Loss of Mfn2 results in progressive, retrograde degeneration of dopaminergic neurons in the nigrostriatal circuit

Abstract

Mitochondria continually undergo fusion and fission, and these dynamic processes play a major role in regulating mitochondrial function. Studies of several genes associated with familial Parkinson's disease (PD) have implicated aberrant mitochondrial dynamics in the disease pathology, but the importance of these processes in dopaminergic neurons remains poorly understood. Because the mitofusins Mfn1 and Mfn2 are essential for mitochondrial fusion, we deleted these genes from a subset of dopaminergic neurons in mice. Loss of Mfn2 results in a movement defect characterized by reduced activity and rearing. In open field tests, Mfn2 mutants show severe, age-dependent motor deficits that can be rescued with L-3,4 dihydroxyphenylalanine. These motor deficits are preceded by the loss of dopaminergic terminals in the striatum. However, the loss of dopaminergic neurons in the midbrain occurs weeks after the onset of these motor and striatal deficits, suggesting a retrograde mode of neurodegeneration. In our conditional knockout strategy, we incorporated a mitochondrially targeted fluorescent reporter to facilitate tracking of mitochondria in the affected neurons. Using an organotypic slice culture system, we detected fragmented mitochondria in the soma and proximal processes of these neurons. In addition, we found markedly reduced mitochondrial mass and transport, which may contribute to the neuronal loss. These effects are specific for Mfn2, as the loss of Mfn1 yielded no corresponding defects in the nigrostriatal circuit. Our findings indicate that perturbations of mitochondrial dynamics can cause nigrostriatal defects and may be a risk factor for the neurodegeneration in PD.

Figure 1.

Growth defect in Mfn2 mutant mice. (A) Mating scheme. Slc6a3-Cre is controlled by the dopamine transporter locus and is expressed in a subset of dopaminergic neurons, particularly the SNc, VTA and retrorubral field (21). The cross also incorporated a cre-induced, mitochondrially targeted Dendra2 (termed PhAM). (B) Representative image of a 22-week-old Mfn2 mutant (KO) compared with a heterozygous littermate (Het). Note the small size and severe kyphosis of the Mfn2 mutant mice. (C) Plot showing weekly weight measurements. Each point represents the average weight ± SEM (n = 10–15 for each genotype and sex). Both male and female Mfn2 mutants are significantly smaller (P < 0.05, two-tailed Student's t-test) than control animals by 5 weeks of age.

Figure 2.

Longitudinal analysis of locomotive activity in Mfn2 mutants. (A) Representative traces from open field analysis. The traces represent spontaneous movement in an open field during a 15-min observation period. The genotypes and ages of the mice are indicated. The open field analysis was quantified to obtain the (B) distance traversed, (C) average velocity, (D) rearing frequency and (E) immobile periods between activities. In all graphs, values from the heterozygous and homozygous animals were normalized to that of the wild-type controls, and error bars represent the propagated standard error. The Student's t-test was used to obtain P-values between Mfn2 mutants and wild-type controls (*P < 0.05; **P < 0.001; n = 6–10 animals per age and genotype).

Figure 3.

Retrograde degeneration of SNc dopaminergic neurons. (A) Dopaminergic projections to the striatum (Str), NAc and OT. The diagram delineates these regions. Sections were stained with TH antibody to label dopaminergic projections (brown pigment). At 3 weeks of age, Mfn2 mutant animals show decreased TH-immunoreactive terminals in the dorsolateral striatum (outlined region). Later time points reveal widespread loss in the striatum. VTA projections to the NAc and OT are still present, albeit reduced, at 11 and 14 weeks (outlined regions in bottom two panels). (B) Dopaminergic neurons at the SNc and VTA. Sections of the midbrain were stained with TH and counterstained with Cresyl violet (blue) to identify dopaminergic neurons. At 11 and 14 weeks, the Mfn2 mutants exhibit reduced staining in the SNc, whereas the VTA is relatively preserved. The SNc and VTA regions are outlined. (C) Quantification of TH-staining. Measured values of the TH-positive signal from heterozygous and homozygous animals were normalized to wild-type controls. For each animal, three sections were measured. The Student's t-test was used to obtain P-values (*P < 0.05; **P < 0.001; n = 3–6), and the error bars represent the propagated error. (D) Quantitation of dopaminergic cell loss in the SNc. Counts from heterozygous and homozygous animals were normalized to wild-type controls (n = 3 for each genotype). For each animal, nine sections spanning the rostro-caudal extent of the midbrain were manually counted. Statistical analysis was performed as in (C). (E) Magnified images of (B) showing the loss of dopaminergic neurons and processes in Mfn2 mutant animals.

Figure 4.

Loss of dopaminergic neurons in the SNc. Coronal midbrain slices were analyzed by fluorescent Nissl staining to highlight neurons. (A) The SNc is indicated by the outlined oval. Decreased neuronal density in the SNc is apparent by 11 weeks in the Mfn2-null mutant. Scale bar is 100 μm. (B) High-magnification images of Nissl-stained cells in the SNc. Scale bar is 20 μm.

Figure 5.

Mitochondrial fragmentation and depletion in slice cultures of Mfn2 mutants. Cre-mediated expression of mito-Dendra2 labels dopaminergic neurons. (A) Slice cultures of heterozygous controls and Mfn2 mutants. Slices were immunostained with TH (red). The first column shows a merged image of TH and mito-Dendra2 fluorescence (green), while the last column is an enlargement of the boxed zone. The asterisks highlight degenerating Mfn2-null neurons that have diminished or absent TH staining. (B) Mito-Dendra2 signal in neuronal projections. Marked depletion of mitochondria in both proximal and distal processes is evident in Mfn2 mutant slices. (C) Quantification of mitochondrial mass normalized to the number of dopaminergic neurons. For each sample, the total mito-Dendra2-positive area in a 5 mm × 5 mm region was measured and normalized to the number of Dendra2-positive neurons. Mitochondrial mass is reported as the percentage area of heterozygous control ± SEM. The Student's t-test was used to evaluate statistical significance (**P < 0.001; n = 5 for mutant slices; n = 7 for control). Scale bar is 10 μm for all images.

Figure 6.

Decreased mitochondrial transport in Mfn2 mutant cultures. (A) A representative tracking experiment of an Mfn2 heterozygous control. A subset of mitochondria in the nerve process was photo-converted to red for time-lapse imaging. The images of the photo-converted signal were processed into a kymograph to visualize mitochondrial movement (binary image). Velocity measurements were calculated from the red tracks that overlay mitochondrial trajectories. (B) Representative tracking experiment in an Mfn2 mutant showing the loss of mitochondrial transport. (C) Quantification of mitochondrial transport in neuronal processes. For each photo-conversion experiment, a positive event was defined as the directed movement of more than 5 μm during the 15 min recording session. The graph shows the frequency of photo-conversion experiments that resulted in at least one positive event. For Mfn2 heterozygous slices, 150 experiments were scored; for Mfn2 mutant slices, 138 experiments were scored. The Student's t-test was used to calculate statistical significance (**P < 0.001). (D) A scatter plot of the average velocity of moving mitochondria. The red dots indicate the population averages ± SD. Statistical significance was calculated as in (C) (n = 138 mitochondria in heterozygous slices, n = 38 mitochondria in mutant slices). Scale bar is 5 μm for all images.

Figure 7.

Increased motor activity after L-DOPA injection. Animals were analyzed by an open field test after a control saline injection to obtain baseline activity. They were tested a second time after an L-DOPA injection. Quantification of open field data for total distance traveled (A) and rearing frequency (B). For each animal, the activity after L-DOPA treatment was normalized to the activity after the saline injection. The Student's t-test was used to obtain P-values (*P < 0.05; **P < 0.001; n = 10–15 per genotype), and error bars represent the propagated error.