Activity-dependent degeneration of axotomized neuromuscular synapses in Wld S mice.

Activity and disuse of synapses are thought to influence progression of several neurodegenerative diseases in which synaptic degeneration is an early sign. Here we tested whether stimulation or disuse renders neuromuscular synapses more or less vulnerable to degeneration, using axotomy as a robust trigger. We took advantage of the slow synaptic degeneration phenotype of axotomized neuromuscular junctions in flexor digitorum brevis (FDB) and deep lumbrical (DL) muscles of Wallerian degeneration-Slow (Wld(S)) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32°C for up to 48 h. About 90% of fibers from Wld(S) mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100 Hz: 1s/100s) reduced synaptic protection in Wld(S) preparations by about 50%. This effect was abolished in reduced Ca(2+) solutions. Next, we assayed FDB and DL innervation after 7 days of complete tetrodotoxin (TTX)-block of sciatic nerve conduction in vivo, followed by tibial nerve axotomy. Five days later, only about 9% of motor endplates remained innervated in the paralyzed muscles, compared with about 50% in 5 day-axotomized muscles from saline-control-treated Wld(S) mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4 weeks prior to axotomy. Surprisingly, exercising Wld(S) mice ad libitum for 4 weeks increased about twofold the amount of subsequent axotomy-induced synaptic degeneration. Together, the data suggest that vulnerability of mature neuromuscular synapses to axotomy, a potent neurodegenerative trigger, may be enhanced bimodally, either by disuse or by hyperactivity.

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld(S) prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld(S) protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.

Differential protection of neuromuscular sensory and motor axons and their endings in Wld(S) mutant mice.

Orthograde Wallerian degeneration normally brings about fragmentation of peripheral nerve axons and their sensory or motor endings within 24-48 h in mice. However, neuronal expression of the chimaeric, Wld(S) gene mutation extends survival of functioning axons and their distal endings for up to 3 weeks after nerve section. Here we studied the pattern and rate of degeneration of sensory axons and their annulospiral endings in deep lumbrical muscles of Wld(S) mice, and compared these with motor axons and their terminals, using neurone-specific transgenic expression of the fluorescent proteins yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) as morphological reporters. Surprisingly, sensory endings were preserved for up to 20 days, at least twice as long as the most resilient motor nerve terminals. Protection of sensory endings and axons was also much less sensitive to Wld(S) gene-copy number or age than motor axons and their endings. Protection of γ-motor axons and their terminals innervating the juxtaequatorial and polar regions of the spindles was less than sensory axons but greater than α-motor axons. The differences between sensory and motor axon protection persisted in electrically silent, organotypic nerve-explant cultures suggesting that residual axonal activity does not contribute to the sensory-motor axon differences in vivo. Quantitative, Wld(S)-specific immunostaining of dorsal root ganglion (DRG) neurones and motor neurones in homozygous Wld(S) mice suggested that the nuclei of large DRG neurones contain about 2.4 times as much Wld(S) protein as motor neurones. By contrast, nuclear fluorescence of DRG neurones in homozygotes was only 1.5 times brighter than in heterozygotes stained under identical conditions. Thus, differences in axonal or synaptic protection within the same Wld(S) mouse may most simply be explained by differences in expression level of Wld(S) protein between neurones. Mimicry of Wld(S)-induced protection may also have applications in treatment of neurotoxicity or peripheral neuropathies in which the integrity of sensory endings may be especially implicated.

Activation of alpha-latrotoxin receptors in neuromuscular synapses leads to a prolonged splash acetylcholine release.

The mechanisms of acetylcholine release in presynaptic terminals of motoneurons induced by mutant alpha-latrotoxin (LT(N4C)) were analyzed. In contrast to wild-type alpha-latrotoxin that causes both continuous and splash secretion of acetylcholine and necessarity block neuromuscular transmission, LT(N4C) causes only splash release lasting over many hours. Thus, activation of alpha-latrotoxin receptors controls long-lasting enhanced secretion of acetylcholine.

Programmed axon death, synaptic dysfunction and the ubiquitin proteasome system.

Axons are essential, vulnerable and often irreplaceable so it is essential to understand how they are lost in neurodegenerative disease. Recent data link the mechanism of injury-induced Wallerian degeneration to that of axon death in CNS and PNS disease. The neuroprotective gene Wld(S) delays Wallerian degeneration, CNS axonal dystrophy, 'dying-back' pathology and to a lesser extent synapse loss, despite the different causes and morphologies of degeneration. These findings validate Wallerian degeneration as a model to understand and prevent mechanisms of axon and synapse loss in neurodegenerative disorders. The existence of a gene that alters Wallerian degeneration suggests it is a regulated program of axon death normally held back by axonal inhibitors, similar in principle to apoptosis. The Wld(S) protein and proteasome inhibitor experiments implicate the ubiquitin proteasome system (UPS) in Wallerian degeneration. However, the site of UPS involvement and the molecular events remain unclear because the UPS is highly compartmentalized in neurons, affecting complex and sometimes conflicting processes in nuclei, axons, growth cones and synapses. Proteasome inhibitors are blunt tools for studying such a complex system and they are also particularly toxic to axons and alter synapse function. In contrast, Wld(S) acts on a specific step, leaving mice healthy with normal development and behavior. This also makes it an attractive drug target. We need to understand which UPS step is blocked in which neuronal compartment, and to define the pathway in order to develop new strategies to block axon pathology.

Compartmental neurodegeneration and synaptic plasticity in the Wld(s) mutant mouse.

This review focuses on recent developments in our understanding of neurodegeneration at the mammalian neuromuscular junction. We provide evidence to support a hypothesis of compartmental neurodegeneration, whereby synaptic degeneration occurs by a separate, distinct mechanism from cell body and axonal degeneration. Studies of the spontaneous mutant Wld(s) mouse, in which Wallerian degeneration is characteristically slow, provide key evidence in support of this hypothesis. Some features of synaptic degeneration in the absence of Wallerian degeneration resemble synapse elimination in neonatal muscle. This and other forms of synaptic plasticity may be accessible to further investigations, exploiting advantages afforded by the Wld(s) mutant, or transgenic mice that express the Wld(s) gene.

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

Competition at silent synapses in reinnervated skeletal muscle.

Synaptic connections are made and broken in an activity-dependent manner in diverse regions of the nervous system. However, whether activity is strictly necessary for synapse elimination has not been resolved directly. Here we report that synaptic terminals occupying motor endplates made electrically silent by tetrodotoxin and alpha-bungarotoxin block were frequently displaced by regenerating axons that were also both inactive and synaptically ineffective. Thus, neither evoked nor spontaneous activation of acetylcholine receptors is required for competitive reoccupation of neuromuscular synaptic sites by regenerating motor axons.

Co-regulation of synaptic efficacy at stable polyneuronally innervated neuromuscular junctions in reinnervated rat muscle.

1. Intracellular recordings and quantal analysis of synaptic transmission were made at neuromuscular junctions receiving stable convergent innervation in reinnervated rat lumbrical muscles, following recovery from chronic nerve conduction block. The polyneuronally innervated motor endplates (pi-junctions) were identified by vital staining of lateral plantar nerve (LPN) and sural nerve (SN) motor terminals, using the activity-dependent staining properties of the aminostyryl dyes RH414 and FM1-43, respectively. 2. Endplate depolarisation and quantal content per unit area varied by more than a factor of ten ( approximately 0.1-1. 4 quanta microm-2) between fibres. However, the stable pi-junctions produced nearly equivalent endplate depolarisations and quantal content per unit area, suggesting that synaptic strengths were co-regulated at these motor endplates. Quantal content per unit area was also independent of the size of individual synaptic inputs, or whether one, both or neither input was judged sufficient to produce suprathreshold or subthreshold endplate depolarisations. 3. Simultaneous excitation of convergent LPN and SN inputs from some pi-junctions resulted in profound non-linear summation, and in some cases complete occlusion of the response of the smaller input. The amplitude of the smaller, test responses recovered with a time constant of 2.1 +/- 0.5 ms (mean +/- s.e.m.) on varying the interval between paired stimuli, of similar order to the time constant of repolarisation of the conditioning endplate potential. 4. The data show that it is not necessary for a motor nerve terminal to occupy most of an endplate, or to produce a suprathreshold response in order to become stable. The occlusion of linear summation, similar to that described previously at polyneuronal junctions in neonates, suggests that convergent inputs comprising interdigitated synaptic boutons evoke self-contained synaptic responses at endplates, and that these are non-co-operative with respect to overall endplate depolarisation or safety margin for synaptic transmission.

Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line-derived neurotrophic factor (GDNF).

1. The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml-1: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), and glial cell line-derived neurotrophic factor (GDNF). In some experiments, the responses to 2 microM LaCl3 and 10 mM K+, or to 2-5 nM purified alpha-latrotoxin (alpha-LTX) were also measured. 2. Neither BDNF, NT-3, NT-4, LIF, IGF-1 or IGF-2 - singly or in combination - caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2-fold increase in neurotransmitter release that was reproduced in fourteen muscles. 3. Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or alpha-latrotoxin. 4. The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non-saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.

Elimination of motor nerve terminals in neonatal mice expressing a gene for slow wallerian degeneration (C57Bl/Wlds).

Degeneration of motor terminals after nerve section occurs much more slowly than normal in young adult mice of the C57Bl/Wlds strain. This observation prompted us to re-examine the possible role of degeneration and intrinsic axon withdrawal during neonatal synapse elimination. Polyneuronal innervation was assayed by two methods: intracellular recording of end-plate potentials in cut-muscle fibre preparations of isolated hemidiaphragm and soleus muscles; and in silver-stained preparations of triangularis sterni and transversus abdominis muscle fibres. No differences in the rate of synapse elimination were detected in unoperated Wlds compared with CBA, C3H/HE and BALB/c mice. At 3 days of age, > 80% of fibres were polyneuronally innervated. By 7 days this declined to approximately 20% of hemidiaphragm, 50% of triangularis sterni and 60% of soleus fibres. Nearly all fibres were mononeuronally innervated by 15 days. The mean number of terminals per triangularis sterni muscle fibre 7 days after birth was 1.55 +/- 0.07 in Wlds and 1.56 +/- 0.09 in wild-type mice. Three to 4 days after sciatic nerve section, near-normal numbers of motor units were evident in isometric tension recordings of the soleus muscle, and intracellular recordings revealed many polyneuronally innervated fibres. Mononeuronally and polyneuronally innervated fibres were also observed in silver-stained preparations of soleus and transversus abdominis muscles made 3-4 days after sciatic or intercostal nerve section. We conclude (i) that the Wlds gene has no direct impact on the normal rate of postnatal synapse elimination, (ii) that Wallerian degeneration and synapse elimination must occur by distinct and different mechanisms, and (iii) that muscle fibres are able to sustain polyneuronal synaptic inputs even after motor axons have become disconnected from their cell bodies.

Persistent polyneuronal innervation in partially denervated rat muscle after reinnervation and recovery from prolonged nerve conduction block.

The contribution of activity to the long-term stability of synaptic connections is a subject of ongoing debate. In the present study we examined the effects of recovery from chronic disuse on the pattern of reinnervation of partially denervated adult rat skeletal muscles, using tension measurements, intracellular recordings, and observations of vital staining with activity-dependent styryl dyes. Fourth deep lumbrical muscles were partially denervated by crushing the lateral plantar nerve (LPN) bilaterally. Denervated muscle fibers became innervated by sprouts from the sural nerve (SN); 17-23 d after LPN crush, conduction in the right sciatic nerve was blocked by connecting an osmotic minipump containing tetrodotoxin to a cuff placed around the nerve. Distal muscles remained continuously paralysed for 10-19 d. After 2 weeks of nerve block the tension produced by stimulating the regenerated LPN axons had reached about 70% of the total. Regenerating axons in contralateral muscles reinnervated only about 55% of the muscle fibers. The level of dual innervation by both regenerating and intact axons reached about 50% of the total muscle fibers after 2 weeks of paralysis, but only about 20% in contralateral controls. We then measured the rate and amount of decline in motor unit tension and polyneuronal innervation in reinnervated muscles during an eight week period of recovery from nerve block. Some LPN and SN connections regressed within 2-4 weeks, but about 35% of the muscle fibers still retained convergent inputs from LPN and SN motor axons 8 weeks after activity had resumed. This was about twice the level observed in contralateral reinnervated muscles. Vital staining with the styryl dyes FM1-43 and RH414 confirmed that many of the reinnervated motor end-plates were convergently supplied by both SN and LPN axons. Intracellular recordings showed that most of the dually innervated fibers in paralyzed muscles were supplied by suprathreshold inputs from both LPN and SN axons. The increased excitability of these muscle fibers was partly explained by their two-fold increased input resistance. Input resistance recovered to control levels within 4 weeks of resumption of activity, but dually innervated fibers in previously blocked muscles still gave mostly suprathreshold responses to stimulation of both the LPN and the SN. We conclude that chronic nerve conduction block generates or sustains a local environment which allows some convergent synaptic inputs on reinnervated muscle fibers to become consolidated and strengthened, independent of subsequent neuromuscular activity.

Persistence of neuromuscular junctions after axotomy in mice with slow Wallerian degeneration (C57BL/WldS).

The present study was undertaken to examine the fate of neuromuscular junctions in C57BL/WldS mice (formerly known as OLA mice) after nerve injury. When a peripheral nerve is injured, the distal axons normally degenerate within 1-3 days. For motor axons, an early event is deterioration of motor nerve terminals at neuromuscular junctions. Previously, the vulnerability of motor terminals has been attributed either to a 'signal' originating at the site of nerve injury and transported rapidly to the terminals or to their continual requirement for essential maintenance factors synthesized in the motor neuron cell body and supplied to the terminals by fast axonal transport. Mice of the WldS strain have normal axoplasmic transport but show an abnormally slow rate of axon and myelin degeneration. Structure and function are retained in the axons of distal nerve stumps for several days or even weeks after nerve injury in these mice. The results of the present study show that WldS neuromuscular junctions are also preserved and continue to release neurotransmitter and recycle synaptic vesicle membrane for at least 3 days and in some cases up to 2 weeks after nerve injury. Varying the site of the nerve lesion delayed degeneration by approximately 1-2 days per centimetre of distal nerve remaining. These findings suggest that the mechanisms of nerve terminal degeneration after injury are more complex than can be accounted for simply by the failure of motor neuron cell bodies to supply their terminals with essential maintenance factors. Rather, the data support the view that nerve section normally activates cellular components or processes already present, but latent, in motor nerve endings, and that in WldS mice either the trigger or the cellular response is abnormal.

Cell viability and laminin-induced neurite outgrowth in cultures of embryonic chick neural tube cells: effects of cytosine-B-D-arabinofuranoside.

We have measured the effects of cytosine-beta-D-arabinofuranoside (AraC) on cell survival and neurite outgrowth in cultures of dissociated 4-6 day embryonic chick neural tube cells. High concentrations of AraC (greater than 100 microM) reduced neuronal cell survival and neurite outgrowth from viable cells. Concentrations normally used to inhibit mitotic cell division (1-10 microM) were toxic to the neurones cultured in serum free medium on a poly-DL-ornithine/laminin substrate. AraC does not appear to have a neurite promoting effect on dissociated neurones that are cultured in the presence of low numbers of non-neuronal cells. This suggests that the neurite promoting effects of AraC reported by others is likely to be through the non-neuronal cells that were an inherent feature of the culturing systems in these studies. AraC cytotoxicity was completely blocked by the addition of the competitive antagonist: 2'deoxycytidine (2'DC) but not by its metabolic precursor cytosine (cyt). We suggest that the acute effects of AraC on neurones which are actively growing neurites are the result of interference with lipid metabolism.

Segmental independence and age dependence of neurite outgrowth from embryonic chick sensory neurons.

Targets in limb regions of the chick embryo are further removed from the dorsal root ganglia that innervate them compared with thoracic ganglion-to-target distances. It has been inferred that axons grow into the limb regions two to three times faster than into nonlimb regions. We tested whether the differences were due to intrinsic properties of the neurons located at different segmental levels. Dorsal root ganglia (DRG) were isolated from the forelimb, trunk, and hind limb regions of stage 25-30 embryos. Neurite outgrowth was measured in dissociated cell culture and in cultures of DRG explants. Although there was considerable variability in the amount of neurite outgrowth, there were no substantive differences in the amount or the rate of outgrowth comparing brachial, thoracic, or lumbosacral neurons. The amount of neurite outgrowth in dissociated cell cultures increased with the stage of development. Overall, our data suggest that DRG neurons express a basal amount of outgrowth, which is initially independent of target-derived neurotrophic influences; the magnitude of this intrinsic growth potential increases with stage of development; and the neurons of the DRG are not intrinsically specified to grow neurites at rates that are matched to the distance they are required to grow to make contact with their peripheral targets in vivo. We present a speculative model based on Poisson statistics, which attempts to account for the variability in the amount of neurite outgrowth from dissociated neurons.

Optical measurements of activity-dependent membrane recycling in motor nerve terminals of mammalian skeletal muscle.

Motor nerve terminals in a variety of rat and mouse skeletal muscles were stained in an activity-dependent fashion using the styryl dyes FM1-43 or FM2-10. Low-light video microscopy and digital image processing techniques were used to evaluate destaining of the preparations during application of depolarizing stimuli. Best results were obtained with the mouse triangularis sterni muscle. Quantitative analysis of the destaining of dye-loaded terminals supports the suggestion that FM1-43 stains a recycling membrane compartment, most probably synaptic vesicles. However, the pattern of staining and destaining were not the same as those reported previously for frog neuromuscular junctions. The pattern of nerve terminal staining was less punctate and the rate and amount of activity-dependent destaining were less than in frog muscle. Part of the explanation may be a more acute susceptibility of mammalian terminals to phototoxicity.

Co-existence and elimination of convergent motor nerve terminals in reinnervated and paralysed adult rat skeletal muscle.

1. Experiments were carried out to determine whether neuromuscular synapse elimination can occur in skeletal muscle in the complete absence of conducted neural activity, using reinnervation of partially denervated adult muscle as a paradigm. Partially denervated rat lumbrical muscles were paralysed with a nerve conduction block applied to the sciatic nerve during regeneration of injured sural nerve motor axons. Both intact (lateral plantar nerve) and regenerating motor axons converging on the same muscle fibres were therefore inactive. 2. Paralysed muscles expressed prolonged twitch contractions, low tetanus-to-twitch ratios, prolonged synaptic potentials and marked post-tetanic potentiation of frequency of miniature endplate potentials compared with control muscles and neuromuscular junctions. 3. Isometric tension and intracellular recording data suggest that regenerating axons reinnervated more muscle fibres in paralysed muscles than in controls. A greater proportion of muscle fibres was polyneuronally innervated in the paralysed muscles, but significant numbers of muscle fibres acquired a mononeuronal innervation by regenerated, inactive motor nerve terminals. 4. The data suggest that muscle paralysis enhances the regeneration of motor axons when they grow into partially denervated muscles, but activity-independent competition may also be important in the mechanism of synapse elimination at neuromuscular junctions. The data further imply that when nerve endings expressing identical patterns of activity converge on a postsynaptic cell, Hebbian rules may not be sufficient to predict the outcome of the competition, contrary to specific postulates of the neurotrophic theory of development and maintenance of neural connections.

Culture of isolated embryonic chick dorsal root ganglia at an air-liquid interface: a simple method for studying the mechanism and control of neurite outgrowth.

Extensive neurite outgrowth occurs within 24 h from explants of embryonic chick dorsal root ganglia floated on the surface of serum-free culture medium. The amount of neurite outgrowth was less in culture medium containing serum albumen and varied systematically with the concentration of nerve growth factor (NGF). Compared with outgrowth from floating ganglia, the NGF-dependent outgrowth of neurites from ganglia stuck to coated substrata was much less on polylysine, but outgrowth was more extensive on a laminin-coated substrate. Neurites growing out from floating ganglia showed more fasciculation than those growing out from adherent ganglia. This new, simple preparation provides a serum- and substrate-independent system for studying mechanisms of neurite outgrowth and for quantitative bioassay for potential neurotrophic factors or for factors which influence neurite fasciculation.