Characterization of Amino Acid Transporters : Transporters expressed in the central nervous system belonging to the Solute Carrier family SLC38

In cells and organelles transporters are responsible for translocation of amino acids, sugars and nucleotides among others. In the central nervous system (CNS), amino acid transporters can function as neurotransmitter transporters and nutrient sensors. The Solute carrier (SLC) superfamily is the largest family of transporters with 395 members divided in 52 families. The system A and system N amino acid transporter family, SLC38, consists of 11 members, SNAT1-11 (SLC38A1-11). The members are expressed in the brain, exclusively in neurons or astrocytes and some in both. Amino acid signaling is mainly regulated via two pathways, the amino acid responsive (AAR) pathway and the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) pathway. These pathways regulate the protein synthesis in opposite directions depending on the amino acid availability. SLC38 members along with other SLCs have been identified to participate in these pathways. In paper I, the regulation of SLC genes after complete amino acid starvation in mouse hypothalamic cells have been studied with microarray and we found that 47 SLC genes were significantly altered at five hours of starvation. Interestingly, we found that Slc38a1 and Slc38a7 were upregulated along with the known starvation responding gene, Slc38a2. A complementary starvation study for the SLC38 genes was performed using primary mouse embryonic cortex cells. We found that Slc38a1, Slc38a2, Slc38a5, Slc38a6 and Slc38a8 were upregulated while Slc38a3, Slc38a7 and Slc38a11 were downregulated. Three members from the SLC38 family, SNAT8 (paper IV), SNAT9 (paper III) and SNAT10 (paper II) have been histologically characterized in mouse brain and all these transporters are exclusively neuronal. SNAT8 and SNAT10 were also functionally characterized and shown to be transporters for alanine and glutamine among others. SNAT8 was shown to mediate sodium dependent transport and was classified to system A. SNAT10 was shown to be a sodium independent bidirectional transporter and displayed characteristics for system A and N. SNAT9 is a lysosomal component of the Ragulator-Rag complex which senses amino acid availability and activates mTORC1. In paper III we also found that Slc38a9 gene expression was upregulated following starvation and downregulated following high-fat diet in mouse brain

Description of the protein data sets used for the phylogenetic analysis.

<p>Description of the protein data sets used for the phylogenetic analysis.</p

Primer sequences used in the quantitative real-time PCR analyses.

<p>Primer sequences used in the quantitative real-time PCR analyses.</p

mRNA expression in wildtype and food controlled mice.

<p>Relative mRNA expression of <i>Mfsd4a</i> and <i>Mfsd9</i>, in central and peripheral tissues from adult C57Bl6/J mice, was analysed using qPCR. Samples were made from tissue collected from five animals per organ. The mRNA was normalized against the geometric mean of the reference genes <i>Gapdh</i>, <i>bTub</i>, <i>Rpl19</i>, <i>Cyclo</i> and <i>Actb</i>. The relative expression levels (±SD) were plotted. <i>Mfsd4a</i> (A) and <i>Mfsd9</i> (B) were detected in both central and peripheral tissues.</p

Neuroanatomical distribution of MFSD9.

<p>MFSD9 histology was displayed using colorimetric staining on 70μm coronal sections from adult mouse brains. Overview micrographs (A-D), with magnifications (E-L) are showed. MFSD9 staining in striatum (CPu) (E), cortex (F) with possible projections stained in layer 4 and 5 (G) and hypothalamic areas around third ventricle (3V) (H). In thalamus cell bodies were marked by MFSD9 (I). In hippocampus, both soma and projections were detected in CA2, while only soma was seen in CA3 (J). A close up of cells in brainstem (K) and the Purkinje cell layer (L) showed prominent staining. Bregma regions were according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186325#pone.0186325.ref051" target="_blank">51</a>], and scale bars represent 100μm.</p

Antibody verification and fluorescent protein staining.

<p>Antibodies used in the histological methods were verified using western blot with proteins from a fractionated mouse brain. (A) Staining for MFSD4A gave two bands,40 and 56 kDa (expected sizes at 40 and 56 kDa), in the brain sample and (B) staining for MFSD9 showed a band at 60 kDa (expected size at 51 kDa) in the mouse brain. 7μm coronal adult mouse brain sections were stained for MFSD4A and MFSD9, together with the neuronal marker NeuN and astrocytic marker GFAP, and representative staining is shown. Both MFSD4A (green in C) and MFSD9 (red in D) staining co-localised with NeuN. No overlay was detected between MFSD4A (green in E) and GFAP (red in E), or MFSD9 (red in F) and GFAP (green in F). DAPI was used as a nuclei marker and stained in blue.</p