As the understanding of human biology advances the inherent diverse structure of the mitochondria follows, increasing the commonality of the variety in clinical presentations of Primary Mitochondrial Diseases (PMD). Parallel to this, so does the demand for new mitochondrial-based therapeutics.
Well what are PMDs? PMDs are mainly defined by mutations in the nuclear deoxyribonucleic acid (DNA) that encode components of the respiratory complex, leading to abnormal mitochondrial respiration.
Mitochonic Acid (MA-5) is by far the most interesting candidate for treating symptoms in PMD. MA-5 is originally derived from the plant hormone Indole-3-Acetic Acid and is currently in phase II trials for the treatment of different PMDs [1].
But, before I break down what MA-5 does, I’m going to give a brief introduction to the general structure and function of the powerhouse of the cell, the mitochondria.
The Structure and Function of the Mitochondria:
Starting with the Cristae lumens, which to simply put are long and slim tunnels inside Cristae membranes where the electron transport chain (ETC) and the adenosine triphosphate (ATP) synthase are located [2]. The Cristae membrane houses well known phospholipids such as Cardiolipin [3].
The geometrical structure of Cristae lumens are very purposeful. They are bent inwards and are relatively steep.
The reasoning behind the geometrical structure of Cristae lumens derives from the irreplaceable and intricate interaction between mitochondrial proteins such as phospholipids, the ATP synthase, and other Mitochondria junction proteins (OPA1, MICOS complex…).
The importance of mitochondrial proteins has been repeatedly highlighted through KO models. The disruption of one mitochondrial protein can destabilise the entire mitochondrial junction. Additionally, Cristae lumens act as electron storage compartments, forcibly pumping down the stored electrons towards the ATP synthase dimers [2].
The Cristae lumen houses the mitochondrial complexes on the flatter side of the Cristae membrane, and the bends are where the ATP synthase dimers reside, where electrons start to accumulate near the proton sink at the ATP synthase dimer.
The mitochondria gathers electrons through metabolic cycles such as the tricarboxylic acid (TCA) cycle in the form of NADH and FADH2. This is where metabolic intermediates such as NADH and FADH2 are shuttled to Complex I and Complex II [5].
NADH enters Complex I to donate two electrons and one proton (H+) and NAD+ and FADH2 enters Complex II to donate two electrons and FAD.
The reason why Complex II cannot donate H+ is because the complex is relatively small and does not span the intermembrane space.
Complex I pumps 4 H+ all together into the intermembrane space.
The two electrons from either Complex I or II are donated to ubiquinone which then reduces ubiquinone to ubiquinol.
Ubiquinol is then oxidised by Complex III generating two electrons, but Cytochrome C only allows the transport of one electron. During this, every electron transported to Cytochrome C, two H+ are pumped into the intermembrane space, and four H+ are pumped per two electrons.
At Complex IV, Cytochrome C ultimately transports four electrons, which are used to reduce oxygen (O2) to water (H2O). During this, four H+ are pumped into the intermembrane space, but at the expense of two H+, meaning that only a net of two H+ are pumped into the intermembrane space of the mitochondria.
This build up of H+ during oxidative phosphorylation (OXPHOS) creates the proton-motive force (Δp), which is the combination of the proton concentration (pH) and mitochondria membrane potential (ΔΨ), also referred to as the electrochemical gradient. The membrane potential is then decreased when H+ re-enter the mitochondria matrix through the ATP synthase, where it powers the rotator to generate ATP through ADP and phosphate.
Something to note though, is that Complex I, Complex III, and Complex IV are usually bound together as a supercomplex, whereas Complex II is usually alone.
The purpose of supercomplex generation is thought to be an evolutionary mechanism to prevent unnecessary leakage of electrons during electron exchange and to improve bioenergetic efficiency [6].
Figure (1) is the illustrated form of the process involved in ATP generation.
Mitofilin and Mitochonic Acid:
Moving over to the main star of this writeup, Mitofilin.
Mitofilin, is a mitochondrial structural protein located in the Cristae lumen where it shapes Cristae morphology, and is also a broad membrane structural protein as it is a core complex in the MICOS complex [7].
Mitofilin has broad roles outside of Cristae Lumen formation, and I will be briefly listing them.
Mitofilin is anchored to the outer-membrane of the mitochondria, where it interacts with other mitochondrial proteins.
Most notably Mitofilin interactions with two outer membrane mitochondrial proteins, Mia40 and Miro.
Mitofilin interacts with Mia40, which is needed for mitochondrial protein synthesis and positions Mia40 close to the protein translocation channel of the outer membrane [8]. This is important as Mia40 prevents incorrect protein folding by facilitating disulfide bond formation.
Mitofilin is also needed for proper mitochondria motility as it anchors another outer-membrane mitochondrial protein named Miro [9]. Miro connects the mitochondria to microtubule motors, which is important for mitochondria retrograde transfer and translocating the mitochondria to areas where metabolic intermediates are higher for ATP generation.
Mitofilin interacts with mitochondrial carrier subfamily of solute genes (SLC25A) and Cyclophilin D which is needed for ATP generation and closure of the mitochondrial permeability transition pore (mPTP) [10].
Mitofilin’s abundance is regulated by the receptor-interacting protein kinase 3 (RIP3) where their interaction promotes Mitofilin degradation, reduction, and contributes to mitochondrial damage [11].
Additionally, IgG complexes from both Long-Covid and ME/CFS patients indicate Mitofilin as a target for treating the presented mitochondrial abnormalities, and that excessive fission through over-expressed Drp1 is not what drives mitochondrial dysfunction, unlike what was previously thought [12].
MA-5 is thought to bind mitofilin and promote the assembly or stabilisation of ATP synthase complexes from individual units into larger multimeric structures.
It is also speculated that MA-5 interacts with heat shock protein 70 (Hsp70), ATP synthase subunits-alpha and -beta, RuvBL1 and -2, voltage-dependent anion-selective channel 1 (VDAC1), and CHCHD3, interacting to the sites of ATP. Although the supposed effect is currently unknown.
MA-5 is believed to facilitate ATP synthase oligomerisation through possible interactions with Su e, Su g, ATPase, and of course, Mitofilin [13]. This is believed to increase proton pressure on the ATP synthase dimer through modulating the localised proton gradient, hence generating ATP without the need for the Δp and the ΔΨ.
Additionally, because ATP itself causes conformational transitions to the F1 head domain of the ATP synthase, ATP’s direct modulatory effect is sufficient to power the motor for ATP hydrolysis, and it is speculated that MA-5 achieves the same outcome [13].
Interestingly enough, MA-5 has a S- (MA-S) and R- enantiomer (MA-R) and both the S- and R- enantiomers increase ATP by binding to Mitofilin.
Surprisingly, an additional mechanism has been attributed to MA-S, where it significantly increases NAD+ levels by binding to the NAD+-producing key enzyme nicotinamide phosphoribosyltransferase (NAMPT) [14]. Additionally, the MA-S suppresses SIRT1 ubiquitination, increasing SIRT1 induced by tripartite motif containing 28 (TRIM28) phosphorylation, which is mediated by DNA-dependent protein kinase (DNA-PK) activation.
NAMPT is the rate-limited recycler in the salvage pathway. It converts NAM into NMN, which then is adenylylated by NMNAT into NAD+. MA-S directly binds to NAMPT, increasing its activity and therefore NAD+ cellular abundance.
TRIM2 increases both ubiquitination and proteasomal degradation of SIRT1, and the phosphorylation of TRIM on serine 824 by DNA-PK activation prevents ubiquitination activity and increases SIRT1 activity.
DNA-PK is involved in the regeneration of DNA double strand breaks, and, if not repaired, double strand breaks can insult the cell, induce apoptosis, commit the cell to cell cycle arrest, or contribute to genome instability and cancer [15]. Following that, mutations in DNA repair genes are linked to cancer susceptibility.
Fortunately, MA-S directly docks at DNA-PK leading to the subsequent activation and, therefore, DNA repair. This activates DNA-PK without the need of DNA repair pathways.
Figure (2) elucidates the diverse cellular action MA-5 has depending on the enantiomer.
Conclusion:
The many mechanisms underlying the function of MA-5 make it an exciting candidate for treating different PMDs [15].
Concluding this, not only does MA-5 stabilise and further improve the geometrical structure of Cristae membranes and the efficiency of the ETC, but it also increases ATP production in an ETC-independent manner. Finally, MA-5 was successful in improving 24 out of 25 mitochondrial disease cellular samples from humans, extending the lifespan of the cells and improving cellular function [13].
It is worth noting that the only source I was able to find is Molecule Frontier, so trailing now is possible.
