Richard R. Ribchester

The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (WldS) nerves

BackgroundThe progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice.ResultsIn wild-type nerves, we directly observed partially fragmented axons (average 5.3%) among a majority of fully intact or degenerated axons 37–42 h after transection and 40–44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all WldS axons were partially fragmented 15–25 days after nerve lesion, WldS axons degenerated anterogradely independent of lesion type, and signs of degeneration increased gradually along the nerve instead of abruptly. Furthermore, the first signs of degeneration were short constrictions, not complete breaks.ConclusionsWe conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of trophic or regulatory factors by axonal transport triggers degeneration. WldS axons, once they finally degenerate, do so by a fundamentally different mechanism, indicated by differences in the rate, direction and abruptness of progression, and by different early morphological signs of degeneration. These observations suggest that WldS axons undergo a slow anterograde decay as axonal components are gradually depleted, and do not simply follow the degeneration pathway of wild-type axons at a slower rate.

Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves

Nerve impulses are propagated at nodes of Ranvier in the myelinated nerves of vertebrates. Internodal distances have been proposed to affect the velocity of nerve impulse conduction; however, direct evidence is lacking, and the cellular mechanisms that might regulate the length of the myelinated segments are unknown. Ramón y Cajal described longitudinal and transverse bands of cytoplasm or trabeculae in internodal Schwann cells and suggested that they had a nutritive function. Here we show that internodal growth in wild-type nerves is precisely matched to nerve extension, but disruption of the cytoplasmic bands in Periaxin-null mice impairs Schwann cell elongation during nerve growth. By contrast, myelination proceeds normally. The capacity of wild-type and mutant Schwann cells to elongate is cell-autonomous, indicating that passive stretching can account for the lengthening of the internode during limb growth. As predicted on theoretical grounds, decreased internodal distances strikingly decrease conduction velocities and so affect motor function. We propose that microtubule-based transport in the longitudinal bands of Cajal permits internodal Schwann cells to lengthen in response to axonal growth, thus ensuring rapid nerve impulse transmission.

Progressive abnormalities in skeletal muscle and neuromuscular junctions of transgenic mice expressing the Huntington's disease mutation.

Huntingtons disease (HD) is a neurodegenerative disorder with complex symptoms dominated by progressive motor dysfunction. Skeletal muscle atrophy is common in HD patients. Because the HD mutation is expressed in skeletal muscle as well as brain, we wondered whether the muscle changes arise from primary pathology. We used R6/2 transgenic mice for our studies. Unlike denervation atrophy, skeletal muscle atrophy in R6/2 mice occurs uniformly. Paradoxically however, skeletal muscles show age‐dependent denervation‐like abnormalities, including supersensitivity to acetylcholine, decreased sensitivity to µ‐conotoxin, and anode‐break action potentials. Morphological abnormalities of neuromuscular junctions are also present, particularly in older R6/2 mice. Severely affected R6/2 mice show a progressive increase in the number of motor endplates that fail to respond to nerve stimulation. Surprisingly, there was no constitutive sprouting of motor neurons in R6/2 muscles, even in severely atrophic muscles that showed other denervation‐like characteristics. In fact, there was an age‐dependent loss of regenerative capacity of motor neurons in R6/2 mice. Because muscle fibers appear to be released from the activity‐dependent cues that regulate membrane properties and muscle size, and motor axons and nerve terminals become impaired in their capacity to release neurotransmitter and to respond to stimuli that normally evoke sprouting and adaptive reinnervation, we speculate that in these mice there is a progressive dissociation of trophic signalling between motor neurons and skeletal muscle. However, irrespective of the cause, the abnormalities at neuromuscular junctions we report here are likely to contribute to the pathological phenotype in R6/2 mice, particularly in late stages of the disease.

Compartmental neurodegeneration and synaptic plasticity in the Wlds 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 Wlds 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 Wlds mutant, or transgenic mice that express the Wlds gene.

WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice

The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration.

Age-dependent synapse withdrawal at axotomised neuromuscular junctions in Wld s mutant and Ube4b/Nmnat transgenic mice

Axons in WldS mutant mice are protected from Wallerian degeneration by overexpression of a chimeric Ube4b/Nmnat (Wld) gene. Expression of Wld protein was independent of age in these mice. However we identified two distinct neuromuscular synaptic responses to axotomy. In young adult Wlds mice, axotomy induced progressive, asynchronous synapse withdrawal from motor endplates, strongly resembling neonatal synapse elimination. Thus, five days after axotomy, 50–90 % of endplates were still partially or fully occupied and expressed endplate potentials (EPPs). By 10 days, fewer than 20 % of endplates still showed evidence of synaptic activity. Recordings from partially occupied junctions indicated a progressive decrease in quantal content in inverse proportion to endplate occupancy. In Wlds mice aged > 7 months, axons were still protected from axotomy but synapses degenerated rapidly, in wild‐type fashion: within three days less than 5 % of endplates contained vestiges of nerve terminals. The axotomy‐induced synaptic withdrawal phenotype decayed with a time constant of ∼30 days. Regenerated synapses in mature Wlds mice recapitulated the juvenile phenotype. Within 4–6 days of axotomy 30–50 % of regenerated nerve terminals still occupied motor endplates. Age‐dependent synapse withdrawal was also seen in transgenic mice expressing the Wld gene. Co‐expression of Wld protein and cyan fluorescent protein (CFP) in axons and neuromuscular synapses did not interfere with the protection from axotomy conferred by the Wld gene. Thus, Wld expression unmasks age‐dependent, compartmentally organised programmes of synapse withdrawal and degeneration.

A rat model of slow Wallerian degeneration (WldS) with improved preservation of neuromuscular synapses

The slow Wallerian degeneration phenotype, WldS, which delays Wallerian degeneration and axon pathology for several weeks, has so far been studied only in mice. A rat model would have several advantages. First, rats model some human disorders better than mice. Second, the larger body size of rats facilitates more complex surgical manipulations. Third, rats provide a greater yield of tissue for primary culture and biochemical investigations. We generated transgenic WldS rats expressing the Ube4b/Nmnat1 chimeric gene in the central and peripheral nervous system. As in WldS mice, their axons survive up to 3 weeks after transection and remain functional for at least 1 week. Protection of axotomized nerve terminals is stronger than in mice, particularly in one line, where 95–100% of neuromuscular junctions remained intact and functional after 5 days. Furthermore, the loss of synaptic phenotype with age was much less in rats than in mice. Thus, the slow Wallerian degeneration phenotype can be transferred to another mammalian species and synapses may be more effectively preserved after axotomy in species with longer axons.

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 α-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.

Neuroprotection after Transient Global Cerebral Ischemia in Wlds Mutant Mice

The Wlds mouse mutant demonstrates a remarkable phenotype of delayed axonal and synaptic degeneration after nerve lesion. In this study, the authors tested the hypothesis that expression of Wld protein is neuroprotective in an in vivo mouse model of global cerebral ischemia. This model is associated with selective neuronal degeneration in specific brain regions such as the caudate nucleus and CA2 hippocampal pyramidal cell layer. The extent of neuronal damage was quantified in Wlds compared to wild-type mice after an identical episode of global cerebral ischemia. The results demonstrated a significant and marked reduction in the extent of neuronal damage in Wlds as compared to wild-type C57Bl/6 mice. In the caudate nucleus, Wld expression significantly reduced the percentage of ischemic neuronal damage after global ischemia (Wlds, 27.7 ± 16.8%; wild-type mice, 58.7 ± 32.3%; P = 0.036). Similarly, in the CA2 pyramidal cell layer, there was a significant reduction of neuronal damage in the Wlds mice as compared to wild-type mice after ischemia (Wlds, 17.7 ± 23.0%; wild-type mice, 41.9 ± 28.0%; P < 0.023). Thus, these results clearly demonstrate that the Wld gene confers substantial neuroprotection after cerebral ischemia, and suggest a new role to that previously described for Wlds.

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 WldS prevent this rise. These data indicate that the mechanism by which NMNAT and the related WldS 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.