Elucidating the role of GODZ-mediated palmitoylation in GABAergic inhibition
- Area of Honors:
- Bachelor of Science
- Document Type:
- Thesis Supervisors:
- Bernhard Luscher, Thesis Supervisor
- Richard W Ordway, Honors Advisor
- Neuroligin 2 (NL2) is a postsynaptic cell adhesion protein that is selectively found at GABAergic but not glutamatergic synapses. Previous unpublished experiments by the Luscher lab have indicated that NL2 interacts with gamma-aminobutyric acid type A receptors (GABAARs) and that this interaction might be important for targeting these receptors to synapses. Another line of research has found that NL2 is palmitoylated when co-expressed in heterologous cells with the palmitoyltransferase, Golgi-specific DHHC zinc finger protein (GODZ). Neurons that have the gene for GODZ deleted show a selective reduction in the density of GABAergic synapses on dendrites of neurons, indicating that GODZ-mediated palmitoylation of NL2 might contribute to synapse formation. The first aim was to assess if palmitoylation of NL2 is required for normal postsynaptic accumulation of GABAARs. Two lentiviral plasmids were constructed for either an HA-tagged WT or an HA-tagged mutant version of NL2 that had the codons of the two cytoplasmic Cys residues representing putative palmitoylation sites mutated to Ala codons. The prediction was that some or all of the mutated HA-NL2 viruses would show reduced GABAARs accumulation at synapses when compared to WT HA-NL2. However, when these lentiviruses were used to transduce neurons they proved to be toxic, which precluded this approach from being informative. As an alternative strategy, the lentiviral plasmids were then directly electroporated into neurons and the neurons immunostained for HA-NL2 and the inhibitory pre-and postsynaptic markers, VGAT (vesicular GABA transporter) and gephyrin, respectively. This method showed efficient transfection of neurons but preliminary immunostaining was weak and not suitable for quantitative analyses of synapses. The second aim was to determine whether GODZ is more localized to the cis or trans face of the Golgi to allow predictions on how GODZ-mediated palmitoylation might affect the trafficking of substrate proteins. HEK 293T cells were transfected with either the cis or the trans Golgi markers, GFP-N3 and GalT-YFP, respectively, and immunostained for these cis and trans markers and GODZ. It was predicted that GODZ is more likely to be localized in the cis face of the Golgi than the trans face, as this would support the theory that GODZ facilitates the trafficking of GABAARs from the endoplasmic reticulum (ER) to the Golgi. This hypothesis was supported when I found that GODZ was significantly more colocalized with the cis-Golgi marker, GFP-N3, than the trans-Golgi marker, GalT-YFP.