Abstract
GABAA receptor-associated protein (GABARAP) was initially identified as a protein that interacts with GABAA receptor. Although LC3 (microtubule-associated protein 1 light chain 3), a GABARAP homolog, has been localized in the dendrites and cell bodies of neurons under normal conditions, the subcellular distribution of GABARAP in neurons remains unclear. Subcellular fractionation indicated that endogenous GABARAP was localized to the microsome-enriched and synaptic vesicle-enriched fractions of mouse brain as GABARAP-I, an unlipidated form. To investigate the distribution of GABARAP in neurons, we generated GFP-GABARAP transgenic mice. Immunohistochemistry in these transgenic mice showed that positive signals for GFP-GABARAP were widely distributed in neurons in various brain regions, including the hippocampus and cerebellum. Interestingly, intense diffuse and/or fibrillary expression of GFP-GABARAP was detected along the axonal initial segments (AIS) of hippocampal pyramidal neurons and cerebellar Purkinje cells, in addition to the cell bodies and dendrites of these neurons. In contrast, only slight amounts of LC3 were detected along the AIS of these neurons, while diffuse and/or fibrillary staining for LC3 was mainly detected in their cell bodies and dendrites. These results indicated that, compared with LC3, GABARAP is enriched in the AIS, in addition to the cell bodies and dendrites, of these hippocampal pyramidal neurons and cerebellar Purkinje cells.
Highlights
GABAA receptor-associated protein (GABARAP) was first isolated as a protein interacting with the intercellular loop of the gamma2 subunit of GABAA receptor [1,2], with the human, mouse, rat and bovine orthologs of GABARAP being 100% identical at the amino acid level [2]
By double staining for GFP and Ankyrin-G or pan voltage gated sodium channels, we have shown here that GFP-GABARAP is enriched in the axonal initial segments (AIS), in addition to dendrites and somata, of hippocampal pyramidal neurons and cerebellar Purkinje cells, while endogenous LC3 is mainly present in the cell bodies and dendrites of these neurons
Previous studies in the neurons of GFPLC3 transgenic mice have suggested that, like endogenous LC3, GFP-LC3 is expressed primarily as diffuse and/or fibrillary signals in the cell bodies and dendrites of neurons [28,31], but is not concentrated in the AIS [30]. These findings indicate that the preferential localization of GFP-GABARAP in the AIS is not due to side effects of its overexpression
Summary
GABAA receptor-associated protein (GABARAP) was first isolated as a protein interacting with the intercellular loop of the gamma subunit of GABAA receptor [1,2], with the human, mouse, rat and bovine orthologs of GABARAP being 100% identical at the amino acid level [2]. PRIP-1 regulates GABAA receptor surface expression by inhibiting the interaction between GABARAP and GABAA receptor. Disruption of the PRIP-1 gene in mice results in the impairment of Zn2+ modulation of GABA-induced Cl2 current in hippocampal neurons, the inhibition of motor coordination and alterations in GABAA receptor pharmacology. Studies have focused on the interaction of GABARAP with GABAA receptor in neurons, GABARAP expression is ubiquitous, being observed in the rat liver, testes, lungs, spleen, thyroid gland, heart, skeletal muscle, and kidneys [10]. GABARAP deficiency has been shown to modulate the expression of NaPiIIa in renal brush borders [9]
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