A Discussion of Recent Work on Allotopic Expression of Mitochondrial Genes at the SENS Research Foundation

A paper published last month outlines recent progress on allotopic expression of mitochondrial genes carried out by the SENS Research Foundation team. Allotopic expression is the name given to the process of putting copies of mitochondrial genes into the nuclear genome, suitably altered to allow proteins to be generated and shipped back to the mitochondria where they are needed. Mitochondria replicate like bacteria, and some forms of stochastic mitochondrial DNA damage can make mitochondria both dysfunctional and able to outcompete their undamaged peers. This is thought to be an important contribution to aging, resulting a small but damaging population of cells that are overtaken by broken mitochondria and which export harmful reactive molecules into the surrounding tissues.

Having a backup supply of mitochondrial proteins can in principle block these consequences of mitochondrial DNA damage, and thus remove this contribution to the aging process. Proof of concept has been demonstrated for a few of the thirteen proteins needed, and work proceeds on the rest. As noted here, one of the challenges in this project is that mitochondrial genetic machinery is of a different evolutionary origin to that of the cell nucleus, and thus the efficient production of equivalent proteins from nuclear genes is a much more challenging process than would otherwise be the case.

While the vast majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondrion, 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be especially severe, as all 13 proteins are core subunits of the oxidative phosphorylation chain, and any disruption to subunit structure, stability, or function may have grave biochemical and physiological consequences. Gene therapy to target affected mitochondrial subunits is a promising alternative strategy which circumvents some of the technical challenges faced by the above approaches. One issue that remains, however, relates to the prokaryotic origin of the organelle. Translation within the mitochondrion deviates from the universal genetic code, utilizing machinery and codon frequencies more similar to its α-proteobacterial ancestry than to the mammalian nuclear genome.

Subsequently, allotopic expression has been suggested as a therapeutic tool to genetically remedy deleterious mitochondrial DNA mutations through nuclear complementation of the affected genes. A critical, but often-overlooked consideration in these nuclear relocation studies is the influence of the primary coding sequence on protein production. The vast majority of these previous studies have utilized what may be considered "minimally-recoded" mitochondrial genes. While making these codon changes is essential to maintain amino acid sequence integrity during cytosolic translation, this minimal approach fails to account for other elements of primary sequence which can critically influence both gene and protein expression.

Many commercial algorithms have therefore been developed to determine the optimal sequence and conditions for expression of a gene from a particular host. Though there are concerns regarding the use of codon optimization to increase homologous expression of a nuclear gene, such as the generation of novel or immunogenic peptides or structural perturbations in the encoded protein, codon optimization continues to be widely utilized for the production of biotherapeutics. Applying this principle to allotopic expression, we hypothesize that, given the bacterial origin of the mitochondrial genome, the coding sequences of minimally-recoded mitochondrial genes are dissimilar from nuclear genes and are inefficiently translated by nuclear machinery, therefore resulting in poor allotopic expression.

Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5-180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation proteins maintained protein expression following stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1, was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.

Link: https://doi.org/10.1016/j.redox.2020.101429


I wonder if it would be possible to apply this changes to germline cells in mice. Apply as many as possible to the same mice and see how long they live.

Posted by: Javier Santos at February 3rd, 2020 6:37 AM

I note that mitochondrial diseases are mentioned in the paper but not aging. It seems that there is still a stigma about mentioning antiaging in papers, so it's better not to talk about combating aging if you want to publish in certain journals.

Posted by: Antonio at February 3rd, 2020 7:41 AM

You can combat are-related decline, though.

@Javier Santos
I am really curious to see such results, but I am afraid it is to early for this. For me migrating genes from mitochondria towards the nucleus is aging l akin of SciFi. Just imagine what it would take. A protein used in a given mitochondrion has to be delivered from some other organelle , be successfully inserted, not interfere with other pathways, be efficient enough and generally work. V then you have to deliver it to every cell of the body. If you use cell ablation and replacement with stem cells then that therapy alone could be enough to rejuvenate the body. But you have to deliver completely reengineered cells. It is truly amazing. But don't hold your breath for the therapies to come near you....

Posted by: Cuberat at February 3rd, 2020 8:42 AM

"I note that mitochondrial diseases are mentioned in the paper but not aging. It seems that there is still a stigma about mentioning antiaging in papers, so it's better not to talk about combating aging if you want to publish in certain journals."

The role of mtDNA mutations in protein coding regions in the course of aging is still controversial; people with mitochondrial myopathies do not exhibit accelerated aging phenotypes, and the mice that have been used in mtDNA aging studies have defective nuclear DNA repair mechanisms.

The true test I think would be engineering wild type mice with allotopic mitochondrial genes and measuring the effect on murine lifespan. If it increases lifespan, then I'm happy to eat crow, especially if it proves workable as a somatic gene therapy instead of a germline intervention, but this is a long-term project, and not necessarily that great in terms of impact even if it does succeed.

Posted by: Dylan Mahd at February 3rd, 2020 10:11 AM

@Dylan Mahd:

Mitochondrial myopathies mutations have nothing to do with age-causing SENS-type mutations. The former are small mutations in the mitochondria of all or almost all cells in the body, while the latter are big mutations (deletions of whole genes) in the mitochondria of a few cells.

Posted by: Antonio at February 3rd, 2020 11:08 AM

And mito mutations are associated with Alzheimer's disease, Parkinson's disease, and
sarcopenia, all of which are definitely diseases of aging.

Posted by: Florin at February 3rd, 2020 6:16 PM
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