Mitochondrial light switches: optogenetic approaches to control metabolism

Abstract

Developing new technologies to study metabolism is increasingly important as metabolic disease prevalence increases. Mitochondria control cellular metabolism and dynamic changes in mitochondrial function are associated with metabolic abnormalities in cardiovascular disease, cancer, and obesity. However, a lack of precise and reversible methods to control mitochondrial function has prevented moving from association to causation. Recent advances in optogenetics have addressed this challenge, and mitochondrial function can now be precisely controlled in vivo using light. A class of genetically encoded, light-activated membrane channels and pumps has addressed mechanistic questions that promise to provide new insights into how cellular metabolism downstream of mitochondrial function contributes to disease. Here, we highlight emerging reagents-mitochondria-targeted light-activated cation channels or proton pumps-to decrease or increase mitochondrial activity upon light exposure, a technique we refer to as mitochondrial light switches, or mt^SWITCH . The mt^SWITCH technique is broadly applicable, as energy availability and metabolic signaling are conserved aspects of cellular function and health. Here, we outline the use of these tools in diverse cellular models of disease. We review the molecular details of each optogenetic tool, summarize the results obtained with each, and outline best practices for using optogenetic approaches to control mitochondrial function and downstream metabolism.

Keywords: AMPK; Parkinson’s; apoptosis; bioenergetics; calcium signaling; diabetes; hypoxia; mitophagy.

Figure 1.. Endogenous protonmotive force (PMF) regulation.

The mitochondrial electron transport chain (ETC) pumps protons from the mitochondrial matrix to the intermembrane space (IMS) to establish the PMF. The PMF drives ATP synthase to phosphorylate ADP to ATP. Other proteins in the inner membrane (IM) regulate mitochondrial function through proton transport which affects the PMF. Uncoupling proteins (UCP) are regulated to dissipate the PMF. Similarly, adenine nucleotide transporters (ANT) can also dissipate the PMF in a regulated manner. Several metabolite and ion exchangers also use the PMF to drive their functions. Cation channels in mitochondria can allow cation (X⁺⁽⁺⁾) accumulation into the matrix. The mitochondrial permeability transition pore (mPTP) is a large conductance channel that can cause total PMF collapse in different physiologic and pathologic conditions.

Figure 2.. mt^SWITCH constructs increase or decrease the PMF in response to light.

mt^SWITCH constructs are shown with their respective mitochondria-targeting sequences (MTS, shown in purple) in the mitochondrial inner membrane. Light-activated proteins are shown in either orange or blue to indicate that they decrease or increase the PMF, respectively. Topology of light-activated proteins are indicated where ions are moved either from the intermembrane space (IMS) to the matrix, or vice versa. N and C termini of peptides are indicated in each construct. A) mitoChR2 uses the COX8A mitochondria-targeting sequence (MTS) repeated in tandem four times to achieve mitochondrial expression. mitoChR2 is activated by 460–480 nm light, and results in decreased PMF. ChR2 is capable of passing protons (H⁺) as well as other cations, potassium (K⁺) the most prevalent in the cytosol. B) ABCB-ChR2 uses the ABCB10 MTS to direct ChR2 to mitochondria, and is topologically oriented the same as mitoChR2. ABCB-ChR2 is also activated by 460–480 nm light and results in decreased PMF. C) mtON is targeted to mitochondria by the IMMT1 MTS and some of the transmembrane protein sequence to flip the topology to direct proton-specific translocation from the matrix to the IMS. The native N terminus of the light-activated proton pump is therefore located on the IMS side, opposite of the MTS N terminus. mtON is activated by 550–590 nm light and results in increased PMF. D) mito-dR uses one COX8A MTS, and the topology and direction of proton translocation is unclear in mitochondria. mito-dR is activated by 550–590 nm light and is proposed to increase the PMF.

Figure 3.. The mt^SWITCH technique affects broad metabolic outputs through PMF manipulation.

mt^SWITCH constructs directly alter mitochondrial functions that impact downstream metabolism. Decreasing or increasing the PMF results in altered ATP production, O₂ consumption by the electron transport chain, Ca⁺⁺ handling and signaling, and cellular redox state. Each of these factors affect cellular signaling. Changing these parameters has impacted physiologic outputs such as apoptosis and mitophagy, glucose-stimulated ATP production in β-cells, AMPK signaling and hypoxia resistance in living animals, and biochemical and behavioral outcomes in neurodegeneration models. These are just a few validated examples of the impact mt^SWITCH will have on many fields of research to understand disease and how metabolism impacts it.