Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy

Abstract

Cardiotrophin-1 (CT-1), an IL-6-related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1-treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1-treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.

Figure 1

Adenovirus-mediated production of biologically active CT-1. Conditioned media from AdCT-1– or AdLacZ-infected human fibroblasts were tested for their trophic activity on cultured motoneurons. The number of surviving motoneurons was determined 48 hours after plating and is represented as a function of the number of infectious particles per fibroblast (PFU/cell) or as a function of recombinant CT-1 protein (rCT-1; inset). Each point represents the mean of duplicate plates.

Figure 2

CT-1 gene expression in pmn mice. (a) Levels of endogenous CT-1 protein in gastrocnemius muscles of 28-day-old normal (lanes 1–3) and pmn (lanes 4–6) mice analyzed by Western blot. (b, c, d, and e) CT-1 expression after AdCT-1 intramuscular injection in neonatal pmn mice. (b) The right gastrocnemius muscle and the lumbar spinal cord were analyzed in AdCT-1–injected (lanes 1–3) and AdLacZ-injected (lanes 4–5) mice. Adenoviral CT-1 transcripts were detected by RT-PCR in all right gastrocnemius muscles of AdCT-1–injected mice at day 25, whereas only weak RT-PCR signals were detected in the lumbar spinal cords of 15-day-old treated mice. C+: cDNA from AdCT-1-infected 293 cells; C–: without template; –, without RT; +, with RT. (c) CT-1 overexpression was revealed by Western blot analysis in AdCT-1–injected muscles (+) but not in AdLacZ-injected muscle (–). Recombinant CT-1 protein (rCT-1) and conditioned media (CM) from AdCT-1–infected (+) or noninfected (–) fibroblasts were used as controls. The 2 bands of apparent molecular mass 23 and 29 kDa might reflect precursor and mature forms of CT-1 or differently glycosylated isoforms. Note that endogenous CT-1 protein in untreated pmn mice was not detectable under these experimental conditions (50 μg of loaded protein, short exposure time). (d) Sera of AdCT-1– and AdLacZ-injected pmn mice were tested for their neurotrophic activity on motoneurons. The number of motoneurons per field (mean ± SEM) was determined and expressed relative to the number of motoneurons surviving in 50 ng/mL CT-1 (100%) and in neurobasal medium (0%). (e) Sera of 25-day-old pmn mice and rCT-1 (inset) were tested in a ciliary ganglion (CG8) neuron survival assay. Elevated serum CT-1 concentrations (in trophic units [TU]/mL) were detected in all 5 tested AdCT-1–injected pmn mice but in none of the 4 AdLacZ-injected mice. One TU corresponds to the serum dilution that allowed half maximal survival of the neurons.

Figure 3

Electromyography in 35-day-old AdCT-1–treated pmn mice. (a) Individual and mean amplitude of CMAPs and motor latency in the calf muscles of normal, untreated pmn, and ACT-1–treated pmn mice. Data of untreated pmn and AdCT-1–treated pmn mice were significantly different (*P < 0.05). (b) Spontaneous electrical activity in the right diaphragm. Note the differences in shape of the inspiratory burst and the frequency of the high-voltage oscillations between untreated and CT-1–treated pmn mice. Asterisks mark ECG recordings.

Figure 4

Terminal innervation of abdominal muscles of 4-week-old mice, stained with the AchE-silver method. (a) Normal mouse (Xt/pmn). Bundles of terminal axons originating from intramuscular nerve branches supply individual endplates. These axons run without branching for up to 400 μm. (b) pmn mouse, untreated. A nerve branch contains few intact, possibly afferent fibers and remnants of degenerated axons. Many endplates are devoid of terminal axons. Between these endplates are numerous fine axonal sprouts that, however, are not visible at this magnification. (c and d) pmn mice, AdCT-1–treated. The nerve branch in c appears well preserved, and all endplates are supplied by relatively long terminal axons. Rare examples of unequivocal sprouting are marked in both micrographs (arrows). A muscle spindle is visible in c (open arrow). Calibration bars: 100 μm for a–c, 50 μm for d. (e) Histograms of the lengths of terminal axons of individual endplates measured from the last axonal branching to the entry into the endplate. The lengths of axons of denervated endplates (example of endplates without terminal axon) were recorded as zero. Note large number of denervated endplates in untreated pmn as compared with CT-1–treated pmn and normal mice.

Figure 5

Light micrographs of phrenic nerves from 25-day-old (a, b, and c) and 45-day-old (d) mice. (a) Untreated pmn; (b) normal littermate; (c and d) AdCT-1–treated pmn. Note the well-preserved nerve of the AdCT-1–treated mice at day 25 and still at day 45 compared with that of the 25-day-old untreated pmn mouse. Scale bar: 50 μm.

Figure 6

Light micrographs of facial nucleus of 35-day-old normal (a and b), untreated pmn (c and d), and AdCT-1–treated pmn (e and f) mice. In untreated pmn mice, the number of facial motoneurons was significantly decreased, and many of them showed extensive chromatolysis. Motoneurons were more numerous in the facial nucleus of AdCT-1–treated mice compared with untreated pmn mice (see also Table 1). Scale bar: 100 μm (a, c, and e), and 50 μm (b, d, and f).

Figure 7

Survival and weight gain of pmn mice after AdCT-1 gene transfer. (a) Survival of AdCT-1–treated mice (filled triangles; n = 18) is significantly improved (log rank test, P = 0.0012) compared with AdLacZ-injected mice (filled squares; n = 20). (b) A dose-dependent reduction in weight gain was observed in AdCT-1–injected pmn mice (filled triangles, 10⁸ PFU; open triangles, 3 × 10⁸ PFU) compared with AdLacZ-injected mice (filled squares, 10⁸ PFU).