Friedreich's ataxia (FRDA) is the most common inherited human ataxia and results from a deficiency of the mitochondrial protein, frataxin (FXN), which is encoded in the nucleus. This deficiency is associated with an iron-sulfur (Fe-S) cluster enzyme deficit leading to progressive ataxia and a frequently fatal cardiomyopathy. There is no cure. To determine whether exogenous replacement of the missing FXN protein in mitochondria would repair the defect, we used the transactivator of transcription (TAT) protein transduction domain to deliver human FXN protein to mitochondria in both cultured patient cells and a severe mouse model of FRDA. A TAT-FXN fusion protein bound iron in vitro, transduced into mitochondria of FRDA deficient fibroblasts and reduced caspase-3 activation in response to an exogenous iron-oxidant stress. Injection of TAT-FXN protein into mice with a conditional loss of FXN increased their growth velocity and mean lifespan by 53% increased their mean heart rate and cardiac output, increased activity of aconitase and reversed abnormal mitochondrial proliferation and ultrastructure in heart. These results show that a cell-penetrant peptide is capable of delivering a functional mitochondrial protein in vivo to rescue a very severe disease phenotype, and present the possibility of TAT-FXN as a protein replacement therapy.

Stringent control of ion and protein transport across the mitochondrial membranes is required to maintain mitochondrial function and biogenesis. In particular, the inner mitochondrial membrane is generally impermeable to proteins entering the matrix except via tightly regulated protein import mechanisms. Recently, cell penetrant peptides have been shown to move across the inner mitochondrial membrane in a manner suggesting an independent mechanism. HIV-1 transactivator of transcription (TAT) is an arginine-rich cell penetrant peptide, 47YGRKKRRQRRR57, which can transduce full-length proteins not only across the cell membrane but also into intracellular organelles. In this study, we investigated the ability of a TAT-containing protein to move into the mitochondrial matrix. Using a novel FACS assay for isolated, purified mitochondria, we show that TAT can deliver a modified fluorescent protein, mMDH-GFP, to the matrix of mitochondria and it is subsequently processed by the matrix peptidases. In addition, transduction of TAT-mMDH-GFP into mitochondria is independent of canonical protein import pathways as well as mitochondrial membrane potential. In direct contrast to published reports regarding the cell membrane where the sodium channel inhibitor, amiloride, blocks endocytosis and inhibits TAT transduction, TAT transduction into mitochondria is markedly increased by this same sodium channel inhibitor. These results confirm that the cell penetrant peptide, TAT, can readily transduce a protein cargo into the mitochondrial matrix. These results also demonstrate a novel role for mitochondrial sodium channels in mediating TAT transduction into mitochondria that is independent of endocytotic mechanisms. The mechanism of TAT transduction into mitochondria therefore is distinctly different from transduction across the cell membrane.

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