Specific labeling of synaptic schwann cells reveals unique cellular and molecular features.

Perisynaptic Schwann cells (PSCs) are specialized, non-myelinating, synaptic glia of the neuromuscular junction (NMJ), that participate in synapse development, function, maintenance, and repair. The study of PSCs has relied on an anatomy-based approach, as the identities of cell-specific PSC molecular markers have remained elusive. This limited approach has precluded our ability to isolate and genetically manipulate PSCs in a cell specific manner. We have identified neuron-glia antigen 2 (NG2) as a unique molecular marker of S100ß+ PSCs in skeletal muscle. NG2 is expressed in Schwann cells already associated with the NMJ, indicating that it is a marker of differentiated PSCs. Using a newly generated transgenic mouse in which PSCs are specifically labeled, we show that PSCs have a unique molecular signature that includes genes known to play critical roles in PSCs and synapses. These findings will serve as a springboard for revealing drivers of PSC differentiation and function.

Attenuating Cholinergic Transmission Increases the Number of Satellite Cells and Preserves Muscle Mass in Old Age.

In addition to driving contraction of skeletal muscles, acetylcholine (ACh) acts as an anti-synaptogenic agent at neuromuscular junctions (NMJs). Previous studies suggest that aging is accompanied by increases in cholinergic activity at the NMJ, which may play a role in neuromuscular degeneration. In this study, we hypothesized that moderately and chronically reducing ACh could attenuate the deleterious effects of aging on NMJs and skeletal muscles. To test this hypothesis, we analyzed NMJs and muscle fibers from heterozygous transgenic mice with reduced expression of the vesicular ACh transporter (VAChT; VKDHet), which present with approximately 30% less synaptic ACh compared to control mice. Because ACh is constitutively decreased in VKDHet, we first analyzed developing NMJs and muscle fibers. We found no obvious morphological or molecular differences between NMJs and muscle fibers of VKDHet and control mice during development. In contrast, we found that moderately reducing ACh has various effects on adult NMJs and muscle fibers. VKDHet mice have significantly larger NMJs and muscle fibers compared to age-matched control mice. They also present with reduced expression of the pro-atrophy gene, Foxo1, and have more satellite cells in skeletal muscles. These molecular and cellular features may partially explain the increased size of NMJs and muscle fibers. Thus, moderately reducing ACh may be a therapeutic strategy to prevent the loss of skeletal muscle mass that occurs with advancing age.

The ALS-inducing factors, TDP43A315T and SOD1G93A, directly affect and sensitize sensory neurons to stress.

There is increased recognition that sensory neurons located in dorsal root ganglia (DRG) are affected in amyotrophic lateral sclerosis (ALS). However, it remains unknown whether ALS-inducing factors, other than mutant superoxide dismutase 1 (SOD1G93A), directly affect sensory neurons. Here, we examined the effect of mutant TAR DNA-binding protein 1 (TDP43A315T) on sensory neurons in culture and in vivo. In parallel, we reevaluated sensory neurons expressing SOD1G93A. We found that cultured sensory neurons harboring either TDP43A315T or SOD1G93A grow neurites at a slower rate and elaborate fewer neuritic branches compared to control neurons. The presence of either ALS-causing mutant gene also sensitizes sensory neurons to vincristine, a microtubule inhibitor that causes axonal degeneration. Interestingly, these experiments revealed that cultured sensory neurons harboring TDP43A315T elaborate shorter and less complex neurites, and are more sensitive to vincristine compared to controls and to SOD1G93A expressing sensory neurons. Additionally, levels of two molecules involved in stress responses, ATF3 and PERK are significantly different between sensory neurons harboring TDP43A315T to those with SOD1G93A in vitro and in vivo. These findings demonstrate that sensory neurons are directly affected by two ALS-inducing factors, suggesting important roles for this neuronal subpopulation in ALS-related pathogenesis.

Fast and slow-twitching muscles are differentially affected by reduced cholinergic transmission in mice deficient for VAChT: A mouse model for congenital myasthenia.

Congenital myasthenic syndromes (CMS) result from reduced cholinergic transmission at neuromuscular junctions (NMJs). While the etiology of CMS varies, the disease is characterized by muscle weakness. To date, it remains unknown if CMS causes long-term and irreversible changes to skeletal muscles. In this study, we examined skeletal muscles in a mouse line with reduced expression of Vesicular Acetylcholine Transporter (VAChT, mouse line herein called VAChT-KDHOM). We examined this mouse line for several reasons. First, VAChT plays a central function in loading acetylcholine (ACh) into synaptic vesicles and releasing it at NMJs, in addition to other cholinergic nerve endings. Second, loss of function mutations in VAChT causes myasthenia in humans. Importantly, VAChT-KDHOM present with reduced ACh and muscle weakness, resembling CMS. We evaluated the morphology, fiber type (myosin heavy chain isoforms), and expression of muscle-related genes in the extensor digitorum longus (EDL) and soleus muscles. This analysis revealed that while muscle fibers atrophy in the EDL, they hypertrophy in the soleus muscle of VAChT-KDHOM mice. Along with these cellular changes, skeletal muscles exhibit altered levels of markers for myogenesis (Pax-7, Myogenin, and MyoD), oxidative metabolism (PGC1-α and MTND1), and protein degradation (Atrogin1 and MuRF1) in VAChT-KDHOM mice. Importantly, we demonstrate that deleterious changes in skeletal muscles and motor deficits can be partially reversed following the administration of the cholinesterase inhibitor, pyridostigmine in VAChT-KDHOM mice. These findings reveal that fast and slow type muscles differentially respond to cholinergic deficits. Additionally, this study shows that the adverse effects of cholinergic transmission, as in the case of CMS, on fast and slow type skeletal muscles are reversible.

Impact of Aging on Proprioceptive Sensory Neurons and Intrafusal Muscle Fibers in Mice.

The impact of aging on proprioceptive sensory neurons and intrafusal muscle fibers (IMFs) remains largely unexplored despite the central function these cells play in modulating voluntary movements. Here, we show that proprioceptive sensory neurons undergo deleterious morphological changes in middle age (11- to 13-month-old) and old (15- to 21-month-old) mice. In the extensor digitorum longus and soleus muscles of middle age and old mice, there is a significant increase in the number of Ia afferents with large swellings that fail to properly wrap around IMFs compared with young adult (2- to 4-month-old) mice. Fewer II afferents were also found in the same muscles of middle age and old mice. Although these age-related changes in peripheral nerve endings were accompanied by degeneration of proprioceptive sensory neuron cell bodies in dorsal root ganglia (DRG), the morphology and number of IMFs remained unchanged. Our analysis also revealed normal levels of neurotrophin 3 (NT3) but dysregulated expression of the tyrosine kinase receptor C (TrkC) in aged muscles and DRGs, respectively. These results show that proprioceptive sensory neurons degenerate prior to atrophy of IMFs during aging, and in the presence of the NT3/TrkC signaling axis.

VAChT overexpression increases acetylcholine at the synaptic cleft and accelerates aging of neuromuscular junctions.

BACKGROUND: Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). METHODS: Chat-ChR2-EYFP (VAChTHyp) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1G93A), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. RESULTS: We show that VAChT is elevated in the spinal cord and at NMJs of VAChTHyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChTHyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChTHyp mice compared to control mice. While the development of NMJs was not affected in VAChTHyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChTHyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChTHyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChTHyp mice. In the SOD1G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice. CONCLUSIONS: The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.

Degeneration of proprioceptive sensory nerve endings in mice harboring amyotrophic lateral sclerosis-causing mutations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily targets the motor system. Although much is known about the effects of ALS on motor neurons and glial cells, little is known about its effect on proprioceptive sensory neurons. This study examines proprioceptive sensory neurons in mice harboring mutations associated with ALS, in SOD1(G93A) and TDP43(A315T) transgenic mice. In both transgenic lines, we found fewer proprioceptive sensory neurons containing fluorescently tagged cholera toxin in their soma five days after injecting this retrograde tracer into the tibialis anterior muscle. We asked whether this is due to neuronal loss or selective degeneration of peripheral nerve endings. We found no difference in the total number and size of proprioceptive sensory neuron soma between symptomatic SOD1(G93A) and control mice. However, analysis of proprioceptive nerve endings in muscles revealed early and significant alterations at Ia/II proprioceptive nerve endings in muscle spindles before the symptomatic phase of the disease. Although these changes occur alongside those at α-motor axons in SOD1(G93A) mice, Ia/II sensory nerve endings degenerate in the absence of obvious alterations in α-motor axons in TDP43(A315T) transgenic mice. We next asked whether proprioceptive nerve endings are similarly affected in the spinal cord and found that nerve endings terminating on α-motor neurons are affected during the symptomatic phase and after peripheral nerve endings begin to degenerate. Overall, we show that Ia/II proprioceptive sensory neurons are affected by ALS-causing mutations, with pathological changes starting at their peripheral nerve endings.