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.

There are currently over 430 known SLC transporters, over 30% of which have an unknown function. Compared to other transporter gene families, the SLC genes are relatively understudied with many orphan genes. SLC transporters have a high disease relevance and can be associated with many different diseases like gout, type 2 diabetes and different forms of cancer. SLC transporters also appear to be very druggable, thus offering a rare opportunity of an underexplored gene family, that can be linked to many diseases and seem to have a general druggability with small organic molecules. This thesis is evaluating three specific SLC transporters of the SLC38 family to discover their different roles and purposes. In this project CRISPR-Cas9 is used to knockout three SLC38 transporters, called SNAT6, SNAT8 and SNAT10. The cell-line used is HEK293 cells, as they are easy to transfect and are thought to express the three genes, however it is not certain that they do express the three SNAT genes. The project aims to optimize the method for best possible transfection by trying different protocols. A literature study is done on what the future experiments of the knocked-out cells could be, including; ensuring the HEK293 cells express the three genes, controlling the effectiveness of the transfection and analyzing the result of such a transfection. To confirm that the HEK293 cells do express the three SNATs a western blot assay could be performed. RT-qPCR is found to be useful in evaluating whether the knockouts are successful, by measuring if the three SNAT transporter proteins are present or not in the knocked-out cells. A metabolic analysis study to determine the result of the knockouts is also described as a future experiment. The experimental finding was a CRISPR-Cas9 transfection method that yielded enough RNA, enabling future experiments such as RT-qPCR.