Rescue of Oxidative Phosphorylation with "Allotopic" mRNA
Age-related accumulation of mutations in mitochondrial DNA (mtDNA) is widely suspected to play an important role in the degenerative aging process, albeit that controversy remains as to the mechanism(s) linking the two. Large deletions in mtDNA seem an especially likely culprit ...
A number of credible proposals have been advanced for rejuvenation biotechnology to restore youthful mitochondrial function in [cells homoplasmic for mitochondria bearing such deletions], reverting their abnormal metabolism and allowing them to resume participation in normal tissue function. The lead candidate approach, first proposed by SENS Foundation Chief Scientific Officer de Grey,(1) is the placement of functioning "backup copies" of the protein-coding mtDNA genes in the cell nucleus ("allotopic expression" (AE)). There has been substantial progress in this area since then,(eg (5-9)), and in recent years SENS Foundation has prioritized funding of AE research ...
But other potential routes to mitochondrial rejuvenation do exist and should also be developed, including the wholesale intraorganellar replacement of mtDNA using "protofection" (2) and the delivery of allotopic RNA to the organelle. The latter possibility was highlighted by work [by Dr. Samit Adhya, of the Division of Molecular and Human Genetics at the Indian Institute of Chemical Biology] targeting tRNA human cell mitochondria with the transgenic use of a transfer RNA import complex [RIC] adapted from the parasitic protozoon Leishmania tropica.(3)
In that work, Dr. Adhya had demonstrated that the Leishmania RIC was efficiently taken up by human cells, where it was targeted to mitochondria and rescued oxidative phosphorylation (OXPHOS) in human cells harboring the same tRNA mutations responsible for the inherited mitochondriopathy Myoclonic epilepsy with Ragged Red Fibers (MERRF).(3) In later work, his lab showed that the RIC could be used to induce the import of antisense oligonucleotides, leading to reduced expression of target mitochondrial mRNA.(4) And in a yet-unpublished preliminary proof-of-principle presented at the third Strategies for Engineered Negligible Senescence (SENS) conference in 2007,(9) Adhya demonstrated that the RIC-based system would work in vivo. Inherited mutations in ND1 subunit of mitochondrial complex I are responsible for the mitochondriopathy mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MERRF). As shown in dramatic video, injection of a RIC-based construct loaded with antisense RNA for ND1 into the rear left paws of wild-type rats lead to muscular degeneration, beginning with swelling and inflammation, and proceeding to severe slackening of the limb muscles, prominent movement impairments, and extensive myocyte necrosis -- a similar phenotype to the original human mitochondrial disease.(9)
Now, Adhya's group reports the next advance in this work, using a RIC complex to deliver large, functional memory RNA transcripts of mitochondrially-encoded electron transport chain (ETC) subunits into to cells lacking the genes those mRNA encode -- and to thereby rescue mitochondrial oxidative phosphorylation.
Kearns–Sayre Syndrome (KSS) is a mitochondriopathy characterized by ophthalmoparesis and ptosis (paralysis or weakness of the muscles that control eye and eyelid movement, respectively) as well as pigmentary retinopathy and abnormalities in cardiac conduction. To test the RIC system using large, mitochondrial mRNA, the investigators used a cybrid line derived from a KSS patient homoplasmic for a mtDNA deletion spanning from within the gene encoding Complex II to within that encoding Complex II. The cybrid line gene expression profile showed no expression of these genes, or of complex I, which the authors hypothesized might be due to destabilization of the transcript secondary to a processing defect brought on by the deletion.(10)
Researchers next generated a ribonucleoprotein complex of polycistronic RNA 1 that included mRNA for the genes along entire stretch of mtDNA from Complexes I through III, along with a mitochondrially-targeted tRNA import signal directly bound to the carrier complex via the tRNA receptors of R8, a functional recombinant subcomplex of RIC. Cells of the cybrid line were then transfected with the construct, termed pcRNA1-R8. Using several mitochondria-specific probes and subcellular fractionation, the researchers demonstrated a high degree of colocalization of fluorescently-labeled pcRNA1 mRNA with cybrid cells' mitochondria, with mitochondrial uptake reaching ~90% within 3 h. Adhya's group were were able to track the process pcRNA1-R8 binding, cellular uptake, cotransport with R8 to mitochondria, and the ultimate import into the mitochondrial matrix. Conversely, they demonstrated that there was no such localization in the case of pcRNA1 deprived of the R8 RIC subunit.(10)
Translation of the Mitochondrial Complex Proteins
The investigators next interrogated the cells for evidence of translation of the pcRNA1-R8-bound mRNA. Because the KSS mitochondrial deletion included the single mitochondrial tRNA for lysine, the cybrid lines suffered from global translational arrest. Exposing the cells to R8 alone restored translation of some mitochondrial genes, presumably by facilitating the import of tRNA from the cytosol, but not those for Complexes I-III. By contrast, pcRNA1-R8 was able to restore the wild-type synthesis pattern of all mitochondrial polypeptides; this was further confirmed for proteins specifically encoded by pcRNA1 using Western blotting, which additionally revealed their specific mitochonrial localization.(10)
The key question, however, is the ability of pcRNA1-R8 to restore the functioning of the electron transport chain revive oxidative phosphorylation in the cybrids.
ETC activity was confirmed in cybrid cells within 24 h of transfection with pcRNA1-R8 with the observation of functioning mitochondrial Complex IV activity; in turn, this implied the proper assembly of both the cybrids' missing subunits, and of the chain as a whole. More importantly, pcRNA1-R8 was also able to restore respiratory capacity, beginning with cellular oxygen uptake in the first 2-3 h of transfection, rising to ~93% of that of HepG2 liver cancer cells (which have wild-type mitochondria) within 24 h, and sustained for ~3d. Contrariwise, there was no stimulative (or inhibitory) effect of pcRNA1-R8 on HepG2 respiration. Inhibition of Complex V by oligomycin substantially inhibited the cybrids' respiratory activity, and the dependence of respiratory activity on translation of the transfected mRNA was supported by treating the cybrids with the mitochondrial protein synthesis inhibitor chloramphenicol, which completely arrested respiration.(10)
Treatment with the membrane-permeating JC-1 dye allows visualization and sorting of cells by the proportion of their mitochondria high (red) and low (green) mitochondrial membrane potentials (ΔΨm). 25% of untreated cybrid lines' mitochondrial populations were entirely composed of fully-depolarized organelles, and none of these cells had were enriched in high-ΔΨm mitochondria. Treatment with R8 alone did not affect the proportion of cells with fully-depolarized mitochondrial populations, though it caused some heterogeneity in the remaining cells' red staining; by contrast, treatment of cybrids with pcRNA1-R8 was able to fully restore the sorting pattern exhibited by mitochondrially wild-type HepG2 cells, and both cell types promptly exhibited high proportions of fully-depolarized mitochondria unpon treatment with an ETC uncoupling agent.(10)
Cells incompetent for OXOHOS depend entirely on glycolysis for ATP production and growth, and thus on glucose as an energy source, whereas OXPHOS-capable cells can generate ATP and maintain growth when deprived of glucose but supplied with galactose. In the presence of glucose, cybrid cells exhibited the same growth rate whether treated with pcRNA1-R8 or not. But provision of galactose alone to untreated cybrid cells did not permit growth and led to some cell death within 2 d, whereas cells transfected with the construct underwent 3 generations of replication, grew to confluence within 3 d, during which they maintained Complex IV activity. pcRNA1-R8 had restored OXPHOS to KSS cybrids to nearly wild-type levels, bypassing and effectively obviating the large deletions in the cells' own mtDNA.(10)
This in vitro experiment is exciting, opening up an alternative means of restoring oxidative phosphorylation to cells homoplasmic for OXPHOS-incompentent mitochondria with large deletions -- cells that accumulate in aging tissues, area associated with age-related diseases such as Parkinson's disease and sarcopenia, and that can strongly be argued to be contributors to the degenerative aging process. The method is rightly described by the authors as "inherently simple, efficient, and fast-acting, and appears to be of general applicability to a wide variety of cells and tissues (data not shown)." Indeed, it shows clear advantages relative to the low targeting to cells and/or mitochondria of previous attempts using pharmacological delivery systems, and appears to show a higher rate of restoration of OXPHOS and normal growth with galactose as the sole energy source than previous efforts using allotopic expression itself.
On the other hand, the effects of the RIC-based construct was transient, as would be predicted from the inherent nature of a therapy based on delivery of mRNA, which are routinely recycled within the cell: after the initial restoration of cybrid cell growth at day 3 of transfection, oxygen consumption rate declined to the basal levels over the ensuing 2 days, and cells began detaching from the vessel surface.(10) One would expect that repeated administration of "booster shots" would extend or restore OXPHOS in cells homoplasmic for mutant mitochondria in mitochondriopathy patients or persons who have undergone age-related mitochondrial deletions, but there is substantial room for skepticism that normal function could be continuously sustained by such means on acceptable booster schedules.
But this work is a major advance, and clearly promising. The therapeutic potential of the RIC-based should now be tested in animal models of inherited mitochondrial disease such as KSS, and if successful, the more ambitious work of using it to restore OXPHOS in the rare cells rendered homoplasmic for somatic mutations as a result of the degenerative aging of wild-type mice. The biomedical rejuvenation of aging human mitochondrial function would not lie far behind, with the promise of muscles maintained, Parkinson's prevented, and an end to the rising systemic metabolic toxicity of reductive cellular hotspots.(1)
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