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APOL1 Kidney Risk Variants Induce Cell Death via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore.

ABSTRACT: BACKGROUND:Genetic Variants in Apolipoprotein L1 (APOL1) are associated with large increases in CKD rates among African Americans. Experiments in cell and mouse models suggest that these risk-related polymorphisms are toxic gain-of-function variants that cause kidney dysfunction, following a recessive mode of inheritance. Recent data in trypanosomes and in human cells indicate that such variants may cause toxicity through their effects on mitochondria. METHODS:To examine the molecular mechanisms underlying APOL1 risk variant-induced mitochondrial dysfunction, we generated tetracycline-inducible HEK293 T-REx cells stably expressing the APOL1 nonrisk G0 variant or APOL1 risk variants. Using these cells, we mapped the molecular pathway from mitochondrial import of APOL1 protein to APOL1-induced cell death with small interfering RNA knockdowns, pharmacologic inhibitors, blue native PAGE, mass spectrometry, and assessment of mitochondrial permeability transition pore function. RESULTS:We found that the APOL1 G0 and risk variant proteins shared the same import pathway into the mitochondrial matrix. Once inside, G0 remained monomeric, whereas risk variant proteins were prone to forming higher-order oligomers. Both nonrisk G0 and risk variant proteins bound components of the mitochondrial permeability transition pore, but only risk variant proteins activated pore opening. Blocking mitochondrial import of APOL1 risk variants largely eliminated oligomer formation and also rescued toxicity. CONCLUSIONS:Our study illuminates important differences in the molecular behavior of APOL1 nonrisk and risk variants, and our observations suggest a mechanism that may explain the very different functional effects of these variants, despite the lack of consistently observed differences in trafficking patterns, intracellular localization, or binding partners. Variant-dependent differences in oligomerization pattern may underlie APOL1's recessive, gain-of-function biology.

SUBMITTER: Shah SS

PROVIDER: S-EPMC6900802 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨM) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 ″super-resolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 s (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 min. The time to first depolarization events was 8.4 min (±1.1 SEM n = 21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 min) and virtually no permanent depolarization events occurred for over 1 h of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 μM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80% and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ± 6 s (n = 478) at 37 °C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca2+. In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨM depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6,7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨM depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool ("Optical Stress-Probe"), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks.

Project description:The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.

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APOL1 Kidney Risk Variants Induce Cell Death via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore.

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