Gene transfer demonstrates that muscle is not a primary target for non-cell-autonomous toxicity in familial amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive paralysis arising from the premature death of motor neurons. An inherited form is caused by a dominant mutation in the ubiquitously expressed superoxide dismutase (SOD1). SOD1 mutant expression within motor neurons is a determinant of onset and early disease, and mutant accumulation within microglia accelerates disease progression. Muscle also is a likely primary source for toxicity, because retraction of motor axons from synaptic connections to muscle is among the earliest presymptomatic events. To test involvement of muscle in ALS, viral delivery of transcription-mediated siRNA is shown to suppress mutant SOD1 accumulation within muscle alone but to be insufficient to maintain grip strength, whereas delivery to both motor neurons and muscle is sufficient. Use of a deletable mutant gene to diminish mutant SOD1 from muscle did not affect onset or survival. Finally, follistatin expression encoded by adeno-associated virus chronically inhibited myostatin and produced sustained increases in muscle mass, myofiber number, and fiber diameter, but these increases did not affect survival. Thus, SOD1-mutant-mediated damage within muscles is not a significant contributor to non-cell-autonomous pathogenesis in ALS, and enhancing muscle mass and strength provides no benefit in slowing disease onset or progression.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Decreasing SOD1 in muscle does not improve grip strength in SOD1G93A mice. (A and B) Genes encoded in AAV are expressed in muscle and in motor neurons after retrograde transport. Genes encoded in lentivirus are expressed in muscle only. One hindlimb of 40-day-old SOD1G93A mice was injected with either lentivirus or AAV containing a siRNA directed against SOD1. The siRNA construct was identical in each case. (C) Protein blots of SOD1 and GFP levels in gastrocnemius from lentivirus- and AAV-injected SOD1G93A mice (three representative animals in each group). (D) Grip strength of the right treated hindlimb in AAV siRNA- vs. lentivirus siRNA-treated animals. (E) Wet weights of 98-day-old SOD1G93A mice treated with AAV siRNA vs. lentivirus siRNA (n = 10, average ± SE).

Fig. 2.

Reduction of mutant SOD1 from skeletal muscle does not affect disease onset or survival of LoxSOD1G37R mice. (A–C) LoxSOD1G37R transgene levels (A and B; Cre+, n = 4; Cre−, n = 3) in quadriceps femoris (A) and gastrocnemius (B) muscle and SOD1 protein levels in gastrocnemius muscle (C) from LoxSOD1G37R/MCK-Cre mice and LoxSOD1G37R mice were determined. Bars represent mean and standard deviation. ∗, P < 0.05; unpaired t test. For B, P = 0.11. (D and E) Onset (D) and survival (E) times of LoxSOD1G37R/MCK-Cre mice (Cre+) and littermate LoxSOD1G37R (Cre−) mice.

Fig. 3.

Follistatin promotes myoblast proliferation in vitro. In vitro testing was performed to confirm the biological activity of follistatin to inhibit myostatin. (A) Schematic of follistatin in AAV vector and plasmids used for AAV production. CMV represents the cytomegalovirus promoter with a splice donor/acceptor sequence. Poly(A) represents the poly-adenylation sequence. The vector is flanked by the two AAV inverted terminal repeats from serotype 2. Rep2Cap1 represents the plasmid used for packaging the AAV into capsid serotype 1 along with the adenoviral helper plasmid. (B) Proliferation assay of myoblasts (C2C12) cultured in growth media in the presence or absence of myostatin (3 μg/ml) mixed with conditioned media from AAV-transduced cells expressing either RFP or follistatin (FS) (n = 4 average ± SE).

Fig. 4.

Follistatin increases muscle mass in SOD1G93A mice. AAV–follistatin or AAV–GFP at 1 × 1011 viral genomes was injected into the hindlimbs (quadriceps and tibialis muscles) of SOD1G93A mice at 40 days of age (n = 15 per group). (A) Representative photograph of end-stage (126 days) SOD1G93A showing the widespread increase in muscle mass in AAV–follistatin-treated animals. (B) Wet weights of tibialis anterior, gastrocnemius, medial quadriceps, and triceps muscle from AAV–follistatin- and AAV–GFP-treated animals (n = 10–15 per group, average ± SE). (C) ELISA for follistatin in blood from AAV–GFP- and AAV–follistatin-treated animals (age, 100 day; n = 8 average ± SE).

Fig. 5.

Follistatin treatment increases the number and size of myofibers. (A) Myofibers were counted in serial sections of gastrocnemius and quadriceps muscle from AAV–follistatin- and AAV–GFP-treated SOD1G93A mice at end stage (126 days) (n = 8 per group, average ± SE). (B and C) Diameters of muscle fibers from quadriceps (B) and gastrocnemius (C) muscle in AAV–follistatin- and AAV–GFP-treated SOD1G93A mice at end stage (126 days) (n = 8 per group, average ± SE).

Fig. 6.

AAV–follistatin increases muscle strength but does not improve rotarod performance or significantly affect survival. (A and B) Grip strength of hindlimbs (A) and forelimbs (B) were recorded in AAV–GFP- and AAV–follistatin-treated SOD1G93A mice. (C) Latency to fall off the rotarod was measured in AAV–follistatin- AAV–GFP-treated SOD1G93A mice. (D) Survival analysis of AAV–follistatin- and AAV–GFP-treated SOD1G93A mice. AAV–GFP, 126 days; AAV–follistatin, 130 days; P = 0.06 (n = 15; litter-matched; the number of female mice equaled the number of male mice).