Current Research

Sympathetic innervation of neuromuscular junctions

The sympathetic nervous system regulates basic body functions such as heartbeat, blood pressure, and gland activities. Whereas hormone secretion from the adrenal medulla modulates these processes systemically, local and fast responses can be mediated by direct sympathetic innervation. Although many effects of the sympathetic system on skeletal muscle physiology and disease are known, direct sympathetic innervation targets in skeletal muscle have been scarcely studied. We are investigating this aspect and have recently described that neuromuscular junctions (NMJs), the synapses between motor neurons and muscle fibers, are innervated by sympathetic neurons (Fig. 1A). This is of crucial importance for the integrity and function of NMJs (Fig. 1B). We aim to further study several aspects of this innervation. These include the precise nature of interaction between sympathetic neurons and muscle fibers, the mechanisms of how sympathetic innervation modulates NMJ formation and maintenance and, finally, the links of sympathetic innervation to neuromuscular transmission disorders. The latter is particularly relevant, because sympathicomimetics have recently been successfully introduced to treat several patients suffering from congenital myasthenic syndromes, a group of inherited neuromuscular transmission disorders.

Ruedolf Fig01

Figure 1: Distribution of sympathetic neurons in skeletal muscle.

(A) Upper panel: Diaphragm muscle of a reporter mouse expressing Tomato protein (red) in sympathetic neurons was costained with anti-tyrosine hydroxylase antibody (catecholaminergic sympathetic neurons, green) and alpha-bungarotoxin (AChR, blue). Three-dimensional maximum projection of a confocal z stack of a representative region is shown. Lower panel: Longitudinal sections of wild-type EDL muscles were labeled against VACHT (cholinergic alpha motor neurons, green), tyrosine hydroxylase (catecholaminergic sympathetic neurons, red), and alpha-bungarotoxin (AChR, blue). Three-dimensional maximum projection of a confocal z stack of a representative region is shown.

(B) Sympathicomimetic treatment rescues NMJ phenotypes of sympathectomized muscle. Muscles of wild-type mice received injections of saline (wt) or 6-hydroxydopamine (sympathicotoxic, SE) on alternate days for 2 weeks. In the last 10 days, one SE group was also treated with the sympathicomimetic clenbuterol (SE+SM). Then, muscles were sectioned and stained with alpha-bungarotoxin and anti-VACHT antibody. Images show projections of representative NMJs. Figures modified from Khan et al. PNAS 2016.

Mechanisms controlling receptor turnover at the NMJ

Age-related loss of muscle mass (sarcopenia) is a feature of increasing clinical relevance in western societies. Sarcopenia is a complex syndrome, but a major hallmark is the loss of functional muscle innervation. The contribution of nerve-dependent and muscle-intrinsic changes that lead to the observed interruption of connectivity is being debated. At the NMJ, nicotinic acetylcholine receptor (AChR) is the major postsynaptic ion channel. This displays a complex activity-dependent intracellular trafficking, which is, in many respects, highly analogous to other ligand-gated channels of the central nervous system. In particular, upon arrival at the postsynaptic membrane, AChR are endocytosed at some point and can then be either recycled or degraded (Fig 2A). The importance of this regulation is shown by the decrease of AChR half-life from roughly 13 days in the innervated to 1-2 days in the denervated muscle. We use a combination of sophisticated expression and imaging protocols to study AChR trafficking in live tissue and can therefore access the large regulatory capacity of nerve-muscle interaction. While the laboratory previously investigated the mechanisms that underlie the process of AChR recycling, we have recently focused on the degradative route. This revealed that particularly under catabolic conditions and the absence of neuronal activity, AChR are degraded by the process of autophagy (Fig. 2B) mediated by a complex of the proteins MuRF1 (E3 ubiquitin ligase), p62/SQSTM1 (selective autophagy adapter) and Bif-1, alias endophilin B1. The latter is a cytosolic protein that can bind to membranes in a phosphorylation-dependent manner and then mediate membrane curvature and formation of autophagosomes. Our current research addresses the precise function and activation of Bif-1 upon muscle atrophy. This includes the detailed in vivo analysis of protein activation mechanisms as well as the search for elucidating the up-stream neuronal or hormonal triggers that decide about AChR recycling or degradation. To that end, the laboratory is in continuous collaboration with a number of internationally leading groups in the field.

Fig1
Figure 2: (A) Scheme depicting the putative life cycle of AChR at the vertebrate NMJ. Upon assembly in the ER and glycosylation in the Golgi apparatus, AChR is delivered by exocytosis to the postsynaptic membrane where it is clustered by means of agrin/ MuSK/ Lrp4/ rapsyn complex, to fulfill its major function, i.e. mediating neuromuscular transmission. Subsequently, AChR is endocytosed and can then be recycled using a cooperative function of myosin Va, PKA type-I and rapsyn, or degraded, presumably via autophagic decay in a MuRF1-dependent manner. Decision-making between recycling or degradation appears to be subject to manifold signals including CaMKII and PKC. Figure from Rudolf et al. Frontiers Aging Neuroscience 2014.

 

Fig2
Figure 2: (B) Upon denervation, AChRs are massively distributed into Bif1-positive autophagsomes. In vivo imaging of denervated muscle showing Bif1-mCherry (red), the autophagosomal marker GFP-LC3 (green), and AChR (blue) reveals massive colocalisation between AChR and Bif-1 (magenta structures). Many of these AChR-Bif1 double-positive vesicles are nicely capped by LC3, indicating their autophagosomal nature. The large blue, arborised structure at the right is the NMJ. Image size: 65x65 µm.
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Latest Revision: 2016-03-14
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