Hypoxia-induced reprogrammed myoblasts enhance the formation of neuromuscular junctions: A pioneer study.

We previously reported that muscle cells could reprogram into progenitors after traumatic injuries. These injury-induced muscle stem cells (iMuSCs) have increased migration and differentiation capacities, including neuronal differentiation. Recent studies in our laboratory suggest that the hypoxia-induced by tissue injury plays an essential role in the reprogramming process of muscle cells. We hypothesize that muscle cells reprogrammed with hypoxia have increased neuronal differentiation potentials and the neuronal differentiation extends into the formation of neuromuscular junction (NMJ)-like structures. In this study, C2C12 myoblasts were cultured under hypoxic conditions and subsequently in neural differentiation media to generate neurospheres, and then with muscle differentiation media to induce NMJ-like structure formation. Hypoxia-induced muscle cells also produced more robust NMJs compared to controls after intramuscular cell transplantation. Our results suggest hypoxia plays a role in the reprogramming of muscle stem cells, which may have the potential to form neuromuscular junctions and ultimately contribute to functional muscle healing.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients' quality of life.

GDNF synthesis, signaling, and retrograde transport in motor neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a 134 amino acid protein belonging in the GDNF family ligands (GFLs). GDNF was originally isolated from rat glial cell lines and identified as a neurotrophic factor with the ability to promote dopamine uptake within midbrain dopaminergic neurons. Since its discovery, the potential neuroprotective effects of GDNF have been researched extensively, and the effect of GDNF on motor neurons will be discussed herein. Similar to other members of the TGF-ß superfamily, GDNF is first synthesized as a precursor protein (pro-GDNF). After a series of protein cleavage and processing, the 211 amino acid pro-GDNF is finally converted into the active and mature form of GDNF. GDNF has the ability to trigger receptor tyrosine kinase RET phosphorylation, whose downstream effects have been found to promote neuronal health and survival. The binding of GDNF to its receptors triggers several intracellular signaling pathways which play roles in promoting the development, survival, and maintenance of neuron-neuron and neuron-target tissue interactions. The synthesis and regulation of GDNF have been shown to be altered in many diseases, aging, exercise, and addiction. The neuroprotective effects of GDNF may be used to develop treatments and therapies to ameliorate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this review, we provide a detailed discussion of the general roles of GDNF and its production, delivery, secretion, and neuroprotective effects on motor neurons within the mammalian neuromuscular system.

The neurochemistry and social flow of singing: bonding and oxytocin.

Music is used in healthcare to promote physical and psychological well-being. As clinical applications of music continue to expand, there is a growing need to understand the biological mechanisms by which music influences health. Here we explore the neurochemistry and social flow of group singing. Four participants from a vocal jazz ensemble were conveniently sampled to sing together in two separate performances: pre-composed and improvised. Concentrations of plasma oxytocin and adrenocorticotropic hormone (ACTH) were measured before and after each singing condition to assess levels of social affiliation, engagement and arousal. A validated assessment of flow state was administered after each singing condition to assess participants' absorption in the task. The feasibility of the research methods were assessed and initial neurochemical data was generated on group singing. Mean scores of the flow state scale indicated that participants experienced flow in both the pre-composed (M = 37.06) and improvised singing conditions (M = 34.25), with no significant difference between conditions. ACTH concentrations decreased in both conditions, significantly so in the pre-composed singing condition, which may have contributed to the social flow experience. Mean plasma oxytocin levels increased only in response to improvised singing, with no significant difference between improvised and pre-composed singing conditions observed. The results indicate that group singing reduces stress and arousal, as measured by ACTH, and induces social flow in participants. The effects of pre-composed and improvised group singing on oxytocin are less clear. Higher levels of plasma oxytocin in the improvised condition may perhaps be attributed to the social effects of improvising musically with others. Further research with a larger sample size is warranted.

Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

GDNF content and NMJ morphology are altered in recruited muscles following high-speed and resistance wheel training.

Glial cell line-derived neurotrophic factor (GDNF) may play a role in delaying the onset of aging and help compress morbidity by preventing motor unit degeneration. Exercise has been shown to alter GDNF expression differently in slow- and fast-twitch myofibers. The aim was to examine the effects of different intensities (10, 20, ~30, and ~40 m·min(-1)) of wheel running on GDNF expression and neuromuscular junction (NMJ) plasticity in slow- and fast-twitch myofibers. Male Sprague-Dawley Rats (4 weeks old) were divided into two sedentary control groups (CON4 week, n = 5 and CON6 week, n = 5), two involuntary running groups, one at a low velocity; 10 m/min (INVOL-low, n = 5), and one at a higher velocity; 20 m/min (INVOL-high, n = 5), and two voluntary running groups with resistance (VOL-R, n = 5, 120 g), and without resistance (VOL-NR, n = 5, 4.5 g). GDNF protein content, determined by enzyme-linked immunosorbent assay (ELISA), increased significantly in the recruited muscles. Plantaris (PLA) GDNF protein content increased 174% (P <0.05) and 161% (P <0.05) and end plate-stained area increased 123% (P <0.05) and 72% (P <0.05) following VOL-R, and VOL-NR training, respectively, when compared to age-matched controls. A relationship exists between GDNF protein content and end plate area (r = 0.880, P < 0.01, n = 15). VOL-R training also resulted in more dispersed synapses in the PLA muscle when compared to age-matched controls (P <0.05). Higher intensity exercise (>30 m/min) can increase GDNF protein content in fast-twitch myofibers as well as induce changes in the NMJ morphology. These findings help to inform exercise prescription to preserve the integrity of the neuromuscular system through aging and disease.

Regional changes in cardiac and stellate ganglion norepinephrine transporter in DOCA-salt hypertension.

Uptake of norepinephrine via the neuronal norepinephrine transporter is reduced in the heart during deoxycorticosterone (DOCA)-salt hypertension. We hypothesized that this was due to reduced norepinephrine transporter mRNA and/or protein expression in the stellate ganglia and heart. After 4 weeks of DOCA-salt treatment there was no change in norepinephrine transporter mRNA in either the right or the left stellate ganglia from hypertensive rats (n=5-7, p>0.05). Norepinephrine transporter immunoreactivity in the left stellate ganglion was significantly increased (n=4, p<0.05) while the right stellate ganglion was unchanged (n=4, p>0.05). Whole heart norepinephrine content was significantly reduced in DOCA rats consistent with reduced uptake function; however, when norepinephrine was assessed by chamber, a significant decrease was noted only in the right atrium and right ventricle (n=6, p<0.05). Cardiac norepinephrine transport binding by chamber revealed that it was only reduced in the left atrium (n=5-7, p>0.05). Therefore, 1) contrary to our hypothesis reduced reuptake in the hypertensive heart is not exclusively due to an overall reduction in norepinephrine transporter mRNA or protein in the stellate ganglion or heart, and 2) norepinephrine transporter regulation occurs regionally in the heart and stellate ganglion in the hypertensive rat heart.

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture.

Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma×neuroblastoma hybrid cells (NG108-15) were used to create nerve-muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha-bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process.

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content.

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.

Effects of polychlorinated biphenyls (PCBs) on expression of neurotrophic factors in C6 glial cells in culture.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect nervous system function. Glial cells are among the first lines of defense in the nervous system and are involved in activities, including production of neurotrophic factors, which maintain an environment optimally suited for neuronal function. In this study, we investigated the effects of a commercial mixture of PCBs, Aroclor 1254 (A1254), on neurotrophic factor secretion by C6 cells in culture. C6 cells were exposed to medium containing 10 ppm A1254, 0.1% dimethyl sulfoxide (DMSO=vehicle), or normal culture medium. Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbant assay. GDNF mRNA was measured by real-time RT-PCR. The role of protein kinase C (PKC) signaling in A1254 effects was investigated using bisindolylmaleimide, a PKC antagonist. Exposure to A1254 increased NGF (8.8x10(-5)+/-8.2x10(-6)pg NGF/cell) and GDNF (1.0x10(-4)+/-6.7x10(-6)pg GDNF/cell) secretion compared to DMSO treated controls (5.0x10(-5)+/-7.5x10(-6)pg NGF/cell and 6.2x10(-5)+/-3.1x10(-6)pg GDNF/cell). The effect of A1254 was long-lived, as GDNF secretion was elevated following 5 days of exposure (4.1x10(-5)+/-1.7x10(-6)pg GDNF/cell in A1254 exposed cells vs. 2.9x10(-5)+/-2.3x10(-6)pg GDNF/cell in DMSO exposed cells). GDNF mRNA expression was also elevated following exposure to A1254 (1.14+/-0.07 gene expression units in A1254 exposed cells vs. 0.8+/-0.07 gene expression units in DMSO exposed cells). Bisindolylmaleimide was able to block the effects of A1254 on GDNF secretion. Thus, one potential mechanism by which PCBs may alter nervous system function is via disruption of neurotrophic factor expression by glial cells. The observation that neurotrophic factor expression was increased following exposure to PCB may suggest that glial cells increase expression of neuroprotective genes following exposure to potentially damaging agents such as PCBs.

GDNF is regulated in an activity-dependent manner in rat skeletal muscle.

Glial cell line-derived neurotrophic factor (GDNF) is produced by skeletal muscle and affects peripheral motor neurons. Elevated expression of GDNF in skeletal muscle leads to hyperinnervation of neuromuscular junctions, whereas postnatal administration of GDNF causes synaptic remodeling at the neuromuscular junction. Studies have demonstrated that altered physical activity causes changes in the neuromuscular junction. However, the role played by GDNF in this process in not known. The objective of this study was to determine whether changes in neuromuscular activity cause altered GDNF content in rat skeletal muscle. Following 4 weeks of walk-training on a treadmill, or 2 weeks of hindlimb unloading, soleus, gastrocnemius, and pectoralis major were removed and analyzed for GDNF content by enzyme-linked immunosorbant assay. Results indicated that walk-training is associated with increased GDNF content. Skeletal muscle from hindlimb-unloaded animals showed a decrease in GDNF in soleus and gastrocnemius, and an increase in pectoralis major. The altered production of GDNF may be responsible for activity-dependent remodeling of the neuromuscular junction and may aid in recovery from injury and disease.