Mitofusin 2 Is Necessary for Transport of Axonal Mitochondria and Interacts with the Miro/Milton Complex

TLDR

Mitofusins are outer mitochondrial membrane proteins that regulate mitochondrial dynamics and interact with the Miro/Milton complex, and mutations in Mfn2 cause Charcot‑Marie‑Tooth type 2A, a disease of long peripheral axons whose tissue selectivity is not yet understood. Here, we present evidence that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Loss or mutation of Mfn2 in neurons causes mitochondria to pause more and move more slowly, selectively impairing mitochondrial transport while sparing other organelles; knockdown of Miro2 reproduces these deficits, whereas disrupting fusion via Opa1 does not affect motility, demonstrating that mitofusins directly regulate axonal mitochondrial transport and elucidating mechanisms underlying CMT2A and optic atrophy.

Abstract

Mitofusins (Mfn1 and Mfn2) are outer mitochondrial membrane proteins involved in regulating mitochondrial dynamics. Mutations in Mfn2 cause Charcot-Marie-Tooth disease (CMT) type 2A, an inherited disease characterized by degeneration of long peripheral axons, but the nature of this tissue selectivity remains unknown. Here, we present evidence that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Live imaging of neurons cultured from Mfn2 knock-out mice or neurons expressing Mfn2 disease mutants shows that axonal mitochondria spend more time paused and undergo slower anterograde and retrograde movements, indicating an alteration in attachment to microtubule-based transport systems. Furthermore, Mfn2 disruption altered mitochondrial movement selectively, leaving transport of other organelles intact. Importantly, both Mfn1 and Mfn2 interact with mammalian Miro (Miro1/Miro2) and Milton (OIP106/GRIF1) proteins, members of the molecular complex that links mitochondria to kinesin motors. Knockdown of Miro2 in cultured neurons produced transport deficits identical to loss of Mfn2, indicating that both proteins must be present at the outer membrane to mediate axonal mitochondrial transport. In contrast, disruption of mitochondrial fusion via knockdown of the inner mitochondrial membrane protein Opa1 had no effect on mitochondrial motility, indicating that loss of fusion does not inherently alter mitochondrial transport. These experiments identify a role for mitofusins in directly regulating mitochondrial transport and offer important insight into the cell type specificity and molecular mechanisms of axonal degeneration in CMT2A and dominant optic atrophy.