Browsing Graduate Student Dissertations & Theses by Subject "SYNAPSES"
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THE ROLE OF THE UBIQUITIN E3/DUB DUAL ENZYME A20 IN NEURONS AND SYNAPSESPosttranslational ubiquitination and deubiquination protein modifications play an essential role in neuronal development, function, and plasticity. This study investigated the role of the E3/deubiquitinase dual ubiquitin enzyme A20 (also called tumor necrosis factor alpha -induced protein 3, TNFAIP3) on neuronal arborization, dendritic spine morphogenesis, and synaptic transmission. The spatial and temporal expression profiles of A20 were investigated in primary neuronal cultures and rodent brains by western blotting and immunofluorescent staining. Several mammalian cDNA expression and shRNA plasmids with the Myc tag or the DsRed or GFP fluorescent reporters were constructed by subcloning, and their efficiency was validated in HEK293FT cell line, cultured neurons, and yeast. These plasmids were used to upregulate or downregulate the A20 level and investigate the effects on neuronal morphology and synaptic function. Overexpressing A20 diminished spine (mushroom, stubby, and thin subtypes) sizes and reduced dendritic spine densities. Sholl analysis showed that A20 upregulation also decreased neurite arborization numbers at medial-distal branches. Consistently, A20 downregulation significantly enlarged mushroom and stubby spine size and modestly increased spine density, which was rescued by an RNAi-resistant A20 cDNA construct. Moreover, electrophysiological recording of mEPSCs from rat hippocampal neurons showed a slight reduction of the mEPSC frequency but a significantly greater mEPSC amplitude when A20 was knocked down by transducing shA20 lentivirus. Finally, in an attempt to identify potential A20 interacting proteins, we conducted a yeast two-hybrid screening assay using a mouse brain cDNA library. Taken together, this study provided strong evidence that A20 regulates neuronal morphology including dendritic spine size, density, and neurite complexity as well as efficacy of synaptic transmission.