Characterization of Activity-Dependent Trafficking and Distribution of Lysosomes in Neurons

My dissertation research focused on studying activity-dependent protein degradation in neurons primarily focusing on lysosomes, a degradative organelle. The role of lysosomes in regulating neuronal activity has been virtually unexplored, but their presence in dendrites was recently confirmed. My thesis work served to try to understand their role in dendrites, showing that lysosomes can be recruited to spines and their inhibition causes mEPSP frequency to decrease. I then expanded on this work to characterize their distribution throughout the entire dendrite, demonstrating that long term depression (LTD) and long term potentiation (LTP) cause lysosomes to shift proximally and distally respectively. This shift is associated with a decrease in surface GluA1 AMPA receptor subunits, suggesting that lysosomes may play a role in regulating the local intensity of LTP and LTD.In Chapter IV, I focus on AMPA receptor half-life by evaluating the effect of the phosphorylation state of serine 845 (s845). This phosphor-site has been shown to regulate internalization of GluA1. I found that phosphorylation of this site increases when neuronal activity is upregulated via bicuculline treatment, and that this phosphorylation changes over time, peaking between 4-8 hours. Inhibiting protein kinase A in conjunction with this treatment causes a synthesis-dependent degradation of GluA1. Chapter V describes the development of a method to purify lysosomes from neurons in preparation for proteomic analysis via mass spectrometry. This method was adapted to neurons from an existing method to purify lysosomes in dividing cells and shown to have the sensitivity to detect autophagy-induced changes in the lysosomal proteome