Exogenous MPP3 is Sufficient to Induce Excitatory Synapse Formation

Brain-derived neurotrophic factor (BDNF) is a secreted growth factor critical for the establishment and maintenance of central nervous system (CNS) synapses. However, the precise mechanisms that allow BDNF signaling to regulate synapse formation are unknown. Interestingly, MAGUK scaffold proteins, a family of proteins critical for organizing the molecular architecture of the synapse, have altered gene expression in the brains of BDNF mutant mice. This suggests that BDNF's in vivo regulation of MAGUK proteins may be important for the process of BDNF-induced synapse formation. Two MAGUKs with the most significantly decreased expression are membrane protein, palmitoylated 3 (MPP3) and postsynaptic density 95 (PSD95). PSD95 is a well-established mediator of synapse formation as demonstrated by PSD95's colocalization with postsynaptic proteins, sufficiency to increase synapse density, and trafficking to synapses in a BDNF-dependent manner. However, little is known about the role of MPP3 in synapse formation. The specific objective for this research project was to determine if MPP3 positively regulates synapse development and to determine the requirement of MPP3 in BDNF-induced excitatory synapse formation. To test the role of MPP3 in synapse formation two MPP3 expression constructs and three short-hairpin RNAs (shRNAs) were created and transfected in neuronal cultures. The neuronal cultures were fixed and stained for excitatory synaptic markers, VGlut1 and PSD95, and finally imaged using laser scanning confocal microscopy to highlight synapses on these neurons. This methodology was used in order to determine if MPP3 1) localizes to postsynaptic areas, 2) is sufficient to increase synapse density, and 3) is required for BDNF-induced synapse formation. Based on our findings, MPP3 localizes to potential postsynaptic regions and is sufficient to increase excitatory synapse formation.