl-2 in the mitochondrial membrane enabling apoptotic cell death.

Ping Gao, Zhi Zhang, Rui Wang, Li Huang, Hao Wu, Zhenzhen Qiao, Xiaohui Wang, Haijing Jin, Jun Peng, Lei Liu, Quan Chen, Jialing Lin.

  Bcl-2 and Bax share a similar structural fold in solution, yet function oppositely in the mitochondrial outer membrane (MOM) during apoptosis. The proapoptotic Bax forms pores in the MOM to trigger cell death, whereas Bcl-2 inhibits the Bax pore formation to prevent cell death. Intriguingly both proteins can switch to a similar conformation after activation by BH3-only proteins, with multiple regions embedded in the MOM. Here we tested a hypothesis that destabilization of the Bcl-2 structure might convert Bcl-2 to a Bax-like perforator. We discovered that mutations of glutamate 152 which eliminate hydrogen bonds in the protein core and thereby reduce the Bcl-2 structural stability. These Bcl-2 mutants induced apoptosis by releasing cytochrome c from the mitochondria in the cells that lack Bax and Bak, the other proapoptotic perforator. Using liposomal membranes made with typical mitochondrial lipids and reconstituted with purified proteins we revealed this perforation activity was intrinsic to Bcl-2 and could be unleashed by a BH3-only protein, similar to the perforation activity of Bax. Our study thus demonstrated a structural conversion of antiapoptotic Bcl-2 to a proapoptotic perforator through a simple molecular manipulation or interaction that is worthy to explore further for eradicating cancer cells that are resistant to a current Bcl-2-targeting drug.

Keywords:  Apoptosis; Bcl-2 protein pore; Cytochrome c release; Mitochondrial membrane

DOI:  https://doi.org/10.1016/j.mitoco.2023.08.001
Cytokine. 2024 Aug 31. pii: S1043-4666(24)00240-0. [Epub ahead of print]183 156737

Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways.

Fei Xie, Yoshinori Kitagawa, Hiroki Ogata, Shingo Yasuhara, Zerong You, J A Jeevendra Martyn.

  BACKGROUND: Opioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway.METHODS: BV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells.
RESULTS: Morphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects.
CONCLUSION: Morphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Keywords:  Inflammation; Morphine; NF-κB; Polarization; TLR4; cGAS-STING

DOI:  https://doi.org/10.1016/j.cyto.2024.156737
J Virol. 2024 Aug 30. e0105524

Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus.

Pheonah Badu, Gabriele Baniulyte, Morgan A Sammons, Cara T Pager.

  Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects of a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated, and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection.IMPORTANCE: Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that co-opts cellular mechanisms to support viral processes that can reprogram the host transcriptional profile. Such viral-directed transcriptional changes and the pro- or anti-viral outcomes remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV-infected mammalian cells, along with other cellular and immune response genes. We now define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. We show that during ZIKV infection, the integrated stress response pathway stimulates ATF3 which enhances the innate immune response to antagonize ZIKV infection. This study establishes a link between viral-induced stress response and transcriptional regulation of host defense pathways and thus expands our knowledge of virus-mediated transcriptional mechanisms and transcriptional control of interferon-stimulated genes during ZIKV infection.

Keywords:  Zika virus; flavivirus; innate immune response; integrated stress response; transcription factor

DOI:  https://doi.org/10.1128/jvi.01055-24
Blood Adv. 2024 Sep 03. pii: bloodadvances.2023012077. [Epub ahead of print]

Mitochondrial dsRNA from B-ALL cells stimulates mesenchymal stromal cells to become cancer associated fibroblasts.

Richard Burt, Aditi Dey, Ayse Akarca, Hermione Allen, Rodothea Amerikanou, Samantha Atkinson, David Auty, Jenny Chatzigerou, Emily Annie Cutler, Jose Afonso Guerra-Assuncao, Kristina Kirschner, Ruchi Kumari, Jiten Manji, Teresa Marafioti, Juma Ward, Adele K Fielding.

Cancer associated fibroblasts (CAF) arising from bone marrow-derived mesenchymal stromal cells (MSC) are prominent in B-precursor acute lymphoblastic leukaemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species-inducing chemotherapy and that CAF support chemoresistance by donating mitochondria to the cancer cells, through tunnelling nanotubes. In the present study, we show that exposure of MSC to ALL cell lines, patient-derived xenografts and primary cells or their conditioned media can also trigger CAF formation. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the take-up of leukaemia cell-derived mitochondrial dsRNA by MSC as a proximate trigger for the MSC to CAF transition. We show that inhibition of dsRNA formation in ALL cells by treatment with low-dose ethidium or the mitochondrial transcription inhibitor IMT1 or degradation of dsRNA in conditioned media by 100°C exposure ablates the ability of the ALL conditioned media to stimulate MSC to CAF transition. Our data reveal a novel and previously undescribed mechanism by which cancer cells induce a CAF phenotype in stromal cells, showing how B-ALL cells can directly induce the previously described niche-mediated protection within the bone marrow.

DOI: https://doi.org/10.1182/bloodadvances.2023012077
PLoS One. 2024 ;19(9): e0304939

Mitochondrial DNA is a sensitive surrogate and oxidative stress target in oral cancer cells.

Jingyu Tan, Xinlin Dong, Haiwen Liu.

Cellular oxidative stress mediated by intrinsic and/or extrinsic reactive oxygen species (ROS) is associated with disease pathogenesis. Oxidative DNA damage can naturally be substituted by mitochondrial DNA (mtDNA), leading to base lesion/strand break formation, copy number changes, and mutations. In this study, we devised a single test for the sensitive quantification of acute mtDNA damage, repair, and copy number changes using supercoiling-sensitive quantitative PCR (ss-qPCR) and examined how oxidative stress-related mtDNA damage responses occur in oral cancer cells. We observed that exogenous hydrogen peroxide (H2O2) induced dynamic mtDNA damage responses, as reflected by early structural DNA damage, followed by DNA repair if damage did not exceed a particular threshold. However, high oxidative stress levels induced persistent mtDNA damage and caused a 5-30-fold depletion in mtDNA copy numbers over late responses. This dramatic depletion was associated with significant growth arrest and apoptosis, suggesting persistent functional consequences. Moreover, oral cancer cells responded differentially to oxidative injury when compared with normal cells, and different ROS species triggered different biological consequences under stress conditions. In conclusion, we developed a new method for the sensitive detection of mtDNA damage and copy number changes, with exogenous H2O2 inducing dynamic mtDNA damage responses associated with functional changes in stressed cancer cells. Finally, our method can help characterize oxidative DNA damage in cancer and other human diseases.

DOI: https://doi.org/10.1371/journal.pone.0304939
J Biol Chem. 2024 Aug 28. pii: S0021-9258(24)02229-4. [Epub ahead of print] 107728

Defective post-transcriptional modification of tRNA disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.

Juanjuan Zhang, Wenxu Li, Zhen Liu, Yingqi Chen, Xiaoyang Wei, Lu Peng, Man Xu, Yanchun Ji.

  Leber's Hereditary Optic Neuropathy (LHON) is a rare, maternally inherited eye disease, predominantly due to the degeneration of retinal ganglion cells (RGCs). It is associated with a mitochondrial DNA (mtDNA) point mutation. Our previous study identified that the m.15927G>A homoplasmic mutation damaged the highly conserved basepairing (28C-42G) in anticodon stem of tRNAThr, caused deficient t6A modification and significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr, and led to mitochondrial dysfunction. Meanwhile, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we manifested that defective nucleotide modification induced by the m.15927G>A mutation interfered with the expression of nuclear genes involved in cytoplasmic proteins essential for oxidative phosphorylation system (OXPHOS), thereby impacting the assemble and integrity of OXPHOS complexes. As a result of these mitochondrial dysfunctions, there was an imbalance in mitochondrial dynamics, particularly distinguished by an increased occurrence of mitochondrial fission. Excessive fission compromised the autophagy process, including initiation phase, formation and maturation of autophagosome. Both Parkin-mediated mitophagy and receptor-dependent mitophagy were significantly impaired in cybrids haboring the m.15927G>A mutation. These changes facilitated intrinsic apoptosis, as indicated by increased cytochrome c release and elevated levels of apoptosis-associated proteins (e.g., BAK, BAX, cleaved caspase 9, cleaved caspase 3, and cleaved PARP) in the mutant cybrids. This study demonstrates that the m.15927G>A mutation contributes to LHON by dysregulating OXPHOS biogenesis, aberrant quality control, increased autophagy, inhibited mitophagy, and abnormal apoptosis.

Keywords:  Leber’s hereditary optic neuropathy (LHON); apoptosis; autophagy; mitochondrial tRNA(Thr) mutation; mitophagy

DOI:  https://doi.org/10.1016/j.jbc.2024.107728
bioRxiv. 2024 Aug 24. pii: 2024.08.23.609473. [Epub ahead of print]

Robustness and reliability of single-cell regulatory multi-omics with deep mitochondrial mutation profiling.

Chen Weng, Jonathan S Weissman, Vijay G Sankaran.

The detection of mitochondrial DNA (mtDNA) mutations in single cells holds considerable potential to define clonal relationships coupled with information on cell state in humans. Previous methods focused on higher heteroplasmy mutations that are limited in number and can be influenced by functional selection, introducing biases for lineage tracing. Although more challenging to detect, intermediate to low heteroplasmy mtDNA mutations are valuable due to their high diversity, abundance, and lower propensity to selection. To enhance mtDNA mutation detection and facilitate fine-scale lineage tracing, we developed the single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM) approach, an integrated experimental and computational framework. Recently, some concerns have been raised about the analytical workflow in the ReDeeM framework. Specifically, it was noted that the mutations detected in a single molecule per cell are enriched on edges of mtDNA molecules, suggesting they resemble artifacts reported in other sequencing approaches. It was then proposed that all mutations found in one molecule per cell should be removed. We detail our error correction method, demonstrating that the observed edge mutations are distinct from previously reported sequencing artifacts. We further show that the proposed removal leads to massive elimination of bona fide and informative mutations. Indeed, mutations accumulating on edges impact a minority of all mutation calls (for example, in hematopoietic stem cells, the excess mutations on the edge account for only 4.3%-7.6% of the total). Recognizing the value of addressing edge mutations even after applying consensus correction, we provide an additional filtering option in the ReDeeM-R package. This approach effectively eliminates the position biases, leads to a mutational signature indistinguishable from bona fide mitochondrial mutations, and removes excess low molecule high connectedness mutations. Importantly, this option preserves the large majority of unique mutations identified by ReDeeM, maintaining the ability of ReDeeM to provide a more than 10-fold increase in variant detection compared to previous methods. Additionally, the cells remain well-connected. While there is room for further refinement in mutation calling strategies, the significant advances and biological insights provided by the ReDeeM framework are unique and remain intact. We hope that this detailed discussion and analysis enables the community to employ this approach and contribute to its further development.

DOI: https://doi.org/10.1101/2024.08.23.609473
J Vis Exp. 2024 Aug 16.

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures.

Qitao Zhang, Daisy Y Shu, Richard A Bryan, John Y S Han, Gillian A Gulette, Kin Lo, Leo A Kim, Jason M L Miller.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

DOI: https://doi.org/10.3791/67038