Selective Uncoupling of Individual Mitochondria within a Cell Using a Mitochondria-Targeted Photoactivated Protonophore

Chalmers, Susan; Caldwell, Stuart T.; Quin, C. E.; Prime, Tracy A.; James, Andrew M.; Cairns, Andrew G.; Murphy, Michael P.; McCarron, John G.; Hartley, Richard C.

doi:10.1021/ja2077922

Cited by 115 publications

(104 citation statements)

References 38 publications

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“…However, 2,4-DNP's narrow therapeutic window led to the abandonment of its use. A recent study found that 2,4-DNP linked to TPP in a covalent manner is ineffective at uncoupling (30). Concerns about the narrow therapeutic window and failure of the covalently linked uncoupler have led to the evaluation of mitochondria-targeted NPs in directing this uncoupler to the mitochondria of cells.…”

Section: Resultsmentioning

confidence: 99%

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

Marrache¹,

Dhar

2012

Proc. Natl. Acad. Sci. U.S.A.

431

340

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Mitochondrial dysfunctions cause numerous human disorders. A platform technology based on biodegradable polymers for carrying bioactive molecules to the mitochondrial matrix could be of enormous potential benefit in treating mitochondrial diseases. Here we report a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various mitochondria-acting therapeutics by blending a targeted poly(D,L-lactic-co-glycolic acid)-block (PLGA-b)-poly(ethylene glycol) (PEG)-triphenylphosphonium (TPP) polymer (PLGA-b-PEG-TPP) with either nontargeted PLGA-b-PEG-OH or PLGA-COOH. An optimized formulation was identified through in vitro screening of a library of charge-and size-varied NPs, and mitochondrial uptake was studied by qualitative and quantitative investigations of cytosolic and mitochondrial fractions of cells treated with blended NPs composed of PLGA-b-PEG-TPP and a triblock copolymer containing a fluorescent quantum dot, PLGA-b-PEG-QD. The versatility of this platform was demonstrated by studying various mitochondria-acting therapeutics for different applications, including the mitochondria-targeting chemotherapeutics lonidamine and α-tocopheryl succinate for cancer, the mitochondrial antioxidant curcumin for Alzheimer's disease, and the mitochondrial uncoupler 2,4-dinitrophenol for obesity. These biomolecules were loaded into blended NPs with high loading efficiencies. Considering efficacy, the targeted PLGA-b-PEG-TPP NP provides a remarkable improvement in the drug therapeutic index for cancer, Alzheimer's disease, and obesity compared with the nontargeted construct or the therapeutics in their free form. This work represents the potential of a single, programmable NP platform for the diagnosis and targeted delivery of therapeutics for mitochondrial dysfunction-related diseases.nanocarrier | organelle | nanomedicine | drug trafficking

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Section: Resultsmentioning

confidence: 99%

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

Marrache¹,

Dhar

2012

Proc. Natl. Acad. Sci. U.S.A.

431

340

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“…For uncouplers to work safely, these compounds should cause uncoupling that increases very little as their concentration rises, potentially widening the difference between therapeutic and toxic doses and giving a wide therapeutic window (62). Recently, however, targeted uncoupling showing precise spatial and temporal control of mitochondrial function have been demonstrated, leading to the selective uncoupling of either individual or small groups of them within a cell (63,64). Such technology, if adapted to SR4 in the future, will increase its safety and potentially lower the effective maximum dose required for its cytotoxic effects on tumors.…”

Section: Discussionmentioning

confidence: 99%

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Figarola

Singhal

Tompkins

et al. 2015

Journal of Biological Chemistry

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Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. Hepatocellular carcinoma (HCC)2 is the most common and severe form of liver cancer, accounting for about 80 -90% of primary liver cancers and 5% of all human cancers. More than 600,000 deaths are attributed to HCC every year with 2:1 ratio for men versus women (1, 2). HCC is a primary cancer of hepatocytes that most typically occurs in the setting of known risk factors, including cirrhosis and chronic hepatitis B virus or hepatitis C virus infections (2, 3), although recently, several lines of evidence suggest that type 2 diabetes is also an independent risk factor for HCC development (4). HCC is an aggressive tumor and represents a major health problem as its incidence is increasing. Systemic chemotherapies have proven ineffective against advanced HCC, so it typically leads to death within 6 -20 months (5). Hepatocarcinogenesis is a multistep process involving inflammation, hyperplasia, and dysplasia that finally leads to malignant transformation. In recent years, mitochondria have been found to provide a novel targeting site for new anticancer drugs (known as "mitocans") that ca...

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“…Techniques to study how mitochondria control specific signals are improving constantly. The recent development [20] of a membrane permeant, caged protonophore (2,4-dinitrophenol), which has been targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system, will improve understanding of the mitochondrial control of local and global Ca 2+ signals in smooth muscle.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial regulation of cytosolic Ca2+ signals in smooth muscle

McCarron

Olson

Chalmers

2012

Pflugers Arch - Eur J Physiol

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The cytosolic Ca²⁺ concentration ([Ca²⁺]c) controls virtually every activity of smooth muscle, including contraction, migration, transcription, division and apoptosis. These processes may be activated by large (>10 μM) amplitude [Ca²⁺]c increases, which occur in small restricted regions of the cell or by smaller (<1 μM) amplitude changes throughout the bulk cytoplasm. Mitochondria contribute to the regulation of these signals by taking up Ca²⁺. However, mitochondria's reported low affinity for Ca²⁺ is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca²⁺]. In cultured smooth muscle, mitochondria are highly dynamic structures but in native smooth muscle mitochondria are immobile, apparently strategically positioned organelles that regulate the upstroke and amplitude of IP₃-evoked Ca²⁺ signals and IP₃ receptor (IP₃R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca²⁺] microdomain arising from an IP₃R cluster to exert significant local control of channel activity. On the other hand, neither the upstroke nor amplitude of voltage-dependent Ca²⁺ entry is modulated by mitochondria; rather, it is the declining phase of the transient that is regulated by the organelle. Control of the declining phase of the transient requires a high mitochondrial affinity for Ca²⁺ to enable uptake to occur over the normal physiological Ca²⁺ range (<1 μM). Thus, in smooth muscle, mitochondria regulate Ca²⁺ signals exerting effects over a large range of [Ca²⁺] (∼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca²⁺ signals.

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Selective Uncoupling of Individual Mitochondria within a Cell Using a Mitochondria-Targeted Photoactivated Protonophore

Cited by 115 publications

References 38 publications

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Mitochondrial regulation of cytosolic Ca2+ signals in smooth muscle

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