Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss.

The muscular dystrophy X-linked mouse (mdx) is the most commonly used preclinical model for Duchenne muscular dystrophy. Although disease progression in the mouse does not perfectly model the human disease, it shares many pathological features. Early characterizations of the model reported severe pathology through early adulthood followed by disease stabilization. As a result, research in the mdx mouse has largely focused on early adulthood. The overarching goal of this study is to improve the understanding of the mdx mouse model by tracking pathological features of the disease throughout life. We performed a thorough characterization of myofiber pathology in mdx mice from 2 weeks to 2 years of age. We report that individual mdx muscle fibers undergo progressive hypertrophy that continues through the lifespan. Despite massive hypertrophy on the myofiber level, we report no hypertrophy on the muscle level. These seemingly contradictory findings are explained by previously underappreciated myofiber loss in mdx mice. We conclude that due to myofiber loss, in combination with the progressive nature of other pathological features, aged mdx muscle tissue provides reliable benchmarks for disease progression that may be valuable in testing the efficacy of therapeutics for Duchenne muscular dystrophy.

Morphological remodeling during recovery of the neuromuscular junction from terminal Schwann cell ablation in adult mice.

Schwann cells (SCs) are integral to the formation and function of the peripheral nervous system (PNS). Exemplifying their importance, the loss or dysfunction of SCs is a feature of a myriad of diseases and conditions that compromise the PNS. Thus, it remains essential to understand the rules that govern the proliferation, differentiation and reconnection of Schwann cells with peripheral axons. Here, we examined the consequences of locally and acutely ablating terminal Schwann cells (tSCs) at the adult mouse neuromuscular junction (NMJ) by using mice expressing diphtheria toxin receptor (DTR) preferentially in tSCs compared to myelinating SCs followed by local application of diphtheria toxin (DTX). After DTX application, tSCs died but, importantly and contrary to expectations, their associated motor axons did not fully degenerate. Within 3 weeks, tSCs returned and reestablished coverage of the synapse with increased numbers. Furthermore, the post-synaptic muscle fibers displayed increased distinct clusters of acetylcholine receptors and axon terminals exhibited numerous terminal varicosities. The lack of degeneration of bare motor axon terminals and the morphological remodeling that occurs upon the return of tSCs to the NMJ may have wider implications for the mechanisms governing tSC occupancy of the adult NMJ and for conditions that adversely affect tSCs.

Exclusive vital labeling of myonuclei for studying myonuclear arrangement in mouse skeletal muscle tissue.

BACKGROUND: The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health, but there is a dearth of in vivo exploration of potential effects of myonuclear organization on the function and regeneration of skeletal muscle because traditional nuclear stains are performed on postmortem tissue. Therefore, we sought a transgenic method to produce a selective and persistent myonuclear label in whole muscles of living mice. METHODS: We bred together a mouse line with skeletal muscle fiber-selective expression of Cre recombinase and a second mouse line with a Cre-inducible fluorescently tagged histone protein to generate a mouse line that produces a myonuclear label suitable for vital imaging and histology of fixed tissue. We tested the effectiveness of this vital label in three conditions known to generate abnormal myonuclear positioning. First, we injured myofibers of young mice with cardiotoxin. Second, this nuclear label was bred into a murine model of Duchenne muscular dystrophy. Finally, we examined old mice from this line that have undergone the natural aging process. Welch's t test was used to compare wild type and transgenic mice. RESULTS: The resulting mouse line transgenically produces a vital red fluorescent label of myonuclei, which facilitates their in vivo imaging in skeletal muscle tissue. Transgenic fluorescent labeling of myonuclei has no significant effect on skeletal muscle function, as determined by twitch and tetanic force recordings. In each muscle examined, including those under damaged, dystrophic, and aged conditions, the labeled myonuclei exhibit morphology consistent with established literature, and reveal a specialized arrangement of subsynaptic myonuclei at the neuromuscular junction. CONCLUSIONS: Taken together, our results demonstrate that this mouse line provides a versatile tool to selectively visualize myonuclei within both living and fixed preparations of healthy, injured, diseased, and aged muscles.

Nerve sprouting capacity in a pharmacologically induced mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.

Schwann cell guidance of nerve growth between synaptic sites explains changes in the pattern of muscle innervation and remodeling of synaptic sites following peripheral nerve injuries.

Terminal Schwann cells (SCs) are nonmyelinating glia that are a prominent component of the neuromuscular junction (NMJ) where motor neurons form synapses onto muscle fibers. These cells play important roles not only in development and maintenance of the neuromuscular synapse but also restoring synaptic function after nerve damage. In response to muscle denervation, terminal SCs undergo dramatic changes in their gene expression patterns as well as in their morphology, such as extending elaborate processes into inter-junctional space. These SC processes serve as a path to guide axon terminal sprouts from nearby innervated junctions, promoting rapid reinnervation of denervated fibers. We studied the role of terminal SCs in synapse reformation by using two different fluorescent proteins to simultaneously label motor axons and SCs; we examined these junctions repeatedly in living animals using a fluorescence microscope. Here, we show that alterations in the patterns of muscle innervation following recovery from nerve injury can be explained by SC guidance of regenerating axons. In turn, this guidance leads to remodeling of the NMJ itself.

Cycles of myofiber degeneration and regeneration lead to remodeling of the neuromuscular junction in two mammalian models of Duchenne muscular dystrophy.

Mice lacking the sarcolemmal protein dystrophin, designated mdx, have been widely used as a model of Duchenne muscular dystrophy. Dystrophic mdx mice as they mature develop notable morphological abnormalities to their neuromuscular junctions, the peripheral cholinergic synapses responsible for activating muscle fibers. Most obviously the acetylcholine receptor aggregates are fragmented into small non-continuous, islands. This contrasts with wild type mice whose acetylcholine receptor aggregates are continuous and pretzel-shaped in appearance. We show here that these abnormalities in mdx mice are also present in a canine model of Duchenne muscular dystrophy and provide additional evidence to support the hypothesis that NMJ remodeling occurs due to myofiber degeneration and regeneration. Using a method to investigate synaptic AChR replacement, we show that neuromuscular junction remodeling in mdx animals is caused by muscle fiber degeneration and regeneration at the synaptic site and is mimicked by deliberate myofiber injury in wild type mice. Importantly, the innervating motor axon plays a crucial role in directing the remodeling of the neuromuscular junction in dystrophy, as has been recorded in aging and deliberate muscle fiber injury in wild type mice. The remodeling occurs repetitively through the life of the animal and the changes in junctions become greater with age.

Schwann cells participate in synapse elimination at the developing neuromuscular junction.

During the initial stages of innervation of developing skeletal muscles, the terminal branches of axons from multiple motor neurons form neuromuscular junctions (NMJs) on a small region of each muscle fiber, the motor endplate. Subsequently, the number of axonal inputs at the endplate region is reduced so that, at maturity, each muscle fiber is innervated by the terminals of a single motor neuron. The Schwann cells associated with the axon terminals are involved in the removal of these synapses but do not select the axon that is ultimately retained on each fiber. Schwann cells perform this function by disconnecting terminal branches from the myofiber surface and by attacking them phagocytically. Here we discuss how this behavior is regulated and argue that such regulation is not unique to development of neuromuscular innervation but is also expressed in the response of the mature NMJ to various manipulations and pathologies.

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

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.

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.

Real-time understanding of lignocellulosic bioethanol fermentation by Raman spectroscopy.

BACKGROUND: A substantial barrier to commercialization of lignocellulosic ethanol production is a lack of process specific sensors and associated control strategies that are essential for economic viability. Current sensors and analytical techniques require lengthy offline analysis or are easily fouled in situ. Raman spectroscopy has the potential to continuously monitor fermentation reactants and products, maximizing efficiency and allowing for improved process control. RESULTS: In this paper we show that glucose and ethanol in a lignocellulosic fermentation can be accurately monitored by a 785 nm Raman spectroscopy instrument and novel immersion probe, even in the presence of an elevated background thought to be caused by lignin-derived compounds. Chemometric techniques were used to reduce the background before generating calibration models for glucose and ethanol concentration. The models show very good correlation between the real-time Raman spectra and the offline HPLC validation. CONCLUSIONS: Our results show that the changing ethanol and glucose concentrations during lignocellulosic fermentation processes can be monitored in real-time, allowing for optimization and control of large scale bioconversion processes.

Changes in aging mouse neuromuscular junctions are explained by degeneration and regeneration of muscle fiber segments at the synapse.

Vertebrate neuromuscular junctions are highly stable synapses, retaining the morphology they achieve in early postnatal development throughout most of life. However, these synapses undergo dramatic change during aging. The acetylcholine receptors (AChRs) change from smooth gutters into fragmented islands, and the nerve terminals change similarly to be varicosities apposed to these islands. These changes have been attributed to a slow deterioration in mechanisms maintaining the synapse. We have used repeated, vital imaging to investigate how these changes occur in the sternomastoid muscle of aging mice. We have found, contrary to expectation, that individual junctions change infrequently, but change, when it occurs, is sudden and dramatic. The change mimics that reported previously for cases in which muscle fibers are deliberately damaged: most of the AChRs present disappear rapidly and are replaced by a new set of receptors that become fragmented. The fiber segment underneath the synapse has centrally located nuclei, showing that this segment has undergone necrosis, quickly regenerated, and been reinnervated with an altered synapse. We show that necrotic events are common in aged muscle and have likely been missed previously as a cause of the alterations in aging because central nuclei are a transient phenomenon and the necrotic events at the junction infrequent. However, the changes are permanent and accumulate over time. Interventions to reduce the neuromuscular changes during aging should likely focus on making muscle fibers resistant to injury.

Nerve terminal growth remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber.

Muscle fibers degenerate and regenerate in response to contractile damage, during aging, and in various muscle diseases that weaken the fibers. It is known that degeneration and regeneration of the segment of the postsynaptic fiber produces dramatic alterations in the neuromuscular junction (NMJ) that forms on the regenerated fiber, but the mechanisms here are incompletely understood. We have used a laser microbeam to damage the postsynaptic fibers at individual NMJs in the sternomastoid muscle of living young adult mice and then followed the synapses vitally over time using fluorescent proteins expressed in motor neurons and glial cells and staining of postsynaptic acetylcholine receptors. We find, in contrast to previous reports, that the mouse nerve terminal retains contact with the synaptic basal lamina marked by cholinesterase staining even in the absence of the target, showing that this terminal does not require a continuous supply of target-derived molecules for its maintenance. Thus, remodeling of the nerve terminal during the period of target absence does not explain the subsequent changes in the new NMJ. Rather, we see that the synapse becomes altered as the new fiber segment regenerates. Mechanisms for remodeling the synapse include failure of the regenerating muscle fiber to contact the old basal lamina and nerve terminal, growth of the nerve terminal and its glia toward the regenerating fiber, and remodeling of the initial contact as the nerve terminal becomes varicose.

Biology and pathology of nonmyelinating Schwann cells.

The CNS contains relatively few unmyelinated nerve fibers, and thus benefits from the advantages that are conferred by myelination, including faster conduction velocities, lower energy consumption for impulse transmission, and greater stability of point-to-point connectivity. In the PNS many fibers or regions of fibers the Schwann do not form myelin. Examples include C fibers nociceptors, postganglionic sympathetic fibers, and the Schwann cells associated with motor nerve terminals at neuromuscular junctions. These examples retain a degree of plasticity and a capacity to sprout collaterally that is unusual in myelinated fibers. Nonmyelin-forming Schwann cells, including those associated with uninjured fibers, have the capacity to act as the "first responders" to injury or disease in their neighborhoods.

A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

Induction of neuregulin signaling in mouse schwann cells in vivo mimics responses to denervation.

Neuregulins play crucial roles in early development of Schwann cells (SCs), but their roles in the activities of SCs during denervation and reinnervation of muscle are less clear. In the present study, the Tet-On system has been used in transgenic mice to enable inducible expression of a mutant, constitutively active neuregulin receptor (ErbB2) in SCs. This induction simulates neuregulin signaling to these cells. Reporter transgenes were used to show a tightly regulated, SC-selective expression in muscle. Induction leads to a number of changes in SCs at neuromuscular junctions that mimic the response to muscle denervation/reinnervation. These include process extension, soma migration, and proliferation. SCs also come to express nestin, a protein characteristic of their reaction to muscle denervation. This activation of SCs results in the sprouting of nerve terminals, and these sprouts follow the extensions of the SCs. However, these sprouts and their associated SCs disappear after the removal of the inducer. Last, induction of the active receptor is sufficient to rescue SCs in neonatal muscle from denervation-induced apoptosis. These findings show that the responses of SCs in muscle to denervation can be explained by induction of an autocrine/paracrine neuregulin signaling cascade suggested by previous molecular studies.

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.

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites.

In partially denervated rodent muscle, terminal Schwann cells (TSCs) located at denervated end plates grow processes, some of which contact neighboring innervated end plates. Those processes that contact neighboring synapses (termed "bridges") appear to initiate nerve terminal sprouting and to guide the growth of the sprouts so that they reach and reinnervate denervated end plates. Studies conducted prior to knowledge of this potential involvement of Schwann cells showed that direct muscle stimulation inhibits terminal sprouting following partial denervation (Brown and Holland, 1979). We have investigated the possibility this inhibition results from an alteration in the growth of TSC processes. We find that stimulation of partially denervated rat soleus muscle does not alter the length or number of TSC processes but does reduce the number of TSC bridges. Stimulation also reduces the number of TSC bridges that form between end plates during reinnervation of a completely denervated muscle. The nerve processes ("escaped fibers") that normally grow onto TSC processes during reinnervation are also reduced in length. Therefore, stimulation alters at least two responses to denervation in muscles: (1) the ability of TSC processes to form or maintain bridges with innervated synaptic sites, and (2) the growth of axons along processes extended by TSCs.