Michelle Mikesh

Fluorescent Proteins Expressed in Mouse Transgenic Lines Mark Subsets of Glia, Neurons, Macrophages, and Dendritic Cells for Vital Examination

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Terminal Schwann Cells Participate in Neuromuscular Synapse Remodeling during Reinnervation following Nerve Injury

Schwann cells (SCs) at neuromuscular junctions (NMJs) play active roles in synaptic homeostasis and repair. We have studied how SCs contribute to reinnervation of NMJs using vital imaging of mice whose motor axons and SCs are transgenically labeled with different colors of fluorescent proteins. Motor axons most commonly regenerate to the original synaptic site by following SC-filled endoneurial tubes. During the period of denervation, SCs at the NMJ extend elaborate processes from the junction, as shown previously, but they also retract some processes from territory they previously occupied within the endplate. The degree of this retraction depends on the length of the period of denervation. We show that the topology of the remaining SC processes influences the branching pattern of regenerating axon terminals and the redistribution of acetylcholine receptors (AChRs). Upon arriving at the junction, regenerating axons follow existing SC processes within the old synaptic site. Some of the AChR loss that follows denervation is correlated with failure of portions of the old synaptic site that lack SC coverage to be reinnervated. New AChR clustering is also induced by axon terminals that follow SC processes extended during denervation. These observations show that SCs participate actively in the remodeling of neuromuscular synapses following nerve injury by their guidance of axonal reinnervation.

Terminal Schwann Cells Participate in the Competition Underlying Neuromuscular Synapse Elimination

The competitive processes that result in elimination/pruning of developing synapses are incompletely understood. Serial electron microscopy was used to image postnatal mouse neuromuscular junctions where elimination is well studied and events at every synaptic contact can be examined. Glial or Schwann cells (SCs) are shown to have two activities during elimination: their processes separate nerve terminals from each other and from the muscle fiber; they contact the plaque of acetylcholine receptors, apposing this surface as closely as the nerve, limiting the area where synaptic transmission occurs. SCs phagocytose nerve terminals contacting the muscle fiber. This phagocytosis involves all axons; SCs are not selecting the winner but rather driving turnover. Previous modeling of stochastic turnover and reoccupation of nerve contacts shows that single innervation of synaptic sites can result. Thus, our study shows roles of SCs in neuromuscular development beyond the previous demonstration of consumption of synaptic inputs after their elimination.

Polyethylene glycol‐fused allografts produce rapid behavioral recovery after ablation of sciatic nerve segments

Restoration of neuronal functions by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed peripheral nerves takes many weeks or months, if it occurs at all, especially after ablation of nerve segments. Distal segments of severed axons typically degenerate in 1–3 days. This study shows that Wallerian degeneration can be prevented or retarded, and lost behavioral function can be restored, following ablation of 0.5–1‐cm segments of rat sciatic nerves in host animals. This is achieved by using 0.8–1.1‐cm microsutured donor allografts treated with bioengineered solutions varying in ionic and polyethylene glycol (PEG) concentrations (modified PEG‐fusion procedure), being careful not to stretch any portion of donor or host sciatic nerves. The data show that PEG fusion permanently restores axonal continuity within minutes, as initially assessed by action potential conduction and intracellular diffusion of dye. Behavioral functions mediated by the sciatic nerve are largely restored within 2–4 weeks, as measured by the sciatic functional index. Increased restoration of sciatic behavioral functions after ablating 0.5–1‐cm segments is associated with greater numbers of viable myelinated axons within and distal to PEG‐fused allografts. Many such viable myelinated axons are almost certainly spared from Wallerian degeneration by PEG fusion. PEG fusion of donor allografts may produce a paradigm shift in the treatment of peripheral nerve injuries.

Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions

Significance Refinement of synaptic connections occurs throughout the nervous system and is essential for its proper function. Significant gaps remain in our understanding of the mechanisms that mediate pruning of synaptic connections—a form of synaptic plasticity known as synapse elimination. Recently it has become clear there is significant glial cell involvement. The present study of the rodent neuromuscular junction addresses two outstanding questions involving this glial involvement: which molecules determine the Schwann cell behavior present during pruning, and whether these behaviors actually alter synaptic structure. Our findings identify axon-tethered neuregulin1 as a molecular determinant for Schwann cell-driven neuromuscular synaptic plasticity. Synaptic connections in the nervous system are rearranged during development and in adulthood as a feature of growth, plasticity, aging, and disease. Glia are implicated as active participants in these changes. Here we investigated a signal that controls the participation of peripheral glia, the terminal Schwann cells (SCs), at the neuromuscular junction (NMJ) in mice. Transgenic manipulation of the levels of membrane-tethered neuregulin1 (NRG1-III), a potent activator of SCs normally presented on motor axons, alters the rate of loss of motor inputs at NMJs during developmental synapse elimination. In addition, NMJs of adult transgenic mice that expressed excess axonal NRG1-III exhibited continued remodeling, in contrast to the more stable morphologies of controls. In fact, synaptic SCs of these adult mice with NRG1-III overexpression exhibited behaviors evident in wild type neonates during synapse elimination, including an affinity for the postsynaptic myofiber surface and phagocytosis of nerve terminals. Given that levels of NRG1-III expression normally peak during the period of synapse elimination, our findings identify axon-tethered NRG1 as a molecular determinant for SC-driven neuromuscular synaptic plasticity.

The curious ability of polyethylene glycol fusion technologies to restore lost behaviors after nerve severance

Traumatic injuries to PNS and CNS axons are not uncommon. Restoration of lost behaviors following severance of mammalian peripheral nerve axons (PNAs) relies on regeneration by slow outgrowths and is typically poor or nonexistent when after ablation or injuries close to the soma. Behavioral recovery after severing spinal tract axons (STAs) is poor because STAs do not naturally regenerate. Current techniques to enhance PNA and/or STA regeneration have had limited success and do not prevent the onset of Wallerian degeneration of severed distal segments. This Review describes the use of a recently developed polyethylene glycol (PEG) fusion technology combining concepts from biochemical engineering, cell biology, and clinical microsurgery. Within minutes after microsuturing carefully trimmed cut ends and applying a well‐specified sequence of solutions, PEG‐fused axons exhibit morphological continuity (assessed by intra‐axonal dye diffusion) and electrophysiological continuity (assessed by conduction of action potentials) across the lesion site. Wallerian degeneration of PEG‐fused PNAs is greatly reduced as measured by counts of sensory and/or motor axons and maintenance of axonal diameters and neuromuscular synapses. After PEG‐fusion repair, cut‐severed, crush‐severed, or ablated PNAs or crush‐severed STAs rapidly (within days to weeks), more completely, and permanently restore PNA‐ or STA‐mediated behaviors compared with nontreated or conventionally treated animals. PEG‐fusion success is enhanced or decreased by applying antioxidants or oxidants, trimming cut ends or stretching axons, and exposure to Ca2+‐free or Ca2+‐containing solutions, respectively. PEG‐fusion technology employs surgical techniques and chemicals already used by clinicians and has the potential to produce a paradigm shift in the treatment of traumatic injuries to PNAs and STAs.

Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves.

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1–3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca2+‐free saline, methylene blue, PEG in distilled water, and finally Ca2+‐containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer‐term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut‐severed axonal ends to Ca2+‐containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut‐severed axons decrease PEG fusion success. Conversely, trimming cut‐severed ends in Ca2+‐free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut‐severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries.

Polyethylene glycol-fusion retards Wallerian degeneration and rapidly restores behaviors lost after nerve severance.

Some biological uses of polyethylene glycol (PEG): The use of PEG as a membrane fusogen was first reported in 1976 with the creation of cell hybrids, formed by suspending two cell lines in a 50% w/w solution of PEG in water. More recently, direct application of PEG has been found to seal off small holes in axons after complete transections of cultured neurites in a procedure referred to as “PEG-sealing” (Spaeth et al., 2012). The term PEG-sealing is also used to describe intravenous injection of PEG or micelles/nano-particles associated with PEG (reviewed by Jin, 2014). The latter “PEG-sealing” in vivo procedures typically produce low concentrations of PEG in body fluids associated with modest increases in behavioral recovery many weeks post injury, presumably by neuroprotective effects that rescue damaged neurons from cell death, rather than by preventing or retarding Wallerian degeneration (Jin, 2014).

Robinson and madison have published no data on whether polyethylene glycol fusion repair prevents reinnervation accuracy in rat peripheral nerve

Robinson and Madison (2016) have recently published an article entitled Polyethylene glycol fusion repair prevents reinnervation accuracy in rat peripheral nerve in the Journal of Neuroscience Research. Unfortunately, the title of the article reflects only one of many possible interpretations of their data. That is, 1) the lack of critical controls in methodologies, and 2) limitations in the experimental design of their protocols lead them to 3) incorrect conclusions that are unfortunately reflected in 4) their very inappropriate and inaccurate title.