Examining the Role of Neuregulin-1 in Synaptogenesis Using Microfluidics

Abstract

Determining the molecular mechanisms behind synaptogenesis (synapse formation and maintenance) is of great importance for understanding higher brain function as well as disease states such as Alzheimer's and muscular dystrophy. Neuregulin-1 (NRG-1), a nerve derived protein, was isolated based on its ability to stimulate new acetylcholine receptor (AChR) formation on muscle. This molecule has been hypothesized to enforce the high density of AChRs on the post-synaptic membrane in neuromuscular synapses, however, its role in vivo has been difficult to study due to the early death of NRG and ErbB mutants. Therefore, we have developed a microfluidic system, mimicking a synapse by focally delivering nerve derived proteins to a cell chamber containing myotubes, to study synaptogenesis. Also, the device's focal delivery capacity coupled with patterning of the culture chamber surface allows us to ask questions with spatial variables. As a platform for our AChR kinetics studies, we have examined complex, aneural AChR clusters found on muscle and first reported by Kummer et al. in 2004. These features are good models of in vivo post-synaptic areas, because they have similar topologies, protein expression and developmental patterns. After staining with fluorescent bungarotoxin, we have found that neuregulin decreases the half-life of receptors in pretzels by 21.4%. We have also confirmed that NRG-1 increases receptor insertion into pretzels. Afterwards, we examined the extent NRG-1 activation travels in the long, multi-nucleated muscle cell using mRNA in situ hybridization.