Mitofusin2 Mutations Disrupt Axonal Mitochondrial Positioning and Promote Axon Degeneration

Axon degeneration caused by MFN2 mutant expression is segmental and dynamic. A–C, Fluo-4 imaging of MFN2 mutant-expressing axons (7 d after infection) visualized by time-lapse microscopy (40× magnification). Axons demonstrated a rapid (minutes) elevation in intra-axonal Ca²⁺, followed by beading of the membrane and finally disappearing, presumably from the inability to retain dye following loss of membrane integrity. B, These frequently appeared as short segments (∼50–100 μm) which swelled to a spheroid like shape before completely degenerating. C, Axons often lost structural integrity at a single point resulting in retraction of then ends from the break point (asterisks label ends of break point). D–G, Corresponding phase contrast and Fluo-4 images illustrate degenerating axon segments.

Disruption of mitochondrial fusion alone is not sufficient to induce axonal degeneration. DRG neurons were infected with lentivirus expressing siRNA to Opa1 (siOpa1) or luciferase (control) to inhibit mitochondrial fusion, together with mito-RFP to visualize mitochondrial dynamics via live cell imaging. A–C, Knock-down of Opa1 leaves mitochondrial transport intact (A, B, kymographs) but results in smaller fragmented mitochondria due to diminished fusion (C). Quantification of mitochondrial flux supported normal mitochondrial mobility in siOpa1-expressing neurons (number of mitochondria/min: siLuc, 2.87 ± 1.87; siOpa1, 2.71 ± 1.51; p = 0.828; n = 10 axons per condition). Unlike expression of MFN2 mutant-expressing neurons, Fluo-4 imaging detected no signs of axonal degeneration in Opa1 deficient neurons (D,D′, E,E′).

Mutant MFN2 and Opa1 knockdown cause similar global suppression of mitochondrial function. A–C, TMRM dye was used to calculate F_m/F_c (an estimate of Δψ that corrects for cytoplasmic background) values of mitochondria in wtMFN2 (A)-, R94Q (B)-, and siOpa1 (C)-expressing cells. Mitochondria in siOpa1 showed a trend toward lower Δψ values, while R94Q expression did not significantly alter Δψ values. D, E, Despite normal levels of ATP in the cultures (D), R94Q- and siOpa1-expressing cultures contained more lactate in the medium, indicating a shift toward the use of glycolysis to maintain ATP levels (E). F, G, H, Treatment of cultures with 15 mm 2DG to inhibit glycolysis for 48 h produced widespread axonal degeneration (as indicated by axonal blebbing seen on phase contrast images) in R94Q- and siOpa1-expressing cultures, supporting the notion that the axons are more reliant on glycolysis to maintain ATP levels. I, MitoTracker Green staining of R94Q- and siOpa1-expressing cultures revealed an ∼20% and 40% decrease in mitochondrial mass, respectively.

Decreasing overall mitochondrial movement alone does not cause axonal degeneration. Synph, which anchors mitochondria to microtubules, was overexpressed by lentivirus infection and mitochondrial motility and axonal viability were examined. A, B, Overall mitochondrial movement was dramatically reduced in Synph-overexpressing neurons (number of mitochondria/min: GFP, 2.71 ± 1.23 vs Synph, 1.09 ± 0.70, p < 0.0001; n = 20 axons per condition). C, A size frequency histogram of mitochondrial lengths demonstrated that Synph overexpression suppressed movement without altering mitochondrial fission/fusion dynamics. D,D′, E,E′, Despite the near complete suppression of mitochondrial movement, degenerating axons were rarely observed in Synph-expressing cultures. Note also that despite lack of movement, the distribution of mitochondria along the axon appeared to be preserved.

Abnormal distribution of axonal mitochondria correlates with axonal degeneration from mutant MFN2 expression. Mitochondria in cultured DRG neurons expressing virally transduced wtMFN2, R94Q, siOpa1 or Synph were labeled with mito-RFP and imaged by time-lapse microscopy 1 week after infection. A–D, Kymographs were used to assist in the identification of fluorescently labeled mitochondria. Individual mitochondria are represented as black dots below kymographs. While mitochondria were evenly distributed along axons of wtMFN2-, Synph-, and siOpa1-expressing neurons (A, C, D), R94Q mutant-expressing axons commonly displayed prolonged segments that were devoid of mitochondria (B, arrowhead). Axons from 10 neurons were divided into 10 μm bins (length of bracket in A) and the number of mitochondria counted per bin. Mitochondrial distributions were compared with a Poisson distribution with a χ² goodness of fit test as described in the text and shown in Table 1.

TTX treatment diminishes axonal degeneration in cultured DRG neurons expressing MFN2 disease mutants. DRG cultures expressing the R94Q or H361Y MFN2 disease mutants to induce axon degeneration were treated with 1 μm TTX on DIV4 through DIV11. A–D, DCFDA imaging after 1 week of treatment showed a marked decrease in the number of degenerating axons (A, C). Fluorescence images were converted into binary format (B, D) and degenerating axons were measured as DCFDA+ pixels per field. E, DRG neurons expressing MFN2 mutants treated with TTX showed a significant decrease in the number of DCFDA-positive degenerating axons 1 week after infection with R94Q or H361Y viral constructs (TTX treatment vs untreated control, *p < 0.01, Student's t test). Control in this experiment refers to MFN2 mutant-expressing cultures, compared with treatment with TTX.