Knockdown of Tim23 <i>In‐Vivo</i> Results in the Activation of the Mitochondrial Unfolded Protein Response

Mitochondria rely heavily on the tight coordination of the nuclear and mitochondrial genomes. Disruption of this regulation has been shown in lower order organisms to result in a mito‐nuclear imbalance in which holenzymes are unable to assume mature stoichiometry. This lack of coordination between nuclear and mitochondrially encoded subunits resulting in orphaned subunits is an activator of the mitochondrial unfolded protein response (UPR mt ). The UPR mt is a highly conserved quality control mechanism that strives to achieve proteostasis by refolding or degrading misfolded proteins as well as misassembled complexes. This process is well characterized in c. elegans however, the UPR mt and its retrograde signals are poorly understood in mammals. Thus we sought to induce the UPR mt in‐vivo‐ in a mammalian model to better characterize this stress response. In order to do so, C57BL/6 mice were injected once a day for 3 days with an antisense oligonucleotide (In‐Vivo Morpholino) targeted to Tim23, the major channel of the inner membrane, to perturb the genomic coordination. Following Tim23 Morpholino treatment, Tim23 protein content was significantly reduced by 50%. The consequence was a 60% reduction of protein import into the mitochondrial matrix. As a result, mitochondrial chaperone cpn10 was significantly elevated by 30% and the transcription factor ATF5 also demonstrated a strong trend to increase, suggesting that the UPR mt was activated following Tim23 knockdown. Mitochondrial respiration and ROS emission was also measured to determine whether Tim23 knockdown resulted in mitochondrial dysfunction. However, no differences were found suggesting that the stress imposed is sufficient to induce the UPR mt without disrupting basic mitochondrial functions. The second aim of this study was to investigate the role of mitochondrial proteolytic byproducts in mediating retrograde signaling. Peptides released from the mitochondria following basal proteolysis were isolated and incubated with import reactions. Our results demonstrate a dose‐ and time‐dependent inhibition of import with the addition of peptides, such that import was reduced by 48% with the addition of 6ug of peptides for 30 minutes. This suggests that mitochondrial proteolytic byproducts can exert an inhibitory effect on the protein import pathway, to inhibit excessive protein import as a negative feedback mechanism. The inhibition of import into the organelle may also serve a retrograde function, to modify nuclear gene expression and improve organelle folding capacity, or alter the expression of protein import machinery components. Support or Funding Information This work is supported by the Natural Science and Engineering Research Council (NSERC) of Canada.

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