Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models

Abstract

Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

Figure 1.

Retrograde transport of mitochondria is first altered in the sciatic nerves of mitoDendra-ALS mice. (A) Time-lapse microscopy of mitoDendra-labeled mitochondria in the intact sciatic nerve of a 45 days old mitoDendra mouse. Frames were acquired every 5 s for a total recording time of 6 min (the panels show some selected frames at 25 s intervals). Anterograde and retrograde mobile mitochondria are identified by solid lines (moving to the right of the panel) and dotted lines (moving to the left), respectively. Note that mitochondria are not always clearly visible in all the selected frames, because of the uneven course of the axon in the nerve, which crosses the focal planes several times. For representative movies from mitoDendra, mitoDendra-SOD1^G93A and mitoDendra-TDP43^A315T mice, see Supplementary Material, Movie S1, S2 and S3. Scale bar, 5 µm. (B) Analysis of mitochondrial transport in mitoDendra-ALS mice over time. n (axons at 45–90 days in 3–4 mice per group) = 39–46 mitoDendra, 22–26 mitoDendra-SOD1^WT, 35–22 mitoDendra-SOD1^G93A and 21–24 mitoDendra-TDP43^A315T. ^#P < 0.01 by Student's t-test, versus mitoDendra littermate controls. (C) Analysis of the direction of mitochondrial transport in mitoDendra-SOD1 mice at 45 days. n (axons in 3 mice per group) = 21 mitoDendra, 22 mitoDendra-SOD1^WT and 35 mitoDendra-SOD1^G93A. (D) Analysis of the direction of mitochondrial transport in mitoDendra-SOD1 mice at 90 days. n (axons in 3–4 mice per group) = 26 mitoDendra, 26 mitoDendra-SOD1^WT and 22 mitoDendra-SOD1^G93A. (E) Analysis of the direction of mitochondrial transport in mitoDendra-TDP43^A315T mice at 45 days. n (axons in 3 mice per group) = 18 mitoDendra, 21 mitoDendra-TDP43^A315T. (F) Analysis of the direction of mitochondrial transport in mitoDendra-TDP43^A315T mice at 90 days. n (axons in 4–5 mice per group) = 20 mitoDendra, 24 mitoDendra-TDP43^A315T. *P < 0.05 and **P < 0.01 by Student's t-test, versus mitoDendra controls. Error bars represent the SEM.

Figure 2.

Mitochondria morphology abnormalities precede transport alterations in the sciatic nerves of mitoDendra-SOD1^G93A mice, but appear later in mitoDendra-TDP43^A315T mice. (A) Representative fields of several mitoDendra-labeled axons from the sciatic nerves of 90 days old FALS mice. Scale bar, 5 µm. (B) Quantification of the length of mitochondria in mitoDendra-SOD1 mice during disease progression. n (axons at 15–45–90 days in 3–4 mice per group) = 36–34–38 mitoDendra, 38–39–35 mitoDendra-SOD1^WT, 37–27–34 mitoDendra-SOD1^G93A. (C) Quantification of the length of mitochondria in mitoDendra-TDP43^A315T mice. n (axons at 45–90 days in 3–4 mice per group) = 26–41 mitoDendra and 31–37 mitoDendra-TDP43^A315T. (D) Details of morphologically abnormal, swollen mitochondria (left, arrow) and abnormal clustering (right, arrow) in the sciatic nerve of a 90 days old mitoDendra-SOD1^G93A mouse. Scale bar, 2 µm. (E) Details of beads-on-string structures (left, top, arrow) and doughnut mitochondria (left, bottom, arrow), and a cluster of mitochondria (right, arrow) in the sciatic nerve of a 90 days old mitoDendra-TDP43^A315T mouse. Scale bar, 2 µm. (F) Quantification of the frequency of clusters of mitochondria in the sciatic nerves during disease progression. n (axons at 45–90 days in 3 mice per group) = 24–32 mitoDendra, 33–40 mitoDendra-SOD1^WT and 28–29 mitoDendra-SOD1^G93A. (G) Quantification of the distribution of clusters of mitochondria in segments of the nerve during disease progression. n (axons at 45–90 days in 3–4 mice per group) = 27–41 mitoDendra, 31–40 mitoDendra-TDP43^A315T. In all panels, **P < 0.01 by Student's t-test, versus mitoDendra controls. Error bars represent the SEM. In (B) and (C), values are expressed as mean percentage ± SEM of mitoDendra controls.

Figure 3.

Abnormal mitochondria are transported in different directions in the sciatic nerves of mitoDendra-SOD1^G93A and mitoDendra-TDP43^A315T mice. (A) Correlation between length and movement of mitochondria in 45 days old mitoDendra-SOD1^G93A mice. n (axons in 3 mice per group) = 9 mitoDendra and 14 mitoDendra-SOD1^G93A. (B) Analysis of length and movement of mitochondria in 45 days old mitoDendra-TDP43^A315T mice. n (axons in 3 mice per group) = 8 mitoDendra and 9 mitoDendra-TDP43^A315T. (C) Correlation with the direction of mitochondrial transport in 45 days old mitoDendra-SOD1^G93A mice. (D) Correlation with the direction of mitochondrial transport in 45 days old mitoDendra-TDP43^A315T mice. *P < 0.05 and **P < 0.01 by Student's t-test, versus mitoDendra controls. n.s., non-significant. Error bars represent the SEM. Values are expressed as mean percentage ± SEM of mitoDendra controls.

Figure 4.

Lack of mitochondrial abnormalities in the proximal segments of motor and sensory axons. (A) Lumbar spinal cord section from a mitoDendra mouse. Mitochondrial lengths were measured from axons projecting from the ventral horn (motor axons, a″) and the dorsal horn (sensory axons, a′). Scale bar, 100 µm. (B) Representative images of motor and sensory axons from 90 days old mitoDendra, mitoDendra-SOD1^G93A and mitoDendra-TDP43^A315T mice. Scale bar, 5 µm. (C) Quantification of the length of mitochondria in motor axons of 90 days old mitoDendra-SOD1 mice. n (axons in 3 mice per group) = 26 mitoDendra and 17 mitoDendra-SOD1^G93A. (D) Quantification of the length of mitochondria in motor axons of 90 days old mitoDendra-TDP43^A315T mice. n (axons in 3 mice per group) = 20 mitoDendra and 29 mitoDendra-TDP43^A315T. (E) Quantification of the length of mitochondria in sensory axons of 90 days old mitoDendra-SOD1 mice. n (axons in 3 mice per group) = 15 mitoDendra and 12 mitoDendra-SOD1^G93A. (F) Quantification of the length of mitochondria in sensory axons of 90 days old mitoDendra-TDP43^A315T mice. n (axons in 3 mice per group) = 17 mitoDendra and 22 mitoDendra-TDP43^A315T. n.s., non-significant. Error bars represent the SEM.

Figure 5.

Mitochondrial morphological abnormalities first appear in the motor terminals of mitoDendra-SOD1^G93A mice. (A) Representative image of the distal nerve segments of a motor neuron in the trapezius muscle of a 45 days old mitoDendra-SOD1^WT mouse. Axons were labeled with NF200 and synaptophysin (in blue), and NMJs identified by fluorescent α-bungarotoxin (BTX, in red). The masks of the axonal labeling (dotted area) and NMJ area were obtained in an unbiased manner, and used to determine the occupancy of mitochondria in the motor axon terminal and synapse, respectively. Scale bar, 20 µm. Detail of mitochondria in the motor terminal; note the tightly packed mitochondria network in the NMJ. Scale bar, 5 µm. (B) Quantification of the length of mitochondria in the nerve terminals during disease progression. Values are expressed as mean percentage ± SEM of mitoDendra controls. n (axons at 15–45–90 days in 3 mice per group) = 34–8–14 mitoDendra, 19–20–11 mitoDendra-SOD1^WT, 28–29–14 mitoDendra-SOD1^G93A. (C) Mitochondrial morphology abnormalities are observed in the nerve terminals of mitoDendra-TDP43^A315T mice at later stages. Quantification of the length of mitochondria in the nerve terminals of mitoDendra-TDP43 mice. n (axons at 45–90 days in 3–4 mice per group) = 21–20 mitoDendra, 30–22 mitoDendra-TDP43^A315T. (D) Quantification of mitochondrial density at motor terminals of mitoDendra-SOD1 mice defined by the occupancy of mitochondria within axon terminals (% of thresholded area). n (axons at 15–45–90 days in 3 mice per group) = 35–21–35 mitoDendra, 34–39–19 mitoDendra-SOD1^WT, 33–55–59 mitoDendra-SOD1^G93A. (E) Abnormal accumulation of mitochondria in the axon terminals of mitoDendra-TDP43^A315T mice. Quantification of the mitochondrial density in the nerve terminals of mitoDendra-TDP43 mice. n (NMJs at 45–90 days in 3–4 mice per group) = 9–11 mitoDendra, 24–21 mitoDendra-TDP43^A315T. (F) Quantification of the mitochondrial density at the NMJs in mitoDendra-SOD1 mice. Data are presented as percentage of co-localization of NMJ area (red fluorescence) over mitochondria (green fluorescence). n (NMJs at 45–90 days in 3 mice per group) = 37–42 mitoDendra, 49–50 mitoDendra-SOD1^WT and 70–65 mitoDendra-SOD1^G93A. (G) No accumulation of mitochondria at the NMJs of mitoDendra-TDP43^A315T mice. Quantification of the mitochondrial density in the NMJ of mitoDendra-TDP43 mice. n (NMJs at 45–90 days in 3–4 mice per group) = 25–42 mitoDendra, 44–30 mitoDendra-TDP43^A315T. *P < 0.05 and **P < 0.01 by Student's t-test, versus mitoDendra controls. Error bars represent the SEM.