Abstract
Two major glutamate decarboxylase isoforms (i.e., GAD65 and GAD67) together synthesize the majority of γ-aminobutyric acid (GABA) in our nervous system. However, the subcellular distribution of these enzymes and their relative impacts on synaptic GABA release remain unclear. To address this important question, here we monitored their synaptic trafficking in male and female mouse brains and dissociated neuronal cultures. We noticed that, unlike some major glutamate-biosynthesizing enzymes, e.g., glutaminase (GLS) and glutamate dehydrogenase (GDH), which were primarily associated with perisomatic mitochondria, both GADs together were highly enriched at GABAergic presynapses. Nevertheless, when expressed separately in GAD-deficient human neurons derived from a male stem cell line, GAD65 exhibited preferential distribution at presynapses over GAD67. Despite these differences in subcellular localization, both GADs produced equivalent levels of intracellular GABA, which adequately diffused to axon terminals, and triggered robust GABAergic activities. These findings raised the question of whether the presynaptic recruitment of GADs is, after all, necessary for reliable GABAergic transmission. To examine this hypothesis, we further swapped or removed the trafficking signals from both GAD isoforms, and even artificially restricted them at non-synaptic compartments, including the cell nucleus. Despite our attempts, the chimeric and mutant GAD variants continued to produce sufficient amount of intracellular GABA for vesicular loading and presynaptic release. These results indicate that GAD65 and GAD67 are functionally redundant in GABA production, if expressed equitably in neurons; and irrespective of GADs’ subcellular trafficking profile, diffusion of GABA molecules from distant sources can effectively supply and replenish the presynaptic terminals for functional activities.
Significance statement The inhibitory transmitter GABA plays a vital role in modulating neuronal excitability. GABA is metabolized from glutamate by two enzymes, GAD65 and GAD67. Interestingly, these proteins contain distinct trafficking signals, exist as soluble vs. membrane-bound forms, and exhibit contrasting subcellular distribution. It is unclear if these differences between GAD isoforms can indirectly influence GABAergic neurotransmission. Here we demonstrate that, when expressed equitably, both GADs synthesized comparable amounts of GABA that diffused throughout the intracellular space, and were adequately packaged into synaptic vesicles for action-potential dependent and independent release. GADs’ N-terminal residues regulated their trafficking without affecting GABA production by their C-terminal domains, which was sufficient for effective synaptic activities. Hence, synapses can successfully utilize principal neurotransmitters manufactured at a distance.
Footnotes
The authors declare no competing interest in relation to the work described in this study. Request for experimental reagents should be addressed to S.C. (soham.chanda{at}colostate.edu).
This work was supported by start-up funding from the Colorado State University to S.C., and grants from the National Institutes of Health to M.A.X. (R01-DC015508) and S.C. (R01-MH126017). We thank Dr. James Bamburg (Colorado State University) for some reagents, including antibodies and cell lines.
↵*These authors contributed equally: O.B. and C.H.K.
