Explorin the role of FIS1 in mitochondrial (patho)physiology

Mitochondria are key players in a plethora of (patho)physiological processes and show a striking morphological and structural versatility, which is ensured by inter-organellar fusion and fission processes mediated by a group of dynamin-related ATPases and their adaptor proteins, the so-called mitochondrial shaping proteins (Friedman and Nunnari, 2014; Hoppins et al., 2007a). Mitochondrial fission requires the translocation of cytosolic dynamin-related protein 1 (DRP1) to the mitochondria and its interaction with FIS1, MFF and MiD49/50, its putative receptors. FIS1 is a tail-anchored protein evenly distributed on the outer mitochondrial membrane and composed by a C-terminus transmembrane (TM) domain and two cytosolic tetratricopeptide repeat (TRP) motifs (James et al., 2003; Stojanovski et al., 2004; Yoon et al., 2003). FIS1 has been proved to mediate mitochondrial-ER tethering, ER-gated apoptosis, autophagy and ischemia/hypoxia-induced fragmentation (Alirol et al., 2006; Iwasawa et al., 2011a; Kim et al., 2011; Shen et al., 2014; Yamano et al., 2014). However, the discovery of MFF and MiD49/50, along with the finding that both DRP1 recruitment and mitochondrial fragmentation take place also in two models of Fis1 in vitro ablation, led to partially discharge FIS1 involvement in such events (Loson et al., 2013; Otera et al., 2010; Palmer et al., 2013). Besides the mechanistic aspects of FIS1-mediated fragmentation, its (patho)physiological relevance is still far from been clarified, and no human pathology has been directly linked to a mutation in Fis1 gene so far. In order to place FIS1 functions into a physiological context and to further define the relevance of mitochondrial fission in vivo, we decided to generate a hypomorphic Fis1 mouse model (Fis1hyp allele) that could be turned into a conditional ablation system (Fis1fl allele). The powerful genetic tool we generated circumvents many of the potential pitfalls of the most commonly used gene targeting approaches. We showed that Fis1 hypomorphism causes a perinatal pleiotropic lethal phenotype, comprising severe progressive muscular atrophy, heart ischemia and compromised vessel integrity. The concomitant muscular atrophy and heart ischemia would suggest (cardio)myopathy and/or neuromuscular defects, whereas extensive blood clots in the thorax, the presence of blood in the lungs and in the intestine and the altered microcircular bed in the retina seem to suggest defects in the maintenance of vessel integrity and/or increased vessel permeability and consequent blood leakage. Remarkably, although reduced, also heterozygous mice display lethality at a later stage and show defects comparable to their Fis1hyp/hyp littermates, thus suggesting a dose dependent relationship between phenotype severity and extent of Fis1 gene ablation. In vitro, we observed mitochondrial elongation and upregulation of the fission machinery as a consequence of Fis1 hypomorphism. Furthermore, we showed that expression of both human and mouse Fis1 genes is controlled by multiple splicing. In particular, an exon-skipping event of conserved exon 2 dictates the preferential expression of variant 2 versus the canonical variant 1 in both species. Remarkably, a non-canonical START codon seems to be responsible for transcription of human variant 2 (hFis1.2). The resulting protein, although lacking only the first 18 amino acids, triggers mitochondrial elongation when overexpressed in mouse. Upon starvation, Fis1 variant 2 expression is up-regulated in a protein kinase A dependent manner and its specific knockdown inhibits autophagy-associated mitochondrial elongation. Thus, Fis1 is alternatively spliced to modulate mitochondrial morphology during autophagy.