Critical period of neuromuscular development: Importance for a new treatment of SMA.

Findings from mice that had their Smn gene deleted and some copies of the human SMN2 gene introduced to produce SMN protein are summarized. Symptoms due to this manipulation can be corrected only by restoring the SMN protein expression in neurones and not in muscle. The changes in muscle and neuromuscular junction (NMJ) in these mutant mice are probably due to the malfunction of the neuronal component of the NMJ i.e. the nerve terminal. The reduction of transmitter release by nerve terminals in animals with reduced SMN protein supports this notion. There is a critical period during which the presence of the SMN protein is mandatory for the survival of the motor unit and the individual. This period coincides with the most important events involved in the development of the motor unit. Results from normal genetically unaffected rats and mice show that during a critical period of development the function of the nerve terminal and the release of transmitter play a crucial role in the development of the motor neurone and muscle. The possibility that targeting the function of the nerve terminal to overcome its inability to release transmitter could benefit patients with the deletion of the SMN gene.

The Contribution of Neuromuscular Stimulation in Elucidating Muscle Plasticity Revisited.

Studies carried out during the past 45 years on the effects of chronic low-frequency stimulation on skeletal muscle have revealed a multiplicity of adaptive changes of muscle fibres in response to increased activity. As reflected by induced changes in the metabolic properties, protein profiles of the contractile machinery and elements of the Ca2+-regulatory system, all essential components of the muscle fibre undergo pronounced changes in their properties that ultimately lead to their reversible transformation from fast-to-slow phenotype. The chronic low-frequency stimulation experiment thus allows exploring many aspects of the plasticity of mammalian skeletal muscle. Moreover it offers the possibility of elucidating molecular mechanisms that remodel phenotypic properties of a differentiated post-mitotic cell during adaptation to altered functional demands. The understanding of the adaptive potential of muscle can be taken advantage of for repairing muscle damage in various muscle diseases. In addition it can be used to prevent muscle wasting during inactivity and aging. Indeed, pioneering studies are still the sound grounds for the many current applications of Functional Electrical Stimulation and for the related research activities that are still proposed and funded.

Sulf1 and Sulf2 expression in the nervous system and its role in limiting neurite outgrowth in vitro.

Sulf1 and Sulf2 are endosulfatases that cleave 6-O-sulphate groups from Heparan Sulphate Proteoglycans (HSPGs). Sulfation levels of HSPGs are critical for their role in modulating the activity of various growth factor receptors. Sulf1 and Sulf2 mRNAs were found to be widely expressed in the rodent nervous system and their full-length proteins were found in many types of neuronal perikarya and axons in the cerebral cortex, cerebellum, spinal cord and dorsal root ganglia (DRG) of adult rats. Sulf1/2 were also strongly expressed by cultured DRG neurons. To determine if blocking Sulf1 or Sulf2 activity affected neurite outgrowth in vitro, cultured DRG neurons were treated with neutralising antibodies to Sulf1 or Sulf2. Blocking Sulf1 and Sulf2 activity did not affect neurite outgrowth from cultured DRG neurons grown on a laminin/polylysine substrate but ameliorated the inhibitory effects of chondroitin sulphate proteoglycans (CSPGs) on neurite outgrowth. Blocking epidermal growth factor receptor (ErbB1) activity also improved neurite outgrowth in the presence of CSPGs, but the effects of ErbB1 antagonists and blocking SULFs were not additive. It is proposed that Sulf1, Sulf2 and ErbB1 are involved in the signalling pathway from CSPGs that leads to inhibition of neurite outgrowth and may regulate structural plasticity and regeneration in the nervous system.

Chemical communication between regenerating motor axons and Schwann cells in the growth pathway.

There are receptors on denervated Schwann cells that may respond to the neurotransmitters that are released from growth cones of regenerating motor axons. In order to ascertain whether the interaction of the transmitters and their receptors plays a role during axon regeneration, we investigated whether pharmacological block of the interaction would reduce the number of motoneurons that regenerate their axons after nerve section and surgical repair. Peripheral nerves in the hindlimbs of rats and mice were cut and repaired, and various drugs were applied to the peripheral nerve stump either directly or via mini-osmotic pumps over a 2-4-week period to block the binding of acetylcholine to nicotinic and muscarinic acetylcholine receptors (AChRs: alpha-bungarotoxin, tubocurarine, atropine and, gallamine) and binding of ATP to P2Y receptors (suramin). In rats, the nicotinic AChR antagonistic drugs and suramin reduced the number of motoneurons that regenerated their axons through the distal nerve stump. In mice, suramin significantly reduced the upregulation of the carbohydrate HNK-1 on the Schwann cells in the distal nerve stump that normally occurs during motor axon regeneration. These data indicate that chemical communication between regenerating axons and Schwann cells during axon regeneration via released neurotransmitters and their receptors may play an important role in axon regeneration.

Lack of myostatin results in excessive muscle growth but impaired force generation.

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Spinal muscular atrophy: a delayed development hypothesis.

Spinal muscular atrophy is an inherited neuromuscular disorder. The gene responsible for the disease has been identified and named the SMN gene. This review is prompted by recent advances in understanding cellular function of the SMN gene and its gene product and by the increasing evidence that maturation of all parts of the neuromuscular system is delayed in spinal muscular atrophy patients. We suggest that the timing of developmental changes in motoneurons and muscles is critical for their survival. Delayed maturation of either motoneuron or muscle can cause these cells to die so the molecules that are involved in controlling their rate of maturation are crucial for normal development. We suggest that SMN gene/protein is one such molecule, because the neuromuscular system develops more slowly in spinal muscular atrophy patients, where SMN protein is absent, and in animals models, where SMN protein is reduced.

Function induced modifications of gene expression: an alternative approach to gene therapy of Duchenne muscular dystrophy.

In Duchenne muscular dystrophy a large gene that codes for dystrophin is altered. The possibility that the defective gene/protein could be at least in part substituted by other molecules that the diseased muscle is able to produce and that have a function similar to that of dystrophin is being discussed. Muscle fibres have a tremendous adaptive potential, and the expression of several protein isoforms can be induced by either stretch or long-term change of activity. The exploitation of this ability of muscle cells to express new genes, which would code for proteins that will not be alien to the individual, for treatment of Duchenne muscular dystrophy is being considered. The argument for this approach is strengthened by results that in patients with Duchenne muscular dystrophy the progress of the disease can be slowed with changes of muscle activity.

Axotomized motoneurons can be rescued from cell death by peripheral nerve grafts: the effect of donor age.

Injury to neonatal nerves, unlike adult nerves, results in poor regeneration and extensive motoneuron death. We examined whether exposure to a more mature nerve environment could rescue axotomized motoneurons following neonatal injury. The sciatic nerve in 1 hindlimb of 3-day-old (P3) rats was transected and the cut end sutured to a nerve graft taken from donor rats, which ranged between P3 and P21. The extent of motoneuron survival and axon regeneration was established 7 days later. Since integrins play an important role in regeneration, we also examined the effect of manipulating integrin binding in nerve grafts. Following axotomy at P3 and implantation of nerve grafts from 3-day-old rats, approximately 38% of motoneurons survived. In contrast, grafts from rats aged 5 days and older resulted in an improvement in regeneration, and over 70% of motoneurons survived. This survival-promoting effect of P5 grafts was prevented by blocking beta1-integrins. In contrast, increasing beta1-integrin levels in grafts from P3 rats dramatically increased motoneuron survival. Thus, following neonatal nerve injury, exposure to a more mature nerve environment significantly increases motoneuron survival, an effect that is dependent upon beta1-integrin signaling. Therefore, pharmacological upregulation of beta1-integrins may significantly improve the outcome of neonatal nerve injuries.

The effect of neonatal nerve injury on the expression of heat shock proteins in developing rat motoneurones.

The expression of the heat shock proteins hsp27 and hsp70 was examined in the spinal cord and sciatic nerves of developing rats. Using immunohistochemistry, we found that hsp27 is present in many motoneurones at birth. With development, the intensity of staining increases, reaching adult levels by 21 days, when all sciatic motoneurones express hsp27. In the sciatic nerve, hsp27 is strongly expressed throughout postnatal development. In contrast, hsp70 immunoreactivity in motoneurones and the sciatic nerve is weak at birth and does not change with development. The expression of heat shock proteins has been shown to increase in cells under conditions of stress, where they have beneficial effects on cell survival. The effect of neonatal nerve injury on hsp27 and hsp70 expression was also examined in this study. Four days after injury, staining for hsp27 increases in motoneurones, whereas hsp70 does not change. However, there is a significant increase in hsp70 staining in glial cells surrounding the injured motor pool, predominantly in astrocytes. Since neonatal nerve injury induces apoptotic motoneurone death, we also studied the co-expression of hsp27 with markers of apoptosis. No hsp27-positive motoneurones were found to be apoptotic, as assessed by both TUNEL and caspase-3 immunoreactivity. Therefore, it is possible that the upregulation of hsp27 observed in injured motoneurones may play a role in protecting motoneurones from apoptotic cell death following nerve injury.

Pseudorabies virus-based gene delivery to rat embryonic spinal cord grafts.

The construction and application of recombinant pseudorabies viruses (PrVs) for the delivery of beta-galactosidase and/or green fluorescent protein (GFP) genes to rat embryonic spinal cord cells are reported here. These viruses were specifically designed to infect embryonic spinal cord neurons, which can be grafted into a lesioned spinal cord in order to restore the lost functions of the host cord. The recombinant viruses were constructed in two steps. The small subunit of the ribonucleotide reductase (RR) gene was first abolished by a frameshift mutation and an expression cassette containing the lacZ gene alone or together with the GFP gene was then inserted in place of the early protein 0 (EP0) gene of PrV. The reporter gene cassettes were positioned downstream from the PrV latency-associated promoter. Using an ex vivo system, we infected embryonic spinal cord explants with these viruses and found that neither vRREP0lac nor vRREP0lacgfp exerted any cytotoxic effect at all. It was also revealed that these viruses infect embryonic cells with high efficiency, and that infected neurons grafted into the spinal cord express the inserted reporter genes for periods of up to 12 weeks. This system offers a new approach for foreign gene transfer to neurons grafted into the CNS.

Electromyographic activity patterns of ankle flexor and extensor muscles during spontaneous and L-DOPA-induced locomotion in freely moving neonatal rats.

In rats, hindlimb postural and locomotor functions mature during the first 3 postnatal weeks. Previous evidence indicates that maturation of descending monoaminergic pathways is important for the postnatal emergence of locomotion with adequate antigravity postural support. Here we have studied the effect of the monoamine precursor L-DOPA on locomotor activity in freely moving postnatal rats (7-9 days old) using electromyographic recordings from ankle extensor (soleus) and flexor (tibialis anterior or extensor digitorum longus) muscles. Before pharmacological treatment, both muscles were usually silent at rest, and during spontaneous movements there was a high degree of coactivation between the two antagonists. This was due to a longer electromyographic (EMG) burst duration in flexors, which partly overlapped with the extensor burst. L-DOPA administration (150 mg/kg) resulted in a marked increase in postural tonic EMG activity in extensors which appeared gradually within 10 min after injection and was sufficient for the pups to maintain a standing posture with the pelvis raised above ground. Thereafter, episodes of locomotion characterized by rhythmic reciprocal bursts of EMG activity in flexor and extensor muscles were seen. The L-DOPA-induced rhythmic EMG pattern was also seen in postnatal rats subjected to a midthoracic spinal cord transection, indicating that the effect of L-DOPA on motor coordination is exerted primarily at the level of the spinal pattern generator. Analysis of EMG burst characteristics showed that the pattern of L-DOPA-induced locomotion in both intact and spinalized postnatal rats resembled in some respects that observed in adults during spontaneous locomotion. The appearance of reciprocal activation during L-DOPA-induced locomotion in neonates was primarily due to a shortening of the EMG burst duration in flexors, which reduced the degree of antagonist coactivation. These results show that the spinal cord has the potential to produce coordinated overground locomotion several days before such movements are normally expressed in the freely moving animal.

EFFECTS OF NEUROMUSCULAR BLOCKING AGENTS ON THE DIFFERENTIATION OF NERVE-MUSCLE CONNECTIONS IN SLOW AND FAST CHICK MUSCLES.

The effects of neuromuscular blocking drugs on the development of slow and fast muscle fibres and their neuromuscular junctions was studied in chick embryos. Treatment of embryos with the depolarizing neuromuscular blocking agent suxamethonium affected the development of muscle fibres of the slow anterior latissimus dorsi (ALD) muscle more than that of muscle fibres of the posterior latissimus dorsi (PLD). The differentiation of the presynaptic elements of the neuromuscular junction was delayed and this was particularly obvious in PLD. Normally the number of axon profiles at individual endplates is reduced by 18 days of incubation, but in suxamethonium treated embryos this reduction took place only at 21 days. During earlier stages of development the axon profiles from treated embryos were small with sparse synaptic vesicles. Nevertheless the subsynaptic site of endplates on ALD and PLD muscle fibres became specialized earlier than normal and to a greater extent. Treatment with hemicholinium (HC-3), a drug that reduces the synthesis of acetylcholine (ACh) in nerve terminals affected the development of PLD muscle fibres more than ALD muscle fibres. Although in HC-3 treated embryos nerve-muscle contacts were formed, the axon terminals look immature and remain small even in 18-day old embryos at both ALD and PLD muscle fibres. The reduction of the number of axon profiles normally seen at 18 days failed to take place in treated embryos. At 18 days of incubation many endplates on PLD muscle fibres showed little sign of postsynaptic specilization and resembled endplates usually seen at this stage on ALD muscle fibres. It is concluded that while neuromuscular activity may be important for the reduction of the number of axon profiles at individual endplates, the specialization of the subsynaptic membrane is brought about by depolarizing effect of ACh.