CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues. CD44 was implicated in the development of peripheral nerves, functioning as a coreceptor for ErbB class of growth factor receptors. However, it is not known whether CD44-ErbB interaction may occur at the adult peripheral synapses. Here we studied, using Fluorescence Lifetime Imaging Microscopy, the proximity between CD44 and ErbB3 at the rat neuromuscular junction (NMJ). This was performed in muscle sections co-immunostained for CD44 and ErbB3, using secondary antibodies coupled to Alexa488 and Alexa647 respectively. Neuromuscular junctions were visualized using Alexa555-bound α-BT. The FRET between Alexa488 (donor) and Alexa647 (acceptor) was judged by measuring an accompanying changes in the donor fl uorescence lifetime. We found that the mean fl uorescence lifetime of the donor fl uorophore labeling CD44 protein was considerably shorter over the NMJ than in nonsynaptic sites. Then we compared normal rat muscle to the muscle affected by denervation in the transgenic model of amyotrophic lateral sclerosis (ALS). Importantly, ALS-like neurodegeneration resulted in signifi cant increase in molecular proximity of CD44 and ErbB3 at the NMJ. The specifi c complex formation between the two proteins was confi rmed using immunoprecipitation analysis. Our study provides novel data on the molecular architecture of the neuromuscular synapse in both health and disease.
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