Intramuscular viral delivery of paraplegin rescues peripheral axonopathy in a model of hereditary spastic paraplegia

Abstract

Degeneration of peripheral motor axons is a common feature of several debilitating diseases including complicated forms of hereditary spastic paraplegia. One such form is caused by loss of the mitochondrial energy-dependent protease paraplegin. Paraplegin-deficient mice display a progressive degeneration in several axonal tracts, characterized by the accumulation of morphological abnormal mitochondria. We show that adenoassociated virus-mediated (AAV-mediated) intramuscular delivery of paraplegin halted the progression of neuropathological changes and rescued mitochondrial morphology in the peripheral nerves of paraplegin-deficient mice. One single injection before onset of symptoms improved the motor performance of paraplegin-deficient mice for up to 10 months, indicating that the peripheral neuropathy contributes to the clinical phenotype. This study provides a proof of principle that gene transfer may be an effective therapeutic option for patients with paraplegin deficiency and demonstrates that AAV vectors can be successfully employed for retrograde delivery of an intracellular protein to spinal motor neurons, opening new perspectives for several hereditary axonal neuropathies of the peripheral nerves.

Figure 1

Peripheral neuropathy in Spg7^–/– mice. (A–D) Semithin sections of the distal sciatic nerve of a 6-month-old WT mouse (A) and Spg7^–/– mice at 6 months (B), 10 months (C), and 18 months (D) of age. Axonal changes, characterized by the accumulation of dense material (arrows), are appreciated in Spg7^–/– mice at 10 months and increase in severity at 18 months. (E and F) Electron micrographs of the sciatic nerve of 6-month-old WT (E) and Spg7^–/– (F) mice. A few axons already contain abnormal mitochondria in Spg7^–/– mice (asterisks). (G and H) Confocal images of neuromuscular junctions from teased muscular fibers of 24-month-old WT (G) and Spg7^–/– (H) mice double-labeled with rhodamine-tagged α-bungarotoxin to stain acetylcholine receptors (red) and antibody to neurofilament NF200 to detect axons (green). Control preparations reveal the typical (1 axon per end plate) pattern of innervation, while in mutant mice the terminal plates are denervated. Scale bar: 15 μm (A–D); 5 μm (E and F). Magnification, ×63 (G and H).

Figure 2

Retrograde transduction of spinal motor neurons by AAV vectors. Expression of β-gal activity in the skeletal muscle (A) and spinal cord (B) 4 weeks after injection of different AAV-LacZ vector serotypes in the gastrocnemius muscle. (A) AAV2/1 shows remarkable transduction of skeletal muscle, followed by AAV2/7 and AAV2/5. Conversely, the muscle transduction level for AAV2/2 and AAV2/8 is low. (B) Longitudinal sections of lumbar spinal cord to visualize targeting of motor neurons by retrograde transport of AAV (left side injected with AAV-LacZ). β-Gal activity restricted to motor neurons is visible only with AAV2/2 and AAV2/1. Magnification, ×10.

Figure 3

Paraplegin expression in skeletal muscle and spinal cord mitochondria after AAV-mediated intramuscular delivery of Spg7 cDNA. Western blot analysis of paraplegin on mitochondrial extracts from the gastrocnemius and the spinal cord of Spg7^–/– mice injected intramuscularly unilaterally with AAV2/1-Spg7 (A) and AAV2/2-Spg7 (B). In both cases, the contralateral side was injected with AAV-LacZ. For control we loaded mitochondrial extracts deriving from WT mice. With both serotypes, paraplegin is absent in muscles injected with AAV-LacZ but is restored upon injection of AAV-Spg7. The level of transduction in the muscle is higher when using AAV2/1, as expected. In the spinal cord, paraplegin is detected only when using AAV2/2-Spg7.

Figure 4

Rescue of the neuropathological phenotype in the peripheral nerve. (A–D) Semithin sections of the distal sciatic nerves in 16-month-old Spg7^–/– mice. Six months before the neuropathological studies, mice were treated with a single intramuscular injection of AAV-LacZ on the left side (A and C) and either AAV2/1-Spg7 (B) or AAV2/2-Spg7 (D) on the right side. Note the decreased number of affected axons (arrows) in the nerves from the side treated with the Spg7 vectors. (E) Morphometric quantification of the percentage of affected axons for animals treated with AAV2/1 and AAV2/2 vectors. The P value of Student’s t test is shown. Scale bar: 15 μm (A–D).

Figure 5

Rescue of the ultrastructural mitochondrial abnormalities. (A–G) Electron micrographs of the sciatic nerves in 16-month-old Spg7^–/– mice. Mice were analyzed 6 months after intramuscular injection with AAV-LacZ on the left side (A, C, and F) and AAV2/2-Spg7 on the right side (B, D, E, and G). A significant reduction in the number of axons containing abnormal mitochondria is observed in the sciatic nerve from the side injected with the AAV2/2-Spg7 vector (B). Giant mitochondria with disrupted cristae and glycogen accumulation (asterisk in F) are detected in the axons from the side treated with the control vector (C and F), while normal mitochondria with well-preserved cristae are present on the contralateral side (D, E, and G). (H) Morphometric quantification of the percentage of sciatic nerve axons with abnormal mitochondria. Upon injection of AAV2/2-Spg7, the number of axons with abnormal mitochondria significantly decreases compared with the contralateral side and drops below the levels that were present at the time of injection. The P values of Student’s t tests are shown. Scale bar: 5 μm (A and B); 1.5 μm (C, D, and F); 1 μm (E); 400 nm (G).

Figure 6

Intramuscular viral delivery of paraplegin improves the motor performance of Spg7^–/– mice. (A) Monthly performance of Spg7^+/+ mice, AAV-LacZ–treated Spg7^–/– mice, and AAV2/2-Spg7–treated Spg7^–/– mice on an accelerating rotarod apparatus beginning at 3 months, the time of the bilateral viral injection. The mice treated bilaterally with the AAV-Spg7 vector showed a progressive better performance compared with Spg7^–/– mice injected bilaterally with the AAV-LacZ vector. Where significant, P values for individual Student’s t test between the 2 groups of treated Spg7^–/– mice are shown. In addition, mice treated with AAV2/2-Spg7 displayed a statistically significant difference in their performance during the course of the experiment (1-way ANOVA, P = 0.0001), as did the control mice (1-way ANOVA, P = 0.008), in contrast to mice injected with AAV-LacZ (1-way ANOVA, P = 0.07). (B) Morphometric quantification of the percentage of affected axons in the sciatic nerves 10 months after treatment with AAV-LacZ or AAV2/2-Spg7 shows a statistically significant decrease due to the treatment. The P value of Student’s t test is shown.