Final Report Summary - NEUROCOMMUNICATION (The Molecular Communication Mechanism of Motor Neuron Survival and Synapse Maintenance)
In this project we gain better mechanistic understanding on motor neuron biochemical and cellular signaling communications that are essential to its survival and synapse maintenance and are associated with impairments in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). To study this communication mechanisms at a subcellular level, we developed a simplified novel compartmental co-culture microfluidic platform with motoneuron cell bodies in one compartment and muscle cells in the other, connected via motor axons extending through microgrooves that form functional neuromuscular junction (NMJs) synapses. This platform allows precise control, monitoring and manipulation of subcellular microenvironments, thus opening new possibilities for experimental analyses of synapse biology. Using this system, we directly demonstrate the vulnerability of NMJs to stress, and tracked the directionality in axon degeneration mechanisms. We further demonstrated a spatial specificity in the effects of the neurotrophic factor- GDNF and directly tracked its transfer from muscle to axons. We also used the rabies virus as a model to study mechanisms of long-distance axonal transport. We demonstrated that Rabies Virus hijacks and accelerates the p75NTR (Neurotrophic factor receptor) retrograde transport machinery. Additionally, we elucidate a signaling pathways essential for trafficking and axon regeneration/degeneration and uncover the underlying molecular mechanism of neurodegeneration in Familial Dysautonomia (FD). We also performed proteomics and genomics screens to identify novel signaling pathways that play a role in synapse stability and neuron survival. A differential proteomics screen for dynein interactors in synapses of ALS mice model, followed by bioinformatic network reconstruction and in vitro and in vivo validations, revealed a role for a novel RNA-binding protein-Staufen1 in ALS pathology. In a follow up study, we investigated the axonal transport and localization mechanism underlying the RNA-induced silencing complex (RISC) along the axon, and demonstrated a novel role for mitochondria in this context, suggesting that mitochondria regulate the localization of RNAi machinery along the axon via a stress-dependent mechanism. We furthered obtain the first subcellular miRNA and mRNA trancriptome profiles of two ALS-linked mutation models and provide an important resource for studies on the roles of local protein synthesis and axon degeneration in ALS. Recently, we have revealed additional critical mechanism that contributes to ALS that involve muscle secretion of toxic factors that can be regulated by miR126. Finally, we study how spatiotemporal organization of receptors along the plasma membrane, can regulate signalling intensity, duration, and fidelity. Using BDNF receptor, TrkB, we demonstrated that TrkB acts as a monomeric receptor at the plasma membrane to activate a weak fast temporal Erk1/2 signaling; while upon its dimerization after internalization to endosomes, it activate long sustain AKT signaling. Thus, we report on a novel mode of monomeric signaling for RTK, and a specific subcellular sorting mechanism that can regulate specific signaling events. All together in this project we demonstrate various levels of subcellular motor neuron communications that are essential to maintain its function, and alter under pathological situations.