Synapse withdrawal at the neuromuscular junction in a mouse with slow Wallerian degeneration (WLDs)

Motor nerve injury results in a sequence of events known as Wallerian degeneration, which takes 1 - 2 days in rodents. Wallerian degeneration is significantly delayed in the naturally occurring C57BL/Wldˢ mutant mouse. The ability to produce compound action potentials and the degradation of axonal material are delayed for up to 3 weeks following nerve injury.This thesis aims to examine the processes that occur after nerve section at neuromuscular junctions of mutant Wldˢ and normal mice. Functional morphological measurement of actively recycling synaptic vesicles at wild type nerve terminals using the vital dye FM1-43 indicated that the loss of the ability to recycle synaptic vesicles occurred within 24 hours, coinciding with disruption of neurofilament and SV2 proteins (stained using immunocytochemical methods). In contrast, the degeneration of nerve terminals in Wldˢ mice following nerve injury was significantly delayed, and once initiated was further slowed, degeneration occurring between 3 and 10 days after axotomy. The morphological appearance of Wldˢ nerve terminals after lesion differed greatly from that of degenerating wild type terminals: Wldˢ terminals appeared to be retracting from the endplate region rather than simply degenerating. Electrophysiological recordings from degenerating junctions demonstrate that the delay of degeneration/withdrawal from nerve terminals of Wldˢ mice is accompanied by a coincident loss of synaptic function. These data indicate that, as in normal mice, the first structural and functional changes to take place after nerve injury in mutant Wldˢ mice is the disruption of nerve terminals at the NMJ. This thesis proposes that nerve terminal retraction in the mutant mouse is caused by a withdrawal mechanism similar to that observed during synapse elimination, a naturally occurring activity dependant developmental phenomenon, rather than by classical Wallerian degeneration as previously assumed. To test this hypothesis, botulinum toxin (which abolishes neurotransmitter release) was administered to the nerve terminal region at the same time as the nerves were lesioned. The results indicate that nerve terminal withdrawal still occurs, but that it is reduced and further delayed in muscles treated with botulinum toxin by 1.14 days, in comparison with denervated alone Wldˢ muscles.Additional studies assessed the role of Schwann cells in nerve terminal withdrawal. Terminal Schwann cells that overly the NMJ sprout in response to denervation, and have been shown to guide axonal processes back to endplates during reinnervation. Experiments were conducted to evaluate the Schwann cell reaction to the absence of degeneration after nerve section, as observed during the first few days after axotomy in the Wldˢ mouse. Schwann cell sprouts were present at various endplates in the mutant mouse after nerve injury. However, when compared with denervated wild-type controls there were fewer Schwann cell sprouts in the Wldˢ and sprouting in general appeared less extensive. Thus, the presence of an intact nerve terminal and spontaneous activity are sufficient to delay, but not abolish, Schwann cell sprouting in the Wldˢ mouse after nerve lesion.In sum, these studies provide an ideal opportunity for the further investigation of the molecular and cellular mechanisms of synapse withdrawal

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

Axonal and neuromuscular synaptic phenotypes in Wld(S), SOD1(G93A) and ostes mutant mice identified by fiber-optic confocal microendoscopy

We used live imaging by fiber-optic confocal microendoscopy (CME) of yellow fluorescent protein (YFP) expression in motor neurons to observe and monitor axonal and neuromuscular synaptic phenotypes in mutant mice. First, we visualized slow degeneration of axons and motor nerve terminals at neuromuscular junctions following sciatic nerve injury in WldS mice with slow Wallerian degeneration. Protection of axotomized motor nerve terminals was much weaker in WldS heterozygotes than in homozygotes. We then induced covert modifiers of axonal and synaptic degeneration in heterozygous WldS mice, by N-ethyl-Nnitrosourea (ENU) mutagenesis, and used CME to identify candidate mutants that either enhanced or suppressed axonal or synaptic degeneration. From 219 of the F1 progeny of ENU-mutagenized BALB/c mice and thy1.2-YFP16/WldS mice, CME revealed six phenodeviants with suppression of synaptic degeneration. Inheritance of synaptic protection was confirmed in three of these founders, with evidence of Mendelian inheritance of a dominant mutation in one of them (designated CEMOP_S5). We next applied CME repeatedly to living WldS mice and to SOD1G93A mice, an animal model of motor neuron disease, and observed degeneration of identified neuromuscular synapses over a 1–4 day period in both of these mutant lines. Finally, we used CME to observe slow axonal regeneration in the ENU-mutant ostes mouse strain. The data show that CME can be used to monitor covert axonal and neuromuscular synaptic pathology and, when combined with mutagenesis, to identify genetic modifiers of its progression in vivo

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

AbstractActivity 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 (WldS) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32°C for up to 48h. About 90% of fibers from WldS mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100Hz: 1s/100s) reduced synaptic protection in WldS preparations by about 50%. This effect was abolished in reduced Ca2+ solutions. Next, we assayed FDB and DL innervation after 7days 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 5day-axotomized muscles from saline-control-treated WldS mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4weeks prior to axotomy. Surprisingly, exercising WldS mice ad libitum for 4weeks 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

Protection of neuromuscular sensory endings by the WldS gene

The compartmental hypothesis of neurodegeneration proposes that the neurone, long recognized to consist of morphologically and functionally distinct compartments, also houses distinct degeneration mechanisms for the soma, axon and nerve endings. Support for this hypothesis is provided by the phenomenon of the WldS (for Wallerian Degeneration, slow) mouse, a mutant in which axons survive several weeks after transection, rather than degenerating within 24-48 hours as in wild type mice, by virtue of expression of a chimeric Nmnat1/Ube4b protein. In this thesis I used the WldS-mouse to re-examine and extend the theory of compartmental neurodegeneration by focusing specifically on sensory axons and endings; and finally by considering a fourth compartment, the dendrites. The first part of this thesis reports that Ia afferent axons and their annulospiral endings are robustly protected from degeneration in WldS mice. Homozygous or heterozygous WldS mice crossbred with transgenic mice expressing fluorescent protein in neurones were sacrificed at various times after sciatic nerve transection. Fluorescence microscopy of whole mount preparations of lumbrical muscles in these mice revealed excellent preservation of annulospiral endings on muscle spindles for at least 10 days after axotomy. No significant difference was detected in the protection with age or gene copy-number in contrast to the protection of motor nerve terminals, which degenerate rapidly in heterozygote and aged homozygote WldS mice. In an attempt to explain the difference in motor and sensory protection by WldS, examination of three hypotheses was undertaken: a) differences in protein expression, tested by western blot and immunohistochemistry; b) differences in the degree of neuronal branching, tested through examination of g-motor axons and endings which have a degree of branching intermediate to motor and sensory neurons; and c) differences in the activity in the disconnected stumps, through primary culture of the saphenous and phrenic nerve, selected because they comprise largely pure sensory and motor axons respectively. The data suggest that none of these hypotheses provides a sufficient explanation for the difference between sensory and motor protection by WldS. The last part of this thesis attempts to extend the theory of compartmental degeneration. I examine a system for investigation of WldS-mediated protection of dendrites. In preliminary experiments retinal explants from transgenic mice expressing YFP in a subset of retinal ganglion-cell neurones were cultured. The dendritic arbours of these cells were shown to be amenable for repeated visualization and accessible to injury and monitoring of degeneration. Overall the data in this thesis suggest that the level of WldS -mediated protection conferred to an axon or axonal endings varies between different neuronal types. This has implications for the potential applications of WldS research to clinical problems. Specifically, the data imply that sensory neuropathies may benefit more than motor neuropathies from treatments based on the protective effects of WldS. These findings in sensory neurones also challenge some of the assumptions made about WldS- mediated protection of neurones, for example the extent of the age-effect on axonal endings. Further investigation of WldS-mediated protection in the CNS could give renewed impetus to attempts to discover targets for treatment in common neurodegenerative diseases. Finally, a system for investigation of dendritic degeneration has been piloted, suggesting that molecules involved in the degeneration of dendrites or in protection from this degeneration may be amenable to investigation in this system, prospectively extending the compartmental hypothesis of neuronal degeneration

Some reminiscences on studies of age-dependent and activity-dependent degeneration of sensory and motor endings in mammalian skeletal muscle

I present here an overview of research on the biology of neuromuscular sensory and motor endings that was inspired and influenced partly by my educational experience in the Department of Zoology at the University of Durham, from 1971 to 1974. I allude briefly to neuromuscular synaptic structure and function in dystrophic mice, influences of activity on synapse elimination in development and regeneration, and activity-dependent protection and degeneration of neuromuscular junctions in Wld(S) mice

Role of activity in neuromuscular synaptic degeneration: insights from Wlds mice

The nervous system is a dynamic structure. Both during development and in the adult, synapses display activity-dependent plasticity which can modify their structure and function. In the neonate, activity influences the stability of functional connections between the muscle and nerve. In adults, the process of neurotransmitter release and the structure of the postsynaptic muscle can also be altered by external stimuli such as exercise. It is important to understand this plasticity of the neuromuscular system, the ways in which it can be modified, and its relationship to the maintenance or degeneration of synapses. After injury, peripheral nerve undergoes Wallerian Degeneration, during which the connections between axons and muscle fibres are lost, followed by the fragmentation of the nerve itself. The primary goal of this thesis was to determine whether activity modulates this process; that is, whether enhancing or reducing neuromuscular activity creates a susceptibility to degeneration or alternatively provides any protection against it. Developing greater understanding of this process is essential in relation to neurodegenerative disorders in which the benefits of activity, in the form of exercise, are controversial. Using Wlds mice, in which synaptic degeneration occurs approximately ten times more slowly than normal after nerve injury, I investigated the influence of both decreased (tetrodotoxin induced paralysis) and increased (voluntary wheel running) activity in vivo on this process. Paralysis prior to axotomy resulted in a significant increase in the rate of synapse degeneration. Using a novel method of repeatedly visualising degenerating synapses and axons in vivo I also established that this effect was specific to the synapse, as it did not affect the degeneration of axons. In contrast, voluntary wheel running had no effect on the rate of either axonal or neuromuscular synapse degeneration, but induced a slight modification of neuromuscular transmission. To provide a more stringent test I developed a novel assay based on overnight, ex vivo incubation of nerve-muscle preparations at 32°C. I first demonstrated that this procedure separates the different degeneration time courses for neuromuscular synapse degeneration in wild-type and Wlds preparations. I then extended the study to investigate further ways of modulating synaptic degeneration. First, I tested the effects of electrical stimulation. Intermittent high frequency (100Hz) stimulation reduced the level of protection. Finally, I tested the effects of NAMPT enzymatic inhibitor FK866 on synaptic degeneration. Interestingly, the synaptic protection observed in Wlds muscles was enhanced in the presence of FK866. The results of my findings are relevant to understanding the plasticity of synapses and its relationship to degeneration. Together, these studies highlight the potential of genetic and epigenetic factors, including activity, to regulate neuromuscular synapse degeneration. My study also provides proof of concept for a novel organotypic culture system in which to identify pharmacological modulators of synaptic degeneration that could form part of a second-line screen for neuroprotective compounds or phenotypes. My findings may be viewed in the wide context of neurodegenerative disease, since synaptic use or disuse is widely thought to influence susceptibility, onset and progression in such disorders