An analysis of membrane fusion between mitochondrial double membranes and MITO-Porter, mitochondrial fusogenic vesicles
Introduction
It has been reported by numerous investigators that genetic defects in mitochondrial DNA (mtDNA) are associated with mitochondrial diseases and that a variety of human disorders, including neurodegenerative diseases, diabetes mellitus and cancer can be attributed to this (Chan, 2006, Schapira, 2006, Taylor and Turnbull, 2005). Mitochondrial genome-targeting nucleic acids are promising candidates for the therapeutic treatment of mitochondrial diseases. Up to the present, a number of systems for the delivery of nucleic acids to the cytosol and the nucleus, including several successful gene therapies, have been reported (Miyata et al., 2012, Nakamura et al., 2012), while much less progress has been made concerning mitochondrial delivery systems (Biswas and Torchilin, 2014, Kajimoto et al., 2014, Weissig, 2011, Yamada and Harashima, 2008, Zhang et al., 2011). It is noteworthy in this respect that mitochondrial gene therapy has never been achieved. An optimal mitochondrial targeting system for regulating intracellular trafficking and the import therapeutic molecules into the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool, are required to accomplish mitochondrial gene therapy.
In a previous study, we reported on the development of a Dual Function (DF)-MITO-Porter, an innovative nanocarrier for achieving mitochondrial delivery, which has the ability to pass through the endosomal and mitochondrial membranes via step-wise membrane fusion (Yamada et al., 2011, Yamada and Harashima, 2012). Octaarginine (R8), a cell penetrating peptide, was used to modify the MITO-Porter system, and was found to function as a useful moiety for cellular uptake via macropinocytosis and mitochondrial targeting via electrostatic interaction (Futaki et al., 2001, Khalil et al., 2006, Yamada et al., 2008). In addition, we succeeded in packaging an oligodeoxynucleotide (a model nucleic acid) in the DF-MITO-Porter and were able to achieve the mitochondrial delivery of nucleic acids to regulate the multiple intracellular processes (Yamada et al., 2012a, Yamada et al., 2012b). More recently, the S2 peptide (Dmt-D-Arg-FK-Dmt-D-Arg-FK-NH2) modified DF-MITO-Porter was found to have a lower cell toxicity compared to the R8 modified carrier, while its mitochondrial targeting activity was similar to that of R8 (Kawamura et al., 2013).
Furthermore, we verified that the MITO-Porter delivered cargoes to the mitochondrial matrix using propidium iodide as a probe to detect mtDNA. As result, we were able to confirm that this system can be used to efficiently visualize mtDNA, not only in isolated mitochondria, but in living cells as well (Yasuzaki et al., 2010). We also attempted the mitochondrial delivery of the DNase I protein using the DF-MITO-Porter to estimate the mitochondrial gene targeting of the carrier (Yamada et al., 2012a, Yamada et al., 2011, Yamada and Harashima, 2012). The results indicated that the use of the DF-MITO-Porter resulted in a decrease in mtDNA-levels followed by a decrease in mitochondrial activity. Based on these previous reports, we conclude that the MITO-Porter system has the ability to deliver cargoes to the mitochondrial matrix and the target mtDNA.
To efficiently import cargoes to the mitochondrial matrix, the MITO-Porter is required to efficiently fuse with both mitochondrial outer and inner membranes. As previously reported, R8-modified envelopes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) showed a high fusogenic activity with the mitochondrial outer membrane (Yamada et al., 2008), however, these lipids may not be the best lipid composition for use in conjunction with the mitochondrial inner membrane. We report herein on an investigation of a fusogenic lipid composition designed for the mitochondrial inner membrane and a relationship analysis of the lipid composition needed for the mitochondrial outer versus the inner membrane. This analysis for mitochondrial membrane fusion was done by monitoring the cancellation of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores, modified on the surface of liposomes (Maier et al., 2002, Struck et al., 1981, Yamada et al., 2012a, Yamada et al., 2008, Yamada and Harashima, 2014). We also evaluated the import of nucleic acids to the mitochondrial matrix by the MITO-Porter system, and investigated the effect of fusion activity for outer and inner membranes on import efficiency.
Section snippets
Materials
Cholesterol (Chol), cardiolipin (CL), 1, 2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), phosphatidyl glycerol (PG), phosphatidyl serine (PS), sphingomyelin (SM), 7-nitrobenz-2-oxa-1, 3-diazole labeled DOPE (NBD-DOPE) and rhodamine-DOPE were purchased from Avanti Polar lipids (Alabaster, AL, USA). Egg yolk phosphatidyl choline (EPC) was obtained from the NOF Corporation (Tokyo, Japan). Cholesteryl hemisuccinate (CHEMS), phosphatidic acid (PA), and phosphatidyl inositol (PI) were
Construction of liposomes with various lipid compositions and an evaluation of mitochondrial outer membrane fusion activity using isolated mitochondria
We previously established a method for evaluating the membrane fusion activity of liposomes with isolated rat liver mitochondria and FRET analysis (Yamada et al., 2012a, Yamada et al., 2008, Yamada and Harashima, 2014), as shown in Fig. 1A. A previous investigation showed that R8-modified envelopes composed of DOPE showed a high fusogenic activity for the mitochondrial outer membrane (Yamada et al., 2008), moreover, the lipid composition containing SM [DOPE/SM/STR-R8 (9:2:1, molar ratio)] was
Conclusion
The findings of this study showed that a MITO-Porter system could effectively fuse with both the mitochondrial outer and inner membranes, and that a combination of R8 and DOPE mainly contributed to the inner membranes, similar to the outer membranes. Moreover, it was confirmed that the MITO-Porter delivered nucleic acids into the mitochondrial matrix, indicating that it holds promise as a mitochondrial vector for nucleic acids. Studies related to this issue are currently in progress.
Acknowledgments
We thank Dr. Y. Shinohara (University of Tokushima, Tokushima, Japan) for kindly supplying primary antibodies from rabbit against VDAC2. This work was supported, in part by, a Grant-in-Aid for Young Scientists (A) [Grant No. 26282131 (to Y.Y.)], a Grant-in Aid for Challenging Exploratory Research [Grant No. 25560219 (to Y.Y.)] and a Grant-in-Aid for Scientific Research (B) [Grant No. 26282131 (to Y.Y.)] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese
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