Axotomy‐dependent and ‐independent synapse elimination in organ cultures of Wld^s mutant mouse skeletal muscle

Abstract

Progressive "dying back" neurodegenerative diseases are debilitating due to loss of connectivity after nerve terminal and axonal withdrawal, which impairs peripheral nerve function and leads ultimately to neuronal cell death. The mutant mouse (Wallerian degeneration slow; Wld s ) provides an accessible model system to understand orthograde and retrograde degeneration, because in these mice axotomy induces slow, progressive withdrawal of nerve terminals from motor endplates. Axon degeneration itself is about 10 times slower than in wildtype mice. We describe an organ culture paradigm that permits direct observation of the progressive changes in morphology of neuromuscular junctions in Wld s mutant mice. Normal nerve terminal and motor endplate morphology were maintained at most Wld s neuromuscular junctions for up to 72 hr in vitro. At others, synaptic boutons were removed from postsynaptic junctional folds in piecemeal fashion, as observed in adults in vivo. By contrast, nerve terminals degenerated rapidly and synchronously in wild-type muscle cultures, resembling Wallerian degeneration in vivo. These observations confirm that in Wld s mice, axotomy triggers a mechanism of nerve-terminal withdrawal that seems qualitatively different from that in wild-type animals. The piecemeal dismantling of presynaptic terminals resembles that occurring during neonatal synapse elimination. Organ cultures of neonatal Wld s muscle maintained for 1-2 days in vitro also showed no evidence of synaptic terminal degeneration, but elimination of polyneuronal innervation progressed in vitro at approximately the same rate as in vivo. Taken together, the data suggest that both natural and axotomy-induced forms of synapse withdrawal may be accessible to continuous observation and analysis, in organ-cultures of Wld S mouse muscles. This offers several advantages over repeated visualization of synaptic remodeling that has thus far been possible only in vivo.

Dynamic remodelling of synapses can occur in the absence of the parent cell body

“…Significantly, during the course of these studies we found no evidence of gross, injury-induced degenerative events up to and including 5 hr after axons were cut during dissection. This agrees with our previous findings which indicated that Wallerian degeneration does not begin until at least 12 hours after dissection [ 6 ]. Specifically, we saw no evidence of terminal Schwann cells engulfing degenerating nerve terminal boutons in-situ, mitochondrial degradation or cytoplasmic vacuolation [ 32 , 38 ], which would indicate degenerative events.…”

“…In these experiments we show that a brief pharmacological signal can trigger rapid and significant morphological events in cell fragments consisting of isolated nerve terminals and attached distal axon stumps. We suggest that the morphology of these events most closely resembles nerve terminal retraction during developmental synapse elimination [ 4 , 6 , 33 , 36 ], following nerve section in the slow Wallerian degeneration mouse [ 6 ] and in motor neurone disease [ 17 ]. We also observed fragmentation of pre and/or postsynaptic sites at a small number of neuromuscular junctions and interestingly similar synaptic responses have also been observed during developmental synapse elimination [ 37 ] and in mouse models of spinal muscular atrophy [ 14 ].…”

“…Retraction of nerve terminals is a fundamental event in the development and maintenance of the nervous system [ 1 , 2 ], occurring when exuberant neuronal projections are pared back by activity-dependent processes. These events have been extensively studied at the neuromuscular junction (NMJ) during development [ 3 , 4 ], re-innervation [ 5 ] and following axotomy [ 6 ]. Here, competing nerve terminals at muscle motor endplates are withdrawn bouton-by-bouton in an apparently controlled, piecemeal fashion until the last bouton withers and is re-absorbed into the parent axon [ 4 , 7 , 8 ].…”

“…Adult axons and terminals appear to contain little if any machinery for protein synthesis, but the first signs of regenerative sprouting occurs within a day of axotomy [ 20 , 21 ], which appears too rapid for communication from the site of injury to the cell body and back, even by fast axonal transport. Neonatal synapse elimination can proceed subsequent to axotomy [ 6 ] and co-ordinated structural changes at presynaptic nerve terminals (retraction) can occur in the absence of parent cell somas. Axons disconnected from their cell bodies in vitro can assemble new growth cones at lesion sites [ 22 - 24 ] and transected axons are able to mount a regenerative response in the absence of cell somas [ 25 ].…”

Rapid loss of motor nerve terminals following hypoxia–reperfusion injury occurs via mechanisms distinct from classic Wallerian degeneration

“…We identified nerve terminal morphologies indicative of Wallerian-like fragmentation processes as well as asynchronous nerve terminal retraction. Although nerve terminal retraction is normally a mechanism of loss associated with developmental synapse elimination (Sanes & Lichtman, 1999;Keller-Peck et al 2001;Gillingwater et al 2002;Walsh & Lichtman, 2003;Parson et al 2004), there are increasing numbers of reports showing that withdrawal can occur in response to injury and disease in adult motor nerve terminals (Gillingwater et al 2002;Fischer et al 2004;Bettini et al 2007;Murray et al 2008). The finding that nerve terminal function may be disrupted in advance of disassembly suggests that future experiments are required to define accurately the spatio-temporal characteristics of hypoxiareperfusion injury.…”