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. 2010 Mar 24;30(12):4232-40.
doi: 10.1523/JNEUROSCI.6248-09.2010.

Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex

Albert Misko  1 , Sirui Jiang, Iga Wegorzewska, Jeffrey Milbrandt, Robert H Baloh
Affiliations

Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex

Albert Misko et al. J Neurosci. .

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.

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Figures

Figure 1.
Figure 1.
CMT2A-associated MFN2 mutants alter the transport of axonal mitochondria. Mitochondria in cultured DRG neurons expressing wtMFN2 or R94Q were labeled with mito-RFP and imaged by time-lapse microscopy. A, Kymograph analysis of mitochondrial movements in R94Q-expressing cells reveal diminished numbers of moving mitochondria. B, Mitochondria from R94Q-expressing neurons spent more time paused between anterograde and retrograde movements than did mitochondria from controls. (*p < 0.005, t test; n = number of axons from which image stacks were created). Each condition contained a total of at least 500 observed mitochondria. C, Velocity distributions representing the amount of time that mitochondria from wtMFN2- or R94Q-expressing neurons spent moving at indicated velocities. Anterograde velocities are above the x-axis, and retrograde velocities are below the x-axis. There was a shift in both the anterograde and retrograde velocity distributions of mitochondria from R94Q-expressing neurons toward slower velocities. The differences were statistically significant as determined by rank-sum test analysis (p < 0.001). D, Size–frequency distributions of axonal mitochondria from wtMFN2-, R94Q-, and H361Y-expressing cells show that CMT2A disease mutants decrease mitochondrial lengths.
Figure 2.
Figure 2.
CMT2A-associated MFN2 mutants specifically disrupt mitochondrial transport. A, B, DRG neurons expressing the H361Y or R94Q MFN2 disease mutants were coinfected with mito-RFP (A) and RhoB-GFP (B) mitochondrial and endosomal markers, respectively. Mutant-expressing neurons revealed diminished mitochondrial mobility (A, kymograph) in the same axons that showed normal endosomal transport (B, kymograph). C, D, The percentage of mobile mitochondria is significantly decreased in R94Q- and H361Y- compared with wtMFN2-expressing neurons (*p < 0.001), whereas the percentage of mobile endosomes in these axons was normal. E, F, Overlay images of phase contrast and EGFP-SKL (which labels peroxisomes) in wtMFN2 (E)- and H361Y (F)-expressing DRG axons. G, Similar to endosomes, the percentage of mobile peroxisomes was unaltered by the expression of MFN2 mutants.
Figure 3.
Figure 3.
Mfn2 is required for normal axonal transport of mitochondria. To assess the effects of loss of Mfn2 on mitochondrial mobility, we analyzed mitochondrial transport in DRG neurons cultured from Mfn2 knock-out animals. A–C, DRG cultures from wild-type (A) or Mfn2−/− (B) mice infected with mito-RFP and imaged 4 d after infection at 10× magnification coupled with corresponding kymographs from single axons. Corresponding axons are presented above each kymograph as an embossed image for clarity. B, Diminished numbers of mitochondria are observed in Mfn2−/− axons due to a delay in the overall outward migration of mitochondria that is corrected by reintroduction of wild-type human MFN2 (C, Mfn2 rescue). D, Size–frequency histogram of mitochondria shows a decrease in the lengths of mitochondria observed in Mfn2−/− axons that is restored with reintroduction of MFN2 (Mfn2 rescue). E–G, Kymograph analysis of mitochondrial movements in individual MFN2−/− axons showed a profound abnormality in transport, resembling the deficit observed with expression of MFN2 disease mutants. The moving mitochondria from Mfn2−/− axons spent more time paused between movements (E, F) (*p < 0.001, t test; n is number of axons from which kymographs were generated), moved at slower velocities in the anterograde and retrograde directions (G), and were completely rescued by expression of either Mfn2 or Mfn1.
Figure 4.
Figure 4.
Mfn2 and Mfn1 interact with Miro and Milton proteins, key components of microtubule-based mitochondrial transport. HEK 293T cells were transiently transfected with the indicated epitope-tagged constructs, followed by immunoprecipitation (IP) and immunoblotting. A, B, Both Mfn2 and Mfn1 were able to interact with Miro1 and Miro2 by coimmunoprecipitation. The Mfn2:Miro2 interaction was consistently more robust than the Mfn2:Miro1 interaction, suggesting there may be selectivity for the formation of this complex. C–F, Mfn2 and Mfn1 interact with the Milton homologues OIP106 and GRIF1, proteins known to function as linkers between mitochondria and kinesins motors. G, H, Neither Mfn2 nor Mfn1 coimmunoprecipitated with Kif5C, indicating that they do not directly link mitochondria to kinesin. I, Additionally, Mfn2 was unable to interact with syntaphilin, an outer mitochondrial membrane protein that anchors mitochondria to microtubules.
Figure 5.
Figure 5.
Depletion of Miro2 produces a mitochondrial transport abnormality similar to that observed with loss of Mfn2 in DRG neurons. A, B, Kymograph analysis reveals that siRNA-mediated knockdown of Miro2 dramatically altered patterns of mitochondrial transport. C, Size–frequency histogram of axonal mitochondria demonstrated that depletion of Miro2 altered mitochondrial transport without changing mitochondrial morphology. D, Similar to Mfn2−/− cultures, mitochondria spent a greater percentage of time paused between anterograde movements in Miro2 knockdown cultures (*p < 0.001, t test; n is the number of axons from which image stacks were created). Pauses between retrograde movements trended toward longer pause times but did not reach statistical significance. E, Mitochondria velocity distributions were also skewed toward slower movements in Miro2 knockdown cultures, similar to effects seen with loss of Mfn2.
Figure 6.
Figure 6.
Disruption of mitochondrial fusion by knockdown of Opa1 does not alter mitochondrial axonal transport. To assess whether disrupting mitochondrial fusion is itself sufficient to alter mitochondrial transport, we used siRNA to knock down Opa1 in DRG neurons. A, B, Embossed images of representative axons and the corresponding kymographs showing Opa1 knockdown (>90%) did not alter mitochondrial movement patterns. C, Mitochondrial size–frequency histogram showed that loss of Opa1 significantly decreased mitochondrial length similar to that seen in Mfn2−/− cultures, reflective of decreased mitochondrial fusion. D, E, Pause times between anterograde or retrograde movements (D) and velocity of movements (E) were not altered by Opa1 knockdown (n is the number of axons from which kymographs were generated), unlike loss of Mfn2, indicating that loss of mitochondrial fusion alone does not alter mitochondrial transport.
Figure 7.
Figure 7.
Fusion-competent, CMT2A-associated MFN2 mutants cannot compensate for the mitochondrial transport abnormality in Mfn2−/− neurons. To compare the ability of fusion-competent (L76P, W740S) and fusion-incompetent (R94Q) MFN2 mutants to mediate mitochondrial transport, we reintroduced these mutants into Mfn2−/− neurons and assessed their ability to rescue the mitochondrial transport abnormality. As compared with wtMFN2, neither the fusion-competent (L76P, W740S) nor fusion-incompetent (R94Q) mutants were able to fully restore mitochondrial pause time (A, B) (n is number of axons from which kymographs were generated) or mitochondrial velocities (C).
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