Philippe Poindron

Progressive Motor Neuron Impairment in an Animal Model of Familial Amyotrophic Lateral Sclerosis

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration.

Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord--human muscle cocultures.

Neurotrophins, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and brain-derived neurotrophic factor (BDNF), were studied in vitro in a coculture model of human skeletal muscle myotubes and rat embryo spinal cord explants, which enables the different steps of functional innervation to be followed, including neurite outgrowth, synapse formation and induction of contractile activity. We found that NT-3, NT-5, BDNF, but not NGF simultaneously induced a significant increase in the number and length of neurites emerging from spinal cord explants, the number of endplates per muscle fiber, and the area of innervated muscle fibers around each spinal cord explant. These results suggest that neurotrophins NT-3, NT-5 and BDNF enhance spinal cord motoneurons potential of innervation.

Muscle could be the therapeutic target in SMA treatment

A nerve‐muscle coculture model (human muscle cells innervated by embryonic rat spinal cord) was used to explore the pathogenesis of spinal muscular atrophy (SMA). Previous studies showed that myofibers from donors with SMA type I or SMA type II (but not SMA type III) undergo a characteristic degeneration 1–3 weeks after innervation (Braun et al. [1995] Lancet 345:694–695). To determine which cells are involved in degeneration, we cloned satellite cells and fibroblasts derived from muscle biopsies of normal (healthy) donors and donors with SMA. We show that fibroblasts are required for successful innervation, that fibroblasts from normal and SMA donors contribute equally well to the establishment of cocultures, and that only SMA satellite cells are responsible for the degeneration of innervated cocultures. We succeeded in preventing the degeneration of cloned satellite cells from SMA donors by adding 50% cloned satellite cells from normal donors to the culture to make heteromyotubes. In mixed cocultures, after innervation, we did not observe degeneration. This result suggests that survival of the cocultures depends on a message derived from the muscle cells. Consequently, we propose that therapeutic approaches for SMA that could repair (or compensate for) the genetic defect in muscle cells (which are otherwise much more accessible for gene therapy than neurons) might prevent motoneuron degeneration. The role of muscle cells in the establishment and the degeneration of neuromuscular junctions deserves further attention and investigation. J. Neurosci. Res. 53:663–669, 1998.

Modulation by prednisolone of calcium handling in skeletal muscle cells.

1 Increased calcium (Ca2+) influx has been incriminated as a potential pathological mechanism in the chronic skeletal muscle degeneration exhibited by Duchenne muscular dystrophy (DMD) patients. We have studied the influence of the glucocorticoid α‐methylprednisolone (PDN), the only drug known to have a beneficial effect on the degenerative course of DMD, on Ca2+ handling in the C2 skeletal muscle cell line. 2 PDN, when added 3 days (when myoblasts start to fuse into myotubes) after cell seeding, led to a 2 to 4 fold decrease in cellular Ca2+ uptake. This decrease was independent of the extracellular Ca2+ concentration applied to cells. The effect took at least 24 h in order to become established (PDN of 10−5 m) and took longer for lower PDN concentrations (EC50 of ca. 10−6 m at day 5, 10−6 5 m at day 7 and 10−7 5 m at day 9 in culture). 3 Cellular calcium accumulation was also decreased in PDN‐treated myotubes exposed to 45Ca2+‐containing medium for 1 to 6 days. 4 No effect of PDN was seen on 45Ca2+ efflux; a decrease in the amount of 45Ca2+released was observed due to the reduction of cellular 45Ca2+ loading. 5 PDN treatment led to an approximately 2 fold decrease in basal cytosolic Ca2+ concentration. 6 Three antioxidant drugs (lazaroids), previously shown to enhance in vitro skeletal muscle cell differentiation to the same extent as PDN, induced a similar decrease in Ca2+ influx. 7 Our results suggest that long‐term incubation of C2 cells with PDN leads to a decrease of the size of the cellular Ca2+ pools and to reduced resting cytosolic Ca2+ levels. Part of the beneficial effect of PDN in DMD patients could be attributed to a reduction of Ca2+ influx and of the size of Ca2+ pools in dystrophic muscle fibres.

Reduced expression of nicotinic AChRs in myotubes from spinal muscular atrophy I patients

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of motoneurons and skeletal muscle atrophy. In its most severe form, it leads to death before the age of 2 years. While primary degeneration of motor neurons is well established in this disease, and this results in neurogenic atrophy of skeletal muscle, we have previously reported evidence for a primary muscle defect. In this study, we used primary cultures of embryonic human skeletal muscle cells from patients with SMA and from controls to examine the effects of muscle fiber differentiation in the absence of a nerve component. Cultured SMA skeletal muscle cells are unable to fuse correctly to form multinuclear myotubes, the precursors of the myofibers. We also show that agrin-induced aggregates of nicotinic acetylcholine receptors, one of the earliest steps of neuromuscular junction formation, cannot be visualized by confocal microscopy on cells from SMA patients. In binding experiments, we demonstrate that this lack of clustering is due to defective expression of the nicotinic acetylcholine receptors in the myotubes of SMA patients whereas the affinity of α-bungarotoxin for its receptor remains unchanged regardless of muscle cell type (SMA or control). These observations suggest that muscle cells from SMA patients have intrinsic abnormalities that may affect proper formation of the neuromuscular junction.

Prevention of mutant SOD1 motoneuron degeneration by copper chelators in vitro.

An animal model of familial amyotrophic lateral sclerosis (FALS) has been generated by overexpression of human CuZn superoxide dismutase (SOD1) containing a substitution of glycine to alanine at position 93 in transgenic G93A mice. The loss of motoneurons shown in this model has been attributed to a dominant gain of function of this mutated enzyme, which might be due to copper toxicity. This hypothesis was tested in purified spinal motoneurons cultures originating from G93A transgenic embryos. Spinal motoneurons were isolated from E13 embryos by several steps including density gradient centrifugation. The effect of copper chelators on survival and neurite growth of motoneurons was investigated. Survival of G93A motoneurons was decreased by 46% as compared to wild-type motoneurons. Moreover, G93A motoneurons showed reduced neurite outgrowth. Copper chelators strikingly increased viability of G93A motoneurons (by over 200%) but had no effect on wild-type cells. Presence of DDC in the medium increases the length of neurites from G93A motoneurons. The present results suggest the capacity of copper chelators to reduce the effect of reverse function of mutated SOD1 on motoneurons.

Internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells

The lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) has been shown previously to behave as a marker of plasma membrane in living cell systems, and it has therefore been widely used in membrane fluidity studies via fluorescence anisotropy measurements. However, progressive internalization of this probe in cells could lead to unsuitable interferences, when long incubations times were required. The mechanism of this internalization had not yet been elucidated. We present here fluorescence-intensity kinetic results and fluorescence micrographic data on L929 cells and on mouse bone-marrow macrophages, which allow us to identify the mechanism as fluid-phase pinocytosis: the probe remains associated with the plasma membrane throughout its internalization-recycling flow and it is finally concentrated in lysosomes. The study was facilitated by the partition equilibrium property of TMA-DPH between plasma membranes and the external aqueous medium, which allowed to immediately distinguish the internalized fraction of the probe from the peripheral labelling, by simply washing cells. This conclusion is confirmed by the features of the influence of temperature on TMA-DPH internalization.

Possible pathogenic role of muscle cell dysfunction in motor neuron death in spinal muscular atrophy

We have previously shown that myofibers formed by fusion of muscle satellite cells from spinal muscular atrophy (SMA) I or II undergo degeneration 1 to 3 weeks after innervation by rat embryonic spinal cord explants, whereas normal myofibers survive for several months. In the “muscle component” of the coculture, the only cells responsible for the degeneration are the SMA muscle satellite cells. Moreover, SMA muscle satellite cells do not fuse as rapidly as do normal muscle satellite cells. To determine whether death of muscle cells precedes that of motor neurons, we studied the origin and kinetics of release of apoptotic microparticles. In SMA cocultures, motor neuron apoptosis occurred before myofiber degeneration becomes visible, indicating that SMA myofibers were unable to sustain survival of motor neurons. In normal cocultures, motor neuron apoptosis occurred 4 days after innervation. However, it did not continue beyond 2 days. These results strengthen the hypothesis that SMA is due to a defect in neurotrophic muscle cell function.

Selective changes in mitochondria respiratory properties in oxidative or glycolytic muscle fibers isolated from G93AhumanSOD1 transgenic mice

Cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in cytosolic copper, zinc superoxide dismutase (SOD1). Total SOD activity and functional mitochondrial properties were studied in muscles and nervous tissues of control and transgenic mice mimicking the disease. It was found that total SOD activity was lower in nervous tissues than in muscles in both transgenic and control mice. In addition SOD activity increased during progression of disease in muscle but not in nervous tissue of transgenic mice. Maximal oxygen consumption and apparent Km for ADP were decreased in mitochondria from transgenic soleus (an oxidative muscle). However there was no difference between control and transgenic mice in respiratory parameters of mitochondria in the EDL muscle (a glycolytic muscle). These findings indicate that oxidative stress due to SOD1 mutations could alter energy metabolism in FALS mice, thereby affecting primarily oxidative muscle of the limbs, independently of motoneuron loss.

The effect of the nonpeptide neurotrophic compound SR 57746A on the progression of the disease state of the pmn mouse

The progressive motor neuronopathy (pmn) mouse is an autosomal recessive mutant, in which the homozygotes suffer caudio‐cranial degeneration of motor axons and die several weeks after birth. This strain provides the opportunity of testing potential therapeutic strategies for the treatment of motor neurone diseases such as amyotrophic lateral sclerosis. We have performed a study of the effects on the pmn mouse of SR 57746A, an orally‐active, non‐peptide compound which has been found to exhibit neurotrophic effects in vitro and in vivo. In order to treat the affected mice from birth, the mothers were administered 2.5 mg kg−1, p.o., SR 57746A every two days until the weaning of the offspring (at day 20); then the offspring were given every two days a dose of 30 μg kg−1, p.o., until their death. Affected mice treated with SR 57746A had a lifespan 50% longer than that of the vehicle‐treated mice (P=0.01). Compared to vehicle‐treated pmn mice, SR 57746A improved the performance of the pmn mice in three different behavioural tasks. SR 57746A also maintained the amplitude of the motor evoked response of the gastrocnemius muscle, reduced the distal motor latency, and delayed the occurrence of the spontaneous denervation activity in this muscle. Histological studies indicated that at 20 days of age the mean surface areas of the fibres of the sciatic nerve were higher in SR 57746A‐treated than in vehicle‐treated mice. At present, SR 57746A is the only orally active, nonpeptide compound known to be capable of delaying the progression of the motor neurone degeneration in pmn mice.