In this study we used quantitative RT-PCR (qPCR) in a large panel of adult rat brain and peripheral tissues (Figure 1), to further refine the expression profile of the Slc6a17 gene. The highest expression levels of Slc6a17 mRNA were found in hindbrain, various brain cross sections, cerebellum, spinal cord, brain stem and hypothalamus, while very low or no expression was seen in the peripheral tissues with the exception of epididymis. Consequently, the Slc6a17 transporter is highly and selectively expressed in the CNS of adult rat.
Abundant mRNA expression of Slc6a17 in adult and embryonic rat CNS has previously been shown using in situ hybridization. Consistent results indicated restricted expression exclusively in neurons, both glutamatergic and subsets of GABAergic [4, 9, 15–17]. Our results from in situ hybridization (Figure 3) shows that the mouse Slc6a17 gene has similar expression pattern as previously seen in rat, with high expression in mouse forebrain and midbrain and lower expression in some parts of the hindbrain such as the basal ganglia. Low mRNA expression was seen in spinal cord. We confirmed Slc6a17gene expression in GABAergic neurons in hippocampus, in the pyramidal and granule cell layer of the dentate gyrus , and in the Purkinje cell layer of cerebellum . The mRNA expression was also found in all layers of cerebral cortex, except layer 1, with strongest expression in cortical layer 5, results that support expression by glutamatergic neurons . The layer-like pattern in cortex gives strength to the conclusion that the expression is neuronal, while a scattered pattern would suggest expression in astrocytes. Hybridization in sections of mouse spinal cord showed expression in subsets of somatic motor neurons and in interneurons. Cortical co-localization of Slc6a17 mRNA with glutamatergic (PAG) and GABAergic (Gad67) mRNA markers confirmed Slc6a17 staining in both excitatory and inhibitory neurons in brain (Figure 2), while co-immuno labeling with the astrocyte protein marker GFAP showed no overlap in spinal cord, suggesting no expression of Slc6a17 in glial cells. The expression in Piris is highly interesting as this area have been shown to be part of a possible amino acid chemo-sensing system, involved in recognition of diets with deficiency of essential amino acids, producing an anorectic response [44, 45]. We also found expression of Slc6a17 in many regions involved in homeostatic control, with pronounced hypothalamic expression specifically in VMH, DMH, LH and Arc. In addition, we found expression in the amygdala (BLA), the pons (LC), and the ventral striatum (NAcc). These are all areas known to be part of the reward network and in regulation of food intake [46–49]. The Slc6a17 mRNA expression pattern in mouse CNS strongly suggests that Slc6a17 play a role in both excitatory and inhibitory neurotransmission and that the transporter could be involved in regulating food intake.
Here, we used a custom-made mouse B0AT3 antibody for further cellular localization of the B0AT3 protein in mouse brain and spinal cord. Immunohistological double labelling on primary cell cultures with the neuronal cell marker (MAP2) and the neuronal terminal marker (synaptophysin) visualized B0AT3 localization to neurons, with expression both in soma and in synapses (Figure 6). In addition, PLA studies showed protein proximity between antibodies targeting B0AT3 and synaptophysin, a marker for synaptic vesicles, in a hypothalamic mouse cell line (Figure 5). Localization to synapses was also confirmed with double labelling on mouse brain and spinal cord sections (Figures 4 and 5). GABAergic markers (GAD67 and VIAAT) were used and showed neuronal B0AT3 localization in GABAergic neurons and in GABAergic synapses. Interestingly, glutamatergic markers (PAG, VGLUT1 and VGLUT2) showed that B0AT3 is also localized at glutamatergic neurons and in glutamatergic synapses. Direct protein interactions between B0AT3 and PAG in glutamatergic neurons were shown with PLA. We further investigated whether B0AT3 was expressed in choroid plexus, by co-staining with pan-cytokeratin to visualizethe cellular layer surrounding the ventricles of the brain. Highly overlapping expression has previously been shown for B0AT2 and the choroid plexus marker (unpublished data), here B0AT3 and pan-cytokeratin did not co-localize. Despite the absence of co-localization within cells, B0AT3 still localized to the choroid plexus, but only to the brain side of choroid plexus cells. Possibly, B0AT3 and B0AT2 function within these cells and contribute to the uptake of circulating amino acids from the blood stream into the brain. B0AT3 has been suggested to be H+-dependent , but another study has shown that it is Na+-dependent . Our results, showing very clear expression in synapses (most likely located in vesicles), support that B0AT3 is H+-driven, a feature typical for other vesicular transporters such as VGLUTs, VIAAT and VMAT[38, 50]. However, some IHC staining presented here suggest that B0AT3 is also found in the soma of neurons, most likely with expression in the plasma membrane. If this expression is functionally relevant, B0AT3 is most likely utilizing the Na+-gradient over the plasma membrane to drive transport. This is supported by previous studies that have shown that, transport through B0AT3 can be driven by H+ as well as Na+ gradients .
Both acute and chronic food restriction (Figure 7) induce activation of synaptic neurotransmission in hypothalamus, which in turn boasts a strong signal to start food seeking and consumption. Starvation increases the levels of circulating ghrelin which activates neurons in hypothalamus to release the excitatory neurotransmitter glutamate [51, 52]. Glutamate can in turn activate the N-methyl-D-aspartate (NMDA) receptor [53, 54] located on GABAergic neurons, also expressing NPY and AgRP, and hence stimulate feeding . Our results show that Slc6a17mRNA is highly regulated in response to starvation, suggesting that up-regulation of the transporter could be part of the enhancement of the excitatory signal during starvation (Figure 7A), while the expression of Slc6a15 appeared to be less affected. The effect was only seen for long term food restriction, and was not observed after acute food deprivation. Up-regulation of the Slc6a17 transporter indicates increasing uptake of amino acids into vesicles, a function that could influence a number of other mechanisms. B0AT3 has a broad transport profile displaying, among others, uptake of glycine. Of all transporters of amino acids, there are a number of known transporters, with high capacity for glycine transport (GlyT1 (SLC6A9), GlyT2 (SLC6A5), SNAT2 (SLC38A2), PAT1 (SLC36A1), VIAAT (SLC32A1) [3, 50, 55, 56]), although only B0AT3 has vesicular expression. Here, we also study the mRNA expression of Slc6a17 in POMC and NPY positive cells. The Slc6a17 transporter showed localization both at hypothalamic POMC and NPY neurons (Figure 2), which is in agreement with the possible role in body weight homeostasis.
When we challenged the 5-HT (fluoxetine) and the DA/NA systems (bupropion) in rats over a treatment period of fourteen days, we found significant changes in the levels of transcripts for Slc6a17 and Slc6a15 (Figure 7C-D). Administration of fluoxetine, an antidepressant of the selective 5-HT reuptake inhibitor (SSRIs) type, resulted acutely in an increase of 5-HT in the synaptic cleft. Both Slc6a17 and Slc6a15 were significantly up-regulated in hippocampus after fluoxetine injections. Similar stimulatory effect on hippocampal and cortex expression is observed for the vesicular glutamate transporter VGLUT1 in response to antidepressant treatment with fluoxetine, and the transporter is linked to increased 5-HT levels [57, 58]. Moreover, mice with reduced VGLUT1 expression show increased anxiety, depressive-like behaviors, and impaired recognition memory . Bupropion acts as a dual NA and DA uptake inhibitor in several mesocorticolimbic areas . The drug inhibit the reuptake of NA and DA through blockage of the NA transporter (NET/SLC6A1) and DA transporter (DAT/SLC6A3) in humans, without affecting release or transport of other neurotransmitters and without binding to other neurotransmitter receptors . We showed that increased levels of DA and NA gave significant up-regulation of both Slc6a17 and Slc6a15 in prefrontal cortex, up-regulation of Slc6a17 in hypothalamus and of Slc6a15 in hippocampus. The results once again demonstrate that the expression levels of Slc6a17 and Slc6a15 are regulated by changes in monoamine levels. We suggest that the increase of these transporters may have a role in regulating the availability of amino acids used for neurotransmitter precursors. The Slc6a15 results are supported by results from a previous study by Kohli et al. where SLC6A15 was associated with major depression. Here it was found, in a whole genome association study, that allelic variants of SLC6A15 increased the risk of acquiring major depression, although the mechanism behind this is not known. Antidepressant drugs are thought to acutely increase the levels of 5-HT and NA/DA in the brain but, in long term antidepressant drugs, it has been shown to induce remodeling of neuronal circuits by strengthening of synapses , involving both glutamatergic and GABAergic neurons and possibly SLC6A15 and SLC6A17 could play a role in these long term effects. It is also known that starvation induces glutamatergic signaling in POMC neurons of the hypothalamus and it is possible that B0AT3 enhances the signaling capabilities of glutamatergic neurons. B0AT3 is unique among the SLC6 family of proteins in that sense that it is expressed on vesicles rather than on the plasma membrane. B0AT3 has been shown to transport, among other amino acids, glycine. Glycine has at least two important functions in glutamatergic neurotransmission; first, glycine can be used for the synthesis of the excitatory neurotransmitter glutamate, and secondly, glycine is a necessary co-factor for the ability of glutamate to activate NMDA receptors [53, 54, 62]. The up-regulation of the Slc6a17 transporter after long term food restriction and activating drugs suggests the transporter to be involved in the increased glutamatergic signaling. It is possible that B0AT3 provides a mechanism to enhance NMDA receptor activation, possibly providing stronger LTP response, by packing glycine into glutamate containing vesicles and hence increase the local concentration of glycine in the synaptic cleft.