Skeletal muscle reprogramming enhances reinnervation after peripheral nerve injury.

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. The onset of PNI is characterized by nerve degeneration distal to the nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generated a mouse model in which NANOG, a pluripotency-associated transcription factor can be expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulated the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression led to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation, and downregulation of key muscle atrophy genes. Further, NANOG mice demonstrated extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice showed greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming the muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle.

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

Blockage of neuromuscular glutamate receptors impairs reinnervation following nerve crush in adult mice.

Motor axons in peripheral nerves are capable of regeneration following injury. However, complete recovery of motor function is rare, particularly when reinnervation is delayed. We have previously found that glutamate receptors play a crucial role in the successful innervation of muscle during mouse development. In particular, blocking N-methyl-D-aspartate (NMDA) receptor activity delays the normal elimination of excess innervation of each neuromuscular junction. Here, we use behavioral, immunohistochemical, electrophysiological, and calcium imaging methods to test whether glutamate receptors play a similar role in the transition from polyneuronal to mono-innervation and in recovery of function following peripheral nerve injury in mature muscle.

The Role of Deimination in Regenerative Reprogramming of Neurons.

Neurons from the adult central nervous system (CNS) demonstrate limited mRNA transport and localized protein synthesis versus developing neurons, correlating with lower regenerative capacity. We found that deimination (posttranslational conversion of protein-bound arginine into citrulline) undergoes upregulation during early neuronal development while declining to a low basal level in adults. This modification is associated with neuronal arborization from amphibians to mammals. The mRNA-binding proteins (ANP32a, REF), deiminated in neurons, have been implicated in local protein synthesis. Overexpression of the deiminating cytosolic enzyme peptidyl arginine deiminase 2 in nervous systems results in increased neuronal transport and neurite outgrowth. We further demonstrate that enriching deiminated proteins rescues transport deficiencies both in primary neurons and mouse optic nerve even in the presence of pharmacological transport blockers. We conclude that deimination promotes neuronal outgrowth via enhanced transport and local protein synthesis and represents a new avenue for neuronal regeneration in the adult CNS.

Neuromuscular NMDA Receptors Modulate Developmental Synapse Elimination.

UNLABELLED: At birth, each mammalian skeletal muscle fiber is innervated by multiple motor neurons, but in a few weeks, all but one of those axons retracts (Redfern, 1970) and differential activity between inputs controls this phenomenon (Personius and Balice-Gordon, 2001; Sanes and Lichtman, 2001; Personius et al., 2007; Favero et al., 2012). Acetylcholine, the primary neuromuscular transmitter, has long been presumed to mediate this activity-dependent process (O'Brien et al., 1978), but glutamatergic transmission also occurs at the neuromuscular junction (Berger et al., 1995; Grozdanovic and Gossrau, 1998; Mays et al., 2009). To test the role of neuromuscular NMDA receptors, we assessed their contribution to muscle calcium fluxes in mice and tested whether they influence removal of excess innervation at the end plate. Developmental synapse pruning was slowed by reduction of NMDA receptor activation or expression and by reduction of glutamate production. Conversely, pruning is accelerated by application of exogenous NMDA. We also found that NMDA induced increased muscle calcium only during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and synaptic pruning during development. SIGNIFICANCE STATEMENT: In normal adult muscle, each muscle fiber is innervated by a single axon, but at birth, fibers are multiply innervated. Elimination of excess connections requires neural activity; because the neuromuscular junction (NMJ) is a cholinergic synapse, acetylcholine has been assumed to be the critical mediator of activity. However, glutamate receptors are also expressed at the NMJ. We found that axon removal in mice is slowed by pharmacological and molecular manipulations that decrease signaling through neuromuscular NMDA receptors, whereas application of exogenous NMDA at the NMJ accelerates synapse elimination and increases muscle calcium levels during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and elimination of excess synaptic input during development.

Dual origins of the mammalian accessory olfactory bulb revealed by an evolutionarily conserved migratory stream.

The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.

Binocular maps in Xenopus tectum: Visual experience and the development of isthmotectal topography.

Xenopus frogs have a prominent binocular field that develops as a consequence of the migration of the eyes during the remodeling of the head during and after metamorphosis. In the optic tectum, a topographic representation of the ipsilateral eye develops during this same period. It is relayed indirectly, via the nucleus isthmi. In the early stages of binocular development, the topographic matching of the ipsilateral input to the retinotectal input from the contralateral eye is largely governed by chemical cues, but the ultimate determinant of the ipsilateral map is binocular visual input. Visual input is such a dominant factor that abnormal visual input resulting from unilateral eye rotation can induce isthmotectal axons to alter their trajectories dramatically, even shifting their terminal zones from one pole of the tectum to the other. This plasticity normally is high only during a 3-4-month critical period of late tadpole-early juvenile life, but the critical period can be extended indefinitely by dark-rearing. N-methyl-D-aspartate (NMDA) receptors are involved in this process; plasticity can be blocked or promoted by chronic treatment with NMDA antagonists or agonists, respectively. Cholinergic nicotinic receptors on retinotectal axons are likely to play an essential role as well. Modifications in the polysialylation of neural cell adhesion molecule are correlated with the state of plasticity. The circuitry underlying binocular plasticity is not yet fully understood but has proved not to be a simple convergence of ipsilateral and contralateral inputs onto the same targets.

Isthmotectal axons maintain normal arbor size but fail to support normal branch numbers in dark-reared Xenopus laevis.

Developing binocular projections to the Xenopus tectum require visual input in order to establish matching topographic maps. In dark-reared Xenopus, the ipsilateral eye's map, relayed via the retino-tecto-isthmotectal pathway, fails initially to acquire normal rostrocaudal order. Moreover, with extended time in the dark, the ipsilateral map becomes progressively less well organized. This phenomenon showed that without binocular cues, the isthmotectal axons are unable to locate proper sites for their terminal zones but left open the issue of whether the axons are able to establish arbors of normal dimensions and/or to sustain normal numbers of branches. In order to test whether dark-rearing modifies isthmotectal axon branching, we have used horseradish peroxidase to examine axons of Xenopus after dark-rearing for periods from 3 to 298 weeks. The results demonstrate that these axons never acquire more than about half the normal numbers of terminals. Surprisingly, however, the dark-reared axons' terminal zones are normal in mediolateral and rostrocaudal extent despite the lack of binocular cues that normally could constrain arbor size by inducing pruning of branches in regions with mismatched visual inputs. The effects of dark-rearing are reversible. After a return to normal lighting conditions, the recovery process begins quickly, with a significant increase in branch numbers within 4 weeks. The terminal zone remains of normal dimensions. These results support the hypothesis that correlated binocular visual input is essential for the maintenance of normal numbers of isthmotectal branches but that normal termination zone size can be established in the absence of visual cues.

The instructive role of binocular vision in the Xenopus tectum.

This review presents the fascinating neurobiology underlying the development of the frog optic tectum, the brain structure where the two separate inputs from the two eye are combined into a single, integrated map. In the species Xenopus laevis, binocular visual information has a dramatic impact on axon growth and connectivity, and the formation of binocular connections in this system provides a rich basis for both theoretical and experimental investigations.

Connections of contralaterally projecting isthmotectal axons and GABA-immunoreactive neurons in Xenopus tectum: an ultrastructural study.

To investigate the circuitry that mediates binocular interactions in the tectum of Xenopus frogs, we have begun to identify the tectal cells that receive ipsilateral eye input relayed via the nucleus isthmi. Isthmotectal axons were labeled with horseradish peroxidase, and thin sections were labeled by postembedding immunogold reaction with antibodies to gamma-aminobutyric acid (GABA). Ultrastructural examination reveals that many isthmotectal axons terminate on GABA-immunoreactive dendrites. Other isthmotectal axons contact postsynaptic structures that are unlabeled but have an appearance consistent with previously described GABA-poor zones of GABA-immunoreactive dendrites. We also examined the unlabeled inputs to the dendrites that were postsynaptic to filled isthmotectal axons. The most common nonisthmic inputs to those dendrites were GABA-immunoreactive processes with symmetric morphology. Surprisingly, we found only one input with the retinotectal characteristics of densely packed round, clear vesicles and minimal GABA immunoreactivity. These results indicate that isthmotectal axons synapse onto inhibitory interneurons, that retinotectal and isthmotectal axons do not synapse close to each other on the same dendrites, and that inhibitory connections are the closest neighbors to isthmotectal synapses.

Melatonin decreases calcium levels in retinotectal axons of Xenopus laevis by indirect activation of group III metabotropic glutamate receptors.

Melatonin is a neuromodulator that binds to receptors in the retinotectal laminae of the amphibian optic tectum. The effect of melatonin on calcium dynamics in Xenopus retinotectal axons was investigated by imaging retinotectal axons labeled with the fluorescent indicator Fluo-4. Melatonin exerted an inhibitory influence on depolarization-evoked calcium increases, and the melatonin receptor antagonist 4-P-PDOT blocked this effect. Blockade of group III metabotropic receptors (mGluRs) counteracted the effect of melatonin on retinotectal axons. Application of the group II/group III mGluR antagonist MSPG or the group III-selective antagonist MSOP abolished the effect of melatonin. Conversely, this effect was not significantly affected by the group I mGluR antagonist LY367385 nor by EGLU or LY341495 at concentrations that specifically inhibit group II mGluRs. Furthermore, a higher concentration of LY341495 that affects group III mGluRs inhibited the effect of melatonin. The data therefore support the hypothesis that, in retinotectal axons, melatonin reduces cAMP levels, thereby relieving PKA-induced inhibition of group III mGluRs; the newly activated mGluRs in turn inhibit voltage-sensitive calcium channels, leading to a decrease in Ca2+ concentrations. The role of GABA(C) receptors in retinotectal responses was also evaluated. GABA(C) receptor antagonists did not block the effects of melatonin but instead were additive. Moreover, while other studies have shown that in Xenopus tectal cells, GABA(C) receptors mediate inhibition, in retinotectal axons, the opposite appears to occur since depolarization-evoked calcium rises in retinotectal axons were inhibited by GABA(C) receptor blockade. This result suggests that activation of GABA(C) receptors produces an increase in the synaptic excitability of retinotectal axon terminals.

Chronic melatonin and binocular plasticity in Xenopus frogs.

The topographic binocular maps in the optic tectum of Xenopus frogs are notable both for their dramatic plasticity during development and for the high expression of melatonin receptors in the circuitry contributing to those binocular maps. The goal of this study was to determine whether melatonin contributes to the control of binocular tectal plasticity. During development, rotation of one eye leads to compensatory rewiring of ipsilateral maps. The effect of 3-4 months of chronic 20 or 200 nM melatonin on this rewiring was tested by electrophysiological mapping. No decrease in plasticity was observed. In adult Xenopus, rotation of one eye normally does not lead to rewiring of the ipsilateral projection, although adults can exhibit plasticity if they have been dark-reared or have been treated as adults with NMDA. We tested whether exposure to 20-200 nM melatonin during and after the normal critical period would similarly extend plasticity. Eye rotation in adults that had been treated with melatonin did not demonstrate retained plasticity. These results show that melatonin does not reduce the normally high plasticity characteristic of young Xenopus nor does it increase the normally low plasticity of adult Xenopus.

Stimulation of melatonin receptors decreases calcium levels in xenopus tectal cells by activating GABA(C) receptors.

To investigate the physiological effects of melatonin receptors in the Xenopus tectum, we have used the fluorescent indicator Fluo-4 AM to monitor calcium dynamics of cells in tectal slices. Bath application of KCl elicited fluorescence increases that were reduced by melatonin. This effect was stronger at the end of the light period than at the end of the dark period. Melatonin increased gamma-aminobutyric acid-C (GABA(C))-receptor activity, as demonstrated by the ability of the GABA(C)-receptor antagonists, picrotoxin and TPMPA, to abolish the effects of melatonin. In contrast, neither the GABA(A)-receptor antagonist bicuculline nor the GABA(B)-receptor antagonist CGP 35348 diminished the effects of melatonin. RT-PCR analyses revealed expression of the 3 known melatonin receptors, MT1 (Mel1(a)), MT2 (Mel1(b)), and Mel1(c). Because the effect of melatonin on tectal calcium increases was antagonized by an MT2-selective antagonist, 4-P-PDOT, we performed Western blot analyses with an antibody to the MT2 receptor; the data indicate that the MT2 receptor is expressed primarily as a dimeric complex and is glycosylated. The receptor is present in higher amounts at the end of the light period than at the end of the dark period, in a pattern complementary to the changes in melatonin levels, which are higher during the night than during the day. These results imply that melatonin, acting by MT2 receptors, modulates GABA(C) receptor activity in the optic tectum and that this effect is influenced by the light-dark cycle.

Localization of Mel1b melatonin receptor-like immunoreactivity in ocular tissues of Xenopus laevis.

The circadian signaling molecule, melatonin, is produced by pinealocytes and retinal photoreceptors. In the retina, melatonin is thought to diffuse into the inner retina to act as a paracrine signal of darkness by binding to specific receptors in retinal neurons. The retinal cell locations of the Mel1a and Mel1c melatonin receptor types have been reported, but the localization of the Mel1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel1b receptor. Western blot analysis of several ocular tissues revealed the presence of one or more immunoreactive bands in the sclera, cornea, lens, retinal pigment epithelium (RPE)/choroid, and neural retina. In the neural retina, the major immunoreactive bands displayed electrophoretic mobilities corresponding to approximately 35, 42, 45, and 80 Kd. Sections of X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, in combination with antibodies against the Mel1a melatonin receptor, a rod photoreceptor-specific protein, opsin, and two amacrine cell-specific markers, tyrosine hydroxylase (TOH; dopaminergic cells) and glutamic acid decarboxylase (GAD; GABA-ergic cells). Mel1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel1b immunoreactivity was observed in both rod and cone photoreceptor inner segments. Presumptive horizontal cells that ramify in the outer plexiform layer (OPL) were immunoreactive for Mel1b, and were exclusive of the Mel1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel1b immunoreactivity was observed in ganglion cells of the retina, a population of cells covering the outer surface of the outer fibrous layer of the sclera, and in lens fibers located in the outer regions of the lens. These results suggest that melatonin may influence retinal function by binding to receptors on RPE and photoreceptor cells, and by acting on neurons of the inner retina that do not use dopamine or GABA as a neurotransmitter. Furthermore, melatonin may bind to receptors on cells located in the sclera and lens, perhaps to modify the growth or function of these ocular tissues.