Raffaella Willmann

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol‐rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve–muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout‐induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src−/−;fyn−/− myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src‐family kinases were strongly enriched in lipid microdomains prepared from wild‐type myotubes. Microdomain disruption by cholesterol‐sequestering methyl‐β‐cyclodextrin disassembled AChR clusters and decreased AChR–rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src−/−;fyn−/− myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol‐rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain‐association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.

Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited

Abstract. Clustering of neurotransmitter receptors in the postsynaptic membrane is critical for efficient synaptic transmission. During neuromuscular synaptogenesis, clustering of acetylcholine receptors (AChRs) is an early sign of postsynaptic differentiation. Recent studies have revealed that the earliest AChR clusters can form in the muscle independent of motorneurons. Neurally released agrin, acting through the muscle-specific kinase MuSK and rapsyn, then causes further clustering and localization of clusters underneath the nerve terminal. AChRs themselves are required for agrin-induced clustering of several postsynaptic proteins, most notably rapsyn. Once formed, AChR clusters are stabilized by several tyrosine kinases and by components of the dystrophin/utrophin glycoprotein complex, some of which also direct postnatal synaptic maturation such as formation of postjunctional folds. This review summarizes these recent results about AChR clustering, which indicate that early clustering can occur in the absence of nerves, that AChRs play an active role in the clustering process and that partly different mechanisms direct formation versus stabilization of AChR clusters.

Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors

Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs) in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using cultured src-/-;fyn-/- myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin in src-/-;fyn-/- myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased in src-/-;fyn-/- myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJs in vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors

ABSTRACT Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrins absence to further increase MuSK phosphorylation and cluster AChRs.

Enhancing translation: guidelines for standard pre-clinical experiments in mdx mice

Duchenne Muscular Dystrophy is an X-linked disorder that affects boys and leads to muscle wasting and death due to cardiac involvement and respiratory complications. The cause is the absence of dystrophin, a large structural protein indispensable for muscle cell function and viability. The mdx mouse has become the standard animal model for pre-clinical evaluation of potential therapeutic treatments. Recent years have seen a rapid increase in the number of experimental compounds being evaluated in the mdx mouse. There is, however, much variability in the design of these pre-clinical experimental studies. This has made it difficult to interpret and compare published data from different laboratories and to evaluate the potential of a treatment for application to patients. The authors therefore propose the introduction of a standard study design for the mdx mouse model. Several aspects, including animal care, sampling times and choice of tissues, as well as recommended endpoints and methodologies are addressed and, for each aspect, a standard procedure is proposed. Testing of all new molecules/drugs using a widely accepted and agreed upon standard experimental protocol would greatly improve the power of pre-clinical experimentations and help identifying promising therapies for the translation into clinical trials for boys with Duchenne Muscular Dystrophy.

Developing standard procedures for murine and canine efficacy studies of DMD therapeutics: report of two expert workshops on “Pre-clinical testing for Duchenne dystrophy”: Washington DC, October 27th–28th 2007 and Zürich, June 30th-July 1st 2008

Report of two expert workshops “Preclinical testing for Duchenne dystrophy”: Washington, October 27th - 28th 2007 and Zurich, June 30th-July 1st 2008

Developing standard procedures for pre-clinical efficacy studies in mouse models of spinal muscular atrophy: report of the expert workshop "Pre-clinical testing for SMA", Zürich, March 29-30th 2010.

A workshop was held in Zurich (Switzerland) to develop standard procedures and protocols for pre-clinical efficacy studies in mouse models of spinal muscular atrophy (SMA). The workshop was organized by the Network of Excellence TREAT-NMD (Translational Research in Europe – Assessment and Treatment of Neuromuscular Disorders), launched 2007, that addresses the fragmentation currently existing in the evaluation, diagnosis and care of neuromuscular diseases on one side, and in the procedures of drug development from research to the design of clinical trials on the other side. Within this frame, one aspect that needs particular care is the design and performance of pre-clinical animal studies that should predict the efficacy of a new treatment in patients. In the absence of commonly accepted guidelines and standardized protocols, data from different laboratories are often not comparable and the result is a duplication of efforts. The workshop was attended by 30 key researchers from Europe and North America, as well as industry representatives (Santhera Pharmaceuticals, Psychogenics, Genyzme, Trophos) and patient organisations (SMA Foundation, AFM-Association Franc aise contre les Myopathies). The goals of the workshop were (1) to select endpoints that best assess drug efficacy in mouse

Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors

BackgroundDevelopment of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs.ResultsWe have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein.ConclusionOur data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.

Best Practices and Standard Protocols as a Tool to Enhance Translation for Neuromuscular Disorders

Recent years witnessed an exciting increase in the number of clinical trials for neuromuscular disorders, in particular for Duchenne Muscular Dystrophy and Spinal Muscle Atrophy. Given the high emotional impact of such developments for devastating diseases with an urgent medical need, it is particularly important to justify human trials on the basis of robust preclinical studies and to avoid a waste of hopes and of funds.This review focuses the discussion on the quality in the conduct clinically-oriented preclinical assessments in rare neuromuscular disease models and on the importance in reporting of preclinical confirmatory studies. Accordingly, it invites scientists, journal publishers and funding agencies to require quality standards to improve translatability of preclinical findings.

212th ENMC International Workshop

The 212th ENMC workshop, Animal models of Congenital Muscular Dystrophies, took place in Naarden, The Netherlands on May 29–31, 2015 and was attended by 13 participants from France, Israel, Italy, Japan, Sweden, Switzerland, United Kingdom and the USA, including clinical and basic science researchers, as well as one PhD student who received support from the ENMC Young Scientist Program.