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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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APOL1 Kidney Risk Variants Induce Cell Death Via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore BASIC RESEARCH www.jasn.org APOL1 Kidney Risk Variants Induce Cell Death via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore Shrijal S. Shah, Herbert Lannon , Leny Dias, Jia-Yue Zhang, Seth L. Alper, Martin R. Pollak, and David J. Friedman Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 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 non- risk G0 variant or APOL1 risk variants. Using these cells, we mapped the molecular pathway from mito- chondrial 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 per- meability 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. JASN 30: 2355–2368, 2019. doi: https://doi.org/10.1681/ASN.2019020114 African Americans develop ESKD much more fre- quently that other groups. A large fraction of this disparity is due to genetic variants in the APOL1 Received February 5, 2019. Accepted August 15, 2019. gene. Inheriting two copies of APOL1 risk variants Published online ahead of print. Publication date available at (RVs), known as G1 and G2, causes high rates of www.jasn.org. FSGS, HIV-associated nephropathy and hypertension- Correspondence: Dr. David J. Friedman, Beth Israel Deaconess associated ESKD.1–4 Medical Center, 330 Brookline Avenue, Boston, MA 02215. Of the six members of the APOL gene family, Email: [email protected] APOL1 is the sole member with a signal peptide Copyright © 2019 by the American Society of Nephrology JASN 30: 2355–2368, 2019 ISSN : 1046-6673/3012-2355 2355 BASIC RESEARCH www.jasn.org permitting cellular export.5,6 Circulating non-risk APOL1 Significance Statement (G0) confers resistance to trypanosome Trypanosoma brucei brucei through ion channel formation and trypanolysis, but Some variants in APOL1 are associated with high CKD rates in cannot protect against the two subspecies Trypanosoma brucei African Americans, but the molecular mechanism of disease re- rhodesiense and Trypanosoma brucei gambiense.7,8 These highly mains elusive. Previous studies demonstrated that expression of APOL1 risk variants is associated with mitochondrial dysfunction. In pathogenic subspecies have evolved multiple mechanisms to this study, the authors show that import of APOL1 protein into inactivate APOL1-mediated lysis.9,10 G1 (two amino acid sub- mitochondria is essential for risk variant–mediated cytotoxicity, and stitutions: S342G and I384M) and G2 (a two-amino-acid de- map the APOL1 import pathway. They found that whereas APOL1 is letion: del388N389Y) mutations originated in sub-Saharan mostly monomeric, risk variant APOL1 can form large oligomers Africa and rose to high frequency because they provided a and cause opening of the mitochondrial permeability transition pore, ultimately leading to cell death. This difference in propensity selective advantage in regions with pathogenic trypano- of different variants to oligomerize could help explain APOL1 risk – somes.1,11 13 Although inheriting a single copy of G1 or G2 variants’ gain-of-function biology despite a recessive mode of in- is sufficient to enhance protection against resistant trypano- heritance. Understanding APOL1 trafficking and interactions could somes, inheriting two copies increases risk of developing kid- help inform new therapeutic approaches. ney disease. Not all individuals with two copies of the RVs develop kid- could explain why multiple modes of cell death have been ney disease, suggesting a second hit is required for disease to observed in association with APOL1 overexpression. develop in high-risk individuals. APOL1 RVs increase risk in a recessive manner, but unlike most variants that cause a reces- sive mode of inheritance, evidence suggests that APOL1 RVs METHODS are toxic, gain-of-function mutations.14 This hypothesis is supported by the fact that most mammals and even some Cells and Antibodies primates have no APOL1 gene, and at least one human is HEK293 cells expressing tetracycline-inducible APOL1 inte- completely null for APOL1 while having no apparent kidney grated at a single locus were generated using the T-REx system dysfunction.6,15 Although the increased risk of kidney disease (Thermo Fisher Scientific). APOL1 sequences used for each due to APOL1 RVs has been well established, it remains un- genotype are shown in Supplemental Table 1. Cells were cul- certain how the APOL1 RVs promote kidney injury. tured at 37°C with 5% CO2 in DMEM (Corning) supple- Our goal was to address several important questions about mented with 10% tetracycline-free FBS (Atlanta Biologicals). APOL1 biology. First, the nature of APOL1 gain-of-function Cells were validated to be free of any Mycoplasma con- toxicity has remained elusive despite many well designed tamination. Anti-APOL1 rabbit polyclonal antibody studies. Evidence supporting many different cell death (HPA018885, lot #E114503) and anti-FLAG (F1804) — mechanisms apoptosis, autophagy, necrosis, pyroptosis, mouse monoclonal antibody were from Sigma. Anti- — 16–32 and necroptosis has been put forth. Second, the reason ACSL4 (SC-365230), anti-TOMM20 (SC-17764), anti- why two RVs are required for kidney disease, if the variants TOMM22 (SC-101286), anti-TOMM70 (SC-390545), are truly gain-of-function mutations, is not yet understood. anti-TIMM23 (SC-514463), anti-TIMM17 (SC-271152), The recessive mode of inheritance rather than an additive anti-HSPA9 (SC-133137), anti-HSP60 (SC-13115), anti- effect of APOL1 RVs has been proposed to involve oligomer ATP5A (SC-136178), anti-ATP5B (SC-55597) mouse mAb, formation or threshold effects, but there is little direct evi- and anti-APOL1 (SC-18759) goat polyclonal antibodies 33 fi denceforthesetheoriestodate. Third, de nitive differ- were from Santa Cruz Biotechnology. Anti-TIMM22 ences in behavior between G0 and RV APOL1 relating to (14927–1-AP) rabbit polyclonal antibody was from Protein- fi subcellular localization and/or af nity for binding partners tech. Anti-SLC25A5 (14671S) anti-calreticulin (12338S), have not been consistently demonstrated.34,35 We explore a anti-GM130 (12480S), anti-Rab7 (9367S), anti-LC3 modelthatmaybegintounifythesequestions. (12741S), anti-myc (2278S) rabbit polyclonal antibodies In this study, we build on the observation that APOL1 RVs were from Cell Signaling Technologies. cause mitochondrial dysfunction27,32,36 by (1) mapping an APOL1 mitochondrial import pathway, (2) demonstrating en- Preparation of Mitochondria-Enriched Fractions hanced APOL1 RV self-aggregation that occurs after transport Mitochondrial enrichment protocol was adapted from to the mitochondria, (3)defining a set of inner mitochondrial Jastroch et al.37 Briefly, cells were scraped in PBS and centri- membrane (IMM) binding partners of APOL1 that include fuged at 5003g for 5 minutes. Cell pellets were resuspended in several major mitochondrial permeability transition pore STE medium (250 mM sucrose, 5 mM Tris, 2 mM EGTA; pH 7.4) (mPTP) constituents and modulators, and (4) activating the and plasma membranes were disrupted using a dounce ho- mPTP, triggering first mitochondrial dysfunction and then mogenizer. The whole homogenate was centrifuged for 10 cell death. Our data on differential oligomer formation may minutes at 10003g and the supernatant was saved in a fresh ultimately help illuminate why both alleles must be RVs for tube. To increase yields, the cell pellet was resuspended again disease to occur, and our findings regarding mPTP activation in STE, dounced, and centrifuged at 10003g for 10 minutes. 2356 JASN JASN 30: 2355–2368, 2019 www.jasn.org BASIC RESEARCH The two supernatants were pooled and filtered through a cells were pretreated with respective dose of drug for 2 hours 5 mMsyringefilter. An aliquot of filtered lysate was saved as and then induced with 50 ng/ml tetracycline for 18 hours. loading control and denoted on figures
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