Characterization of Centrally Expressed Solute Carriers

Home , Glycine transporter, Passive transport, RHCG, Sialin, SLC15A4, SLC22A1

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1215

Histological and Functional Studies with Transgenic Mice

SAHAR ROSHANBIN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9555-8 UPPSALA urn:nbn:se:uu:diva-282956 2016 Dissertation presented at Uppsala University to be publicly examined in B:21, Husargatan. 75124 Uppsala, Uppsala, Friday, 3 June 2016 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Biträdande professor David Engblom (Institutionen för klinisk och experimentell medicin, Cellbiologi, Linköpings Universitet).

Abstract Roshanbin, S. 2016. Characterization of Centrally Expressed Solute Carriers. Histological and Functional Studies with Transgenic Mice. (. His). Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1215. 62 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9555-8.

The Solute Carrier (SLC) superfamily is the largest group of membrane-bound transporters, currently with 456 transporters in 52 families. Much remains unknown about the tissue distribution and function of many of these transporters. The aim of this thesis was to characterize select SLCs with emphasis on tissue distribution, cellular localization, and function. In paper I, we studied the leucine transporter B0AT2 (Slc6a15). Localization of B0AT2 and Slc6a15 in mouse brain was determined using in situ hybridization (ISH) and immunohistochemistry (IHC), localizing it to neurons, epithelial cells, and astrocytes. Furthermore, we observed a lower reduction of food intake in Slc6a15 knockout mice (KO) upon intraperitoneal injections with leucine, suggesting B0AT2 is involved in mediating the anorexigenic effects of leucine. In paper II, we studied the postnatal, forebrain-specific deletion of Slcz1, belonging to the SLC18 family, in conditional KO mice (cKO). We observed a decreased response to diazepam and a higher neuronal activity in cortex and hippocampus of cKO mice, as well as an impairment in short-term recognition memory. Intracellular expression was found in neurons but not astrocytes with IHC, indicating SLCZ1 is implicated in neuronal regulation of locomotion and memory. In paper III, we performed the first detailed histological analysis of PAT4, a transporter belonging to the SLC36 family, involved in the activation of mTOR complex 1 on lysosomes. We found abundant Slc36a4 mRNA and PAT4 expression in mouse brain, using ISH and IHC. We used IHC to localize PAT4 to both inhibitory and excitatory neurons and epithelial cells. We also found both intracellular- and plasmalemmal expression and partial colocalization of PAT4 with lysosomal markers. Lastly, in paper IV, we provided the first tissue mapping of orphan transporter MCT14 (SLC16A14). Using qPCR, we detected moderate to high Slc16a14 mRNA in the central nervous system and kidney. We found widespread Slc16a14 and MCT14 in mouse brain using ISH and IHC. We also found MCT14 to have intracellular and plasmalemmal expression in mainly excitatory but also inhibitory neurons, as well as epithelial cells. We found MCT14 to be most closely related to MCT8, MCT2 and MCT9, suggesting a similar role for this transporter.

Keywords: SLC, Solute carriers, Amino acid transporter, PAT, B0AT2, mTORC1

Sahar Roshanbin, Department of Neuroscience, Functional Pharmacology, Box 593, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-554-9555-8 urn:nbn:se:uu:diva-282956 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-282956)

To my driving school instructor who told me to use my brain

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Hägglund, MG., Roshanbin, S., Löfqvist, E., Hellsten, SV., Nilsson, VC., Todkar, A., Zhu, Y., Stephansson, O., Drgonova, J., Uhl, GR., Schiöth, HB., Fredriksson, R. (2013) B0 AT2 (SLC6A15) Is Localized to Neurons and Astrocytes, and Is Involved in Mediating the Effect of Leucine in the Brain. PLOS one. 8(3): e58651

II Roshanbin, S., Westerfors, E., Sreedharan, S., Schiöth, HB., Fredriksson, R. :(2016) Postnatal forebrain-specific deletion of neuronal transporter Slcz1. Manuscript

III Roshanbin, S., Hellsten, SV., Tafreshiha, A., Zhu, Y., Raine, A., Fredriksson. R. (2014) PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells. Brain research. 1557: 12-25

IV Roshanbin, S., Lindberg, FA., Lekholm, E., Eriksson, MA., Perland, E., Raine, A., Åhlund, J., Fredriksson, R. (2016) His- tological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney. Submitted manuscript

Reprints were made with permission from the respective publishers.

Additional Publications

V Schiöth HB, Roshanbin S, Hägglund MG, Fredriksson R: Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects. Mol Aspects Med 2013, 34(2-3):571-585.

VI Roshanbin S, Classical Mouse Genetics. In: Stanley Maloy and Kelly Hughes, editors. Brenner's Encyclopedia of Genet- ics, 2nd edition, Vol 2. San Diego: Academic Press; 2013. p. 680-681

VII Caruso V, Hägglund MG, Badiali L, Bagchi S, Roshanbin S, Ahmad T, Schiöth HB, Fredriksson R: The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas. Gene 2014, 553(1):1-6.

VIII Rottkamp DM, Rudenko IA, Maier MT, Roshanbin S, Yulyaningsih E, Perez L, Valdearcos M, Chua S, Koliwad SK, Xu AW: Leptin potentiates astrogenesis in the developing hypothalamus. Mol Metab 2015, 4(11):881-889.

Contents

Introduction ...... 11 Solute Carriers ...... 12 Nomenclature and classification ...... 13 Structure ...... 15 SLC substrates ...... 16 Localization of SLCs ...... 17 SLCs in drug transport ...... 17 SLCs as drug targets ...... 18 SLC-mediated transport in vesicles, peroxisomes and lysosomes ...... 20 SLCs in vesicles ...... 20 SLCs in peroxisomes and lysosomes ...... 21 SLCs and amino acid sensing: the mTOR and GCN pathways ...... 23 The mTOR pathway ...... 23 The GCN2 pathway ...... 26 The monocarboxylate transporter family ...... 27 Transport of monocarboxylates ...... 29 Transport of aromatic amino acids ...... 30 Transport of thyroid hormones ...... 30 Orphan transporters ...... 31 Aims ...... 32 Paper I ...... 32 Paper II ...... 32 Paper III ...... 33 Paper IV ...... 33 Methodological considerations: mouse genetics ...... 34 Results and discussion ...... 38 Paper I ...... 38 Paper II ...... 39 Paper III ...... 40 Paper IV ...... 41

Closing remarks: conclusions and perspectives ...... 42 Paper I ...... 42 Paper II ...... 42 Paper III ...... 43 Paper IV ...... 44 Acknowledgments...... 45 References ...... 48

Abbreviations

AARE amino acid responsive elements ABC ATP-bindig cassette ATPase Adenosine triphosphatase B0AT System B0 amino acid transporter CAT cationic amino acid transporter CNS central nervous system DBI Diazepam binding inhibitor DEPTOR DEP-domain containing mTOR interacting protein eIF2α eukaryotic initiation factor 2 α GABA γ-amino-butyric acid GCN general control non-derepressible ICC immunocytochemistry IHC immunohistochemistry ISH in situ hybridization LAT L-type amino acid transporter MCT monocarbocylate transporter MFS Major Facilitator Superfamily mLST8 mammalian lethal with SEC13 protein 8 mTOR mechanistic target of rapamycin mTORC1 mechanistic target of rapamycin complex 1 mTORC2 mechanistic target of rapamycin complex 2 PAT proton-dependent amino acid transporter PRAS40 40 kDa proline-rich protein of mTOR qPCR quantitative real-time PCR RAPTOR regulatory-associate protein of mTOR Rheb Ras homolog enriched in brain SLC Solute Carrier TM transmembrane tRNA transfer RNA

Introduction

Membranes are specialized barriers enclosing cells and organelles. Their unique properties and composition maintain the integrity of the cellular contents and homeostasis. They do so by separating vital metabolic processes within organelles and by controlling the influx and efflux of solutes. The innate properties of the amphipatic lipid bilayer that constitutes the membrane allows the aggregation and dispersion of proteins for optimal membrane organization. The functionality of the membrane relies largely on the membrane protein component. Its central role in physiological processes makes it the key target for approximately 60% of FDA-approved drugs [1]. The integral membrane proteins are gatekeepers that convey signals from the surroundings and act as receptors, transporters or enzymes. These proteins constitute 27% of the entire human proteome [2, 3], and the transporters make up the second largest group of membrane proteins. Transporters move substrates across membranes, and are divided into three classes depending on their mode of transport: passive transport, and primary- and secondary active transport [4]. Ion- and water-channel proteins are passive transporters (also called facilitated transporters) as they allow diffusion of solutes along their electrochemical gradient [4]. Active transporters translocate substrates using energy from chemical reactions: primary active transporters use energy derived directly from ATP hydrolysis [4] whereas secondary active transporters use an independently established electrochemical gradient to drive transport against a concentration gradient [4]. An example of a primary active transporter is the mammalian ATP-binding cassette (ABC) transporter P-glycoprotein 1, a protein involved in multidrug resistance [5]. ATP-driven ion pumps (ATPases) such as the Na+/K+ pump [6], are also primary active transporters; they pump ions out of cells or into organelles and thereby cre- ate an electrochemical gradient that can be used by the secondary active transporters. The secondary active transport Solute Carrier (SLC) superfamily is by far the largest group of human transporters and currently comprises 456 human solute carriers divided into 52 subfamilies [7-11]. Transporters from the SLC superfamily will be the main focus of this thesis.

11 Solute Carriers SLCs display a broad substrate profile with specificity for structurally diverse compounds, ranging from amino acids and neurotransmitters to fatty acids, drugs and metal ions, amongst others [12]. SLCs primarily function as co-transporters, meaning they couple the transport of the substrate against a concentration gradient with the movement of another molecule or ion down its electrochemical gradient, either in the same direction (symporters) or in the opposite direction (antiporters) [4]. They can also transport solutes in a facilitated manner, where the solute is transported along a concentration gradient [8] (Fig. 1). SLC-mediated transport can be electronegative, elec- tropositive or electroneutral depending on the substrate and ions coupled to transport, and the subsequent intracellular net charge. Generally, the greater the electrochemical gradient, the greater the rate of solute entry [13]; however, this can be affected by the membrane permeability, the number of transporters, and the mode of transport [14]. For facilitated diffusion, the net transport of solutes depends on the sum of electrical and chemical gradients acting on the solute, and the direction of transport is determined by the direction of the larger force [14]. For coupled transport, the rate is determined by the number of available transporters and the rate of transport by the individual transporters. The direction of transport depends on the direction of the electrochemical gradient [14]. When all the solute binding-sites are occu- pied, the SLCs are saturated, reaching the maximum rate of transport, or Vmax, and are thus subject to inhibition [13]. Primary active transporters un- dergo conformational changes to an open state upon hydrolysis of ATP and subsequent phosphorylation of the transporter [13]. In contrast, SLCs under- go conformational changes upon binding substrates and co-substrates [15]. Besides transport of substrates, some SLC family members also play an important role in amino acid sensing, to assess the status of tissue nutrient sup- plies [16]. A "remote sensing and signaling" hypothesis has been postulated wherein transporters work together to sense fluctuations in substrate levels [17, 18].

12 PLASMA MEMBRANE Glucose SLC2, SLC50 Amino acids SLC7. SLC43 Thyroid hormones SLC16 Organic cations SLC20 Acetyl-CoA SLC33 Bile acids SLC51

PLASMA MEMBRANE Amino acids SLC1.SLC38,SLC36 Bicarbonate SLC5 Fatty acids SLC5. SLC16 Bile acids SLC10 Metal ions SLC11 Dicarboxylates SLC13 Peptides SLC15 Monocarboxylates SLC16 Inorganic phosphates SLC20, SLC34 Ascorbic acid SLC23 Nucleosides SLC28 Glucose SLC45 Folates SLC45 Heme SLC48

LYSOSOMES Amino acids SLC36, SLC38

PLASMA MEMBRANE Amino acids SLC7, SLC3 Ions SLC8 Inorganic phosphates SLC37

MITOCHONDRIA Ions SLC24, SLC35

VESICLES Neurotransmitters SLC18, SLC32

PEROXISOMES Co-factors SLC25

GOLGI Nucleotide sugars SLC35 Figure 1. Schematic representation of SLC localization, transport mechanisms and substrates. SLC transporters are membrane-bound proteins divided into uniporters (blue), symporters (orange) or antiporters (grey). Symporters and antiporters couple the transport of substrates against their concentration by co-transporting a substrate, usually Na+ and H+, along its concentration gradient. The main substrates and the sub-cellular localizations of each SLC family are listed in the boxes to the right. Source: www.bioparadigms.org. Credit to Somersault18:24 (www.somersault1824.com) for figure components, shared under a creative commons license (CC-BY-NC-SA 4.0).

Nomenclature and classification The current nomenclature of SLCs was adopted in the 1990s by the HUGO Gene Nomenclature Committee (HGNC) [19]. The denotation of a human gene starts with the SLC root, followed by a numeral denoting the family (e.g. 1 for the SLC1 family), and a letter that acts as a divider between the numerals. This letter is usually A, but for families with further subdivisions, the other families are denoted in an alphabetical order. Lastly, a number designates the individual member. Families and members have been named in the order of their discovery. Orthologs of the human genes commonly use the same designation, although the letter case may vary, e.g. Slc16a14 is the rodent ortholog of SLC16A14 [19]. The inclusion criterion for the SLC superfamily is functional, i.e. integral membrane proteins that transport solutes. However, in accordance with the criterion set for human transporters by

13 HGNC, the encoded proteins within a specific family have to share at least 20% sequence similarity [8]. Currently, there are several other classifications proposed for transport proteins. The Transporter Classification (TC) system is a database in which all transporters, including SLCs across all organisms, are divided into a functional/phylogenetic five-level hierarchal system of classes, subclasses, families, subfamilies, and transport systems (http://www.tdcb.org/) [20-22]. The Protein family (Pfam) database is another classification system, aimed at the entire proteome of all organisms assessed manually, and those Pfam entries judged to be likely homologs are compiled into clans [23]. To date, there are three clans that populate more than one SLC family: the major facilitator superfamily (MFS), the amino acid - polyamine - organocation (APC) superfamily and the cation:proton antiporter (CPA)/anion transporter (AT) clan [24], while the other SLCs belong to Pfam families that are not part of any clans. The MFS clan is the largest clan of membrane transporters found in humans, and transports a variety of substrates by different transport routes [25, 26]. The APC clan primarily contains amino acid transporters, including some SLCs, and due to their relatively high degree of sequence similarity, they are also considered a superfamily [27]. The CPA/AT clan of transporters contains ion transporters. The first comprehensive phylogenetic analysis of the entire SLC superfamily resulted in the clustering of the 15 of the SLC families in to four major phylogenetic groups: the α, β, γ, and δ groups [28]. The α-group is the largest cluster and contains nine SLC families, including the related non- SLC Synaptic Vesicle 2 (SV2) proteins. The β-group is the second largest cluster and contains three SLC families of amino acid transporters while the γ- and δ-groups contain two SLC-families each [28, 29]. The respective clus- ters of SLCs are believed to have had a common evolutionary origin in mammals and they share sequence and structural similarities [28]. Subse- quent analysis of the SLC family has shown that the α-group is equivalent to a sub-branch of the MFS clan, denoted as MFS_1 [24]. The β-group of amino acid transporters constitutes the APC superfamily clan. The γ-group is represented in the CPA/AT clan, while the δ-group belongs to the Na_Ca_Ex family according to Pfam nomenclature, and is not part of any clan [24]. An additional 18 SLC families are classified as Pfam families that are not a part of any clan [24]. An analysis of whole genome sequences from 17 eukaryotic species revealed that SLCs were present in the common ancestor of eu- karyotes, eubacteria and archaea [24], suggesting a long evolutionary history for these transporters. Yet another classification scheme has been proposed that uses so called similarity networks [30]. To depict distant relationships between proteins, this method categorizes protein-protein interactions on a large scale [31] rather than on the basis of their sequence-sequence alignments [9]. With this classification method, the division of transporters is similar to that of the HGNC and the phylogenetic classifications discussed previously, albeit with

14 some diverging families. The similarity network also shows that functional traits, such as the mode of transport and substrate type, are conserved within most families, and that the function of a given SLC family is weakly linked to the complexity of the organism [9]. It is important to establish the relationship between SLC families, as transporters with a common evolutionary origin tend to adopt similar structures and molecular function.

Structure A powerful tool in the study of novel SLCs and their substrates is to use the prediction of structural and functional features of uncharacterized transport proteins based on their alignment with characterized, aligned homologs. One example is the theoretical functional overlap between the SLC22 and SLC2 families, predicted by similarity network analysis. The overlap was later confirmed through kinetic measurements, when representative members from each family were found to have a common substrate, ureic acid [9]. The functional role of SLCs are determined by their structure and the physiochemical properties of the binding site, which in turn determines the types of substrates that can bind and be transported and mode of transport. SLCs can have similar functions despite a low degree of sequence similarity, due to the evolution of different tertiary folds performing similar functions [9]. There are a number of common traits shared by the SLC transporters: they are integral transmembrane proteins, and are composed of a large domain consisting of 10-14 hydrophobic transmembrane α-helices connected by hydrophilic intra- or extracellular loops. Most SLCs contain evolutionarily related, inverted structural repeats [20]. These sequences are highly diver- gent, and the first helix of each repeat is often involved in substrate binding [32, 33]. The functional characterization of SLCs has been hampered by the fact that they are complex integral membrane proteins with hydrophobic domains, making them difficult to express. Consequently, there are very few crystal structures of eukaryotic SLCs. Some structures from lower organisms such as bacteria have been determined, and have served as templates for the modeling of human transporters [34, 35]. However, due to the low degree of identity between the human SLC proteins and their prokaryotic homologs, this has proved to be difficult [9]. To date, the only crystals structures for human transporters are transporter Rh type C (RHCG, SLC43A3), a glycoprotein involved in blood group compatibility [36], and the glucose transporters, GLUT1 (SLC2A1) and GLUT3 (SLC2A3) [37, 38].

Figure 2. Schematic image of a 12 transmembrane (TM) transporter, containing 12 distinct domains and an intracellular N- and C-terminal. Drawn using Protter [39].

SLC substrates The basic notion of a transporter is a protein complex that changes the loca- tion but not the chemical nature of its substrate [15, 32]. SLCs transport a wide array of endogenous and xenobiotic compounds in biological systems: essential biomolecules, environmental toxins, and therapeutic drugs, amongst others (Fig. 1). The mode of transport is a conserved trait within families that are evolutionarily related and this also applies to their substrates [9]. The structures of SLCs reveal a number of key features that determine their substrate binding specificities: the presence of polar groups, hydrophobic or aromatic residues, and charged groups [10]. SLCs, like all proteins, are made of chiral amino acids, which gives them a more rigid structure than the fluid membrane into which they are integrated. As a result, they exhibit stereospecific transport of molecules [40], which is of particular interest in drug transport. The current view is that SLCs transport substrates through an alternating access mechanism [15, 41], i.e., conformational changes of the transporter make the substrate-binding site accessible from either side of the membrane [6]. The transfer of the solute occurs through reversible conformational changes in the transporter that take place when a substrate is bound or dissociated; however, the underlying mechanisms are unknown [6]. SLCs share similar structural core machinery responsible for binding substrates, i.e. inverted structural symmetry motifs [32, 42]. These motifs commonly form the distinct conformations for moving solutes: the outward-open for substrate upload, substrate-bound occluded, and inward-open for substrate release [42, 43]. The physiochemical properties of the binding site, as

16 well as the shape and size of the substrate, affect the transporter-substrate interactions and potential transport. For symporters and antiporters, the substrate binding is coupled to the transport of a co-substrate, usually Na+ or H+. However, the principles behind the specific ions or molecules being co- or counter-transported remain unclear [32]. Multiple transporter types act together to regulate the overall membrane transport and the final concentration of substrates in a specific tissue. This is of particular interest in drug transport across the epithelia lining the kidney, intestine and liver, because of the uptake of drugs and elimination of me- tabolites [44]. There are several approaches to finding substrates for orphan SLCs, including uptake assays and structure-based substrate discovery. In the latter, a large virtual library of small organic molecules is computational- ly screened against the atomic structure of the protein. For SLCs where no determined structures are available, the screen can be performed with substrates against a comparative protein structure model [10, 45, 46].

Localization of SLCs The availability of crystal structures for transporters has contributed greatly to the understanding of their involvement in both normal- and pathophysiol- ogy. The structures allow analysis of binding sites and mode of transport in different conformational states, as well as insights into substrate specificity and possible inhibitors. To obtain deeper knowledge about functional roles, however, the cellular and subcellular localization of SLCs in different tissues must be studied. SLC family members do not appear to cluster by tissue, i.e. most SLCs seem to be distributed over a variety of tissues, and most families exhibit a mix of tissue-specific and ubiquitous members [9]. A number of SLCs are differentially expressed in organs with specialized functions such as liver, kidney, intestines and brain, i.e. structures that do not exist in lower organisms. Analysis of the evolutionary origin of these families indicates that they appeared late in evolution as organisms became more complex and the need for specialized transport arose [9]. SLCs are expressed in the plasma membrane [8] and membranes enclosing various subcellular compartments such as synaptic vesicles [47], peroxisomes [48], lysosomes [49], Golgi bodies [50], and mitochondria [51].

SLCs in drug transport The localization of SLCs on the cell surface makes them targetable for therapeutic agents, where they also partake in drug disposition, metabolism and elimination. For drugs that need to pass through membranes to reach their site of action, the permeability of the membranes is a key factor in the drug

17 delivery. For orally ingested drugs, the primary route for membrane permeation is believed to be through passive diffusion [52, 53]. However, many drugs are limited in this sense due to their biochemical properties, and must therefore rely on alternate pathways to enter the cell. The use of intrinsic transport systems in the body is a way to circumvent limited passive diffusion, and a growing number of membrane transporters are known to transport various drugs [54, 55], including SLCs [56]. In many cases, the drugs are subject to simultaneous active and passive transport, although the weight of contribution from each route is under debate for each drug and tissue [57-59]. Carrier-mediated transport is important for drugs that display low passive permeation, especially for distribution in liver and brain, and in renal excretion as these are the principal organs for drug metabolism [58]. Consequently, when considering SLCs in drug development, the focus has been on drug transporters expressed in the epithelia lining the intestine, kidney and liver and the endothelium in the blood-brain barrier. Pharmacokinet- ic studies suggest that transporters work in concert with drug-metabolizing enzymes, thereby affecting the overall metabolism and subsequent elimination of drugs [60]. SLC members are also subject to drug-drug interactions in the liver, kidney and blood-brain barrier [60, 61]. Drug-drug interactions occur when the uptake or efflux of a drug by a transporter is affected by a second drug, and the phenomenon is of utmost clinical relevance. Conse- quently, the FDA advises investigating interactions with drug-transporting SLCs during early drug development. [58]. The involvement of specific SLCs in drug distribution in vivo has been studied using knockout mice [62] and through loss-of-function studies in humans [63, 64]. Humanized transporter animal models are another useful tool to establish the contribution of the transporter to the disrupted pathways and for studying the absorption, distribution, metabolism and excretion (ADME) properties of the compounds [60, 65, 66].

SLCs as drug targets Mutations in roughly 20% of genes coding for known human SLCs have been linked to monogenic diseases, also known as Mendelian diseases, i.e. conditions that result from the modification of a single gene [67]. The mutations can result in altered expression or dysfunction of transporter proteins, due to misfolding. Many of the SLC polymorphisms involved in common diseases have been identified through Genome-wide Association Studies (GWAS). These studies have revealed several examples where common polymorphisms lead to less serious phenotypes whereas more severe defects in the same gene cause a monogenic disease. As an example, simultaneous polymorphisms in the genes coding for the bilirubin transporters OATP1B1 (SLC0B1) and OATP1B3 (SLC01B3) may lead to jaundice and

18 hyperbilirubinaemia, while more severe mutations or deletion of the transporters result in the rare condition known as Rotor syndrome [67, 68]. Depending on the character of the gene defect, inhibition, enhancement or circumvention of transporter function are strategies for targeting SLCs that cause monogenic diseases [67]. Inhibitor molecules can be identified in high-throughput screening (HTS) using cell lines that overexpress the transporter [69, 70]; they can be further modified to produce more potent and selective inhibitors [71, 72]. Most monogenic diseases linked to SLCs are caused by a loss of transporter function, and enhancers of affected transporters represent a promising approach in drug development. Several advances have been made in the identification of enhancers. One is detection of upregulated reporter gene transcript levels in HTS assays of prescribed drugs, where the SLC gene promoter is placed upstream of the reporter gene [73]. Another approach is screening compound libraries for molecules that induce translation of silenced SLC gene transcripts [74]. Circumvention of mutated SLC transporters can be achieved by developing essential substrates that are modified to enter cells without the affected transporter [75]. Another important aspect to consider in drug development is pharmacogenetics; mutations in SLCs can lead to a differential drug responses and pharmacokinetic profiles in a population [76]. Many SLCs have been found to be druggable, that is, they contribute to a disease phenotype and their function can be modulated by drugs [77]. SLCs are, for instance, widely used drug targets in CNS disorders; the serotonin reuptake inhibitors (SSRIs) are inhibitors for several members in the SLC6 family of monoamine transporters [78-80]. Members of the SLC18 family of vesicular monoamine transporters in the CNS are targeted in psychoses and Huntington's disease [81]. Inhibitors are also under development for the treatment of addiction and psychostimulant abuse [82]. Nevertheless, the diverse SLCs are still underexplored, despite the rapid advances in understanding their roles as drug targets and in the absorption, distribution and elimination of drugs. Little attention has been paid to their biological relevance and many of the transporters are orphans with regard to function and substrates [7, 83]. As well as determining the expression profile of transporters, a functional characterization, would aid in understanding the functional networks in which they act, and their relevance to drug discovery

19 SLC-mediated transport in vesicles, peroxisomes and lysosomes SLCs in vesicles Vesicles are small intracellular structures with specialized biological functions. They are enclosed by membranes, which allows for a self-contained cytosolic environment that is completely different from that of the cell in which they are localized [84]. The composition of the vesicle membrane is similar to that of the plasma membrane, but differs in the membrane protein component. Vesicles are involved in the uptake, storage and secretion of both endogenous and exogenous compounds, and constitute isolated cham- bers for metabolic enzymatic processes [84]. SLCs associated with vesicles are expressed on different types of vesicles, but their main substrates are amino acids and neurotransmitters. Neurotransmitters are released in the synaptic cleft by synaptic vesicles, a type of secretory vesicles. The loading of neurotransmitters into vesicles occurs through carrier-mediated transport, mainly by three SLC families, named 17, 18 and 32. The SLC17 family transporters consist of nine members, of which five are involved in vesicular transport [85]. Sialin, encoded by SLC17A5, is a transporter of glutamate and aspartate in to synaptic vesicles [86]. SLC17A6, 7 and 8 code for the vesicular glutamate transporters (VGLUT) 2, 1 and 3 respectively [85]. VGLUT1 and VGLUT2 are both almost exclusively localized in glutamatergic neurons in the CNS, and their regional distribution in the brain complements that of the other [87]. Both VGLUT1 and VGLUT2, but primarily the latter, are also associated with the plasma membrane [88]. VGLUT3, on the other hand, is expressed in synaptic vesicles and dendrites. However, its expression in the brain is not limited to glutamatergic neurons; it is also found in dopaminergic, serotonergic and cholinergic cells [87]. Lastly, the vesicular nucleotide transporter VNUT, encoded by SLC17A9, is expressed in synaptic vesicles and is responsible for ATP release at purinergic synapses [89]. The bioenergetics underlying the transport of glutamate by these transporters is still under debate [90]. Another important SLC family in vesicular transport is the SLC18 family, also known as the vesicular monoamine transporter (VMAT) family. The SLC18 family consists of four members and transports single positively- charged amine neurotransmitters from the cytoplasm into synaptic vesicles and large dense-core granules containing neuromodulating peptides. VMAT1, encoded by SLC18A1, is preferentially expressed in large, dense core granules in peripheral neuroendocrine cells [91, 92], while VMAT2, encoded by SLC18A2, is mainly expressed in synaptic vesicles in the CNS [93, 94]. Both are pH-dependent antiporters, coupling two protons to the inward transport of one monoamine, such as either serotonin or dopamine, into vesicles [95-97]. VAChT, coded for by SLC18A3, is a vesicular acetyl-

20 choline transporter [98], and is primarily expressed in the synaptic vesicles of CNS cholinergic neurons [94]. VAChT exhibits the same 2:1 stoichiome- try transport mechanism as VMAT1 and VMAT2 [95, 96]. The fourth member of the SLC family is 18B1, a phylogenetically more distant transporter [99]. Human SLC18B1 is a transporter of polyamines such as spermidine and spermine [100] with an unknown transport mechanism (Paper II). The secondary active transport of neurotransmitters described is fueled by a V- ATPase that acidifies the vesicle lumen by proton pumping. These luminal protons are used as co-substrates during the influx of neurotransmitters into vesicles. Lastly, the vesicular inhibitory amino acid transporter (VIAAT), also known as vesicular GABA transporter (VGAT), encoded by SLC32A1, is central in the delivery of neurotransmitters to the synaptic cleft. As the name implies, VIAAT transports the inhibitory amino acid and neurotransmitters GABA and glycine [101-103]. The mechanism underlying the uptake of neurotransmitters is unknown, but a hypothesis is that VIAAT is a chloride/neurotransmitter symporter [104]. Given the fundamental role of vesicular neurotransmitter transporters in neurotransmission, it is not surprising that they are implicated in CNS disorders. The VGLUTs are necessary for the vesicular accumulation and exocytosis of glutamate in synapses. Knockout studies of Slc17a6 result in mice that die at birth [105, 106]; Slc17a7 knockouts have reduced lifespans [107]; and Slc17a8 knockouts have altered pain sensation and hearing [108]. Phar- macologically, VGLUTs are relevant targets for treatment of diseases where chronic excessive glutamate is implicated, such as Alzheimer's and Hunting- ton's [85]. The VMATs are associated with the development of anxiety- related personality traits [109], and VMAT2 is believed to have a protective effect against Parkinson's disease through the packaging of neurotoxic dopamine into vesicles [110]. VMAT1 and VMAT2 can be inhibited by reser- pine and tetrabenzine. These two compounds were previously used as anti- psychotics, but are now used for the treatment of Huntington's disease and in rare cases, hypertension [111]. Precise synaptic communication in neuronal circuits requires a balance between inhibitory and excitatory input. The vesicular neurotransmitter transporters work together in a complex machinery to deliver neurotransmitters to the synaptic cleft in a fast and accurate manner in response to an action potential.

SLCs in peroxisomes and lysosomes SLCs are also central in vesicular transport of solutes into the functionally distinct peroxisomes and lysosomes. Peroxisomes are the major site for a number of catabolic processes, like the breakdown of fatty acids and polyamines [112] and the reduction of reactive oxygen species like hydrogen peroxide [113]. Thus far, only one SLC transporter has been found expressed in the peroxisome membrane and that is peroxisomal protein 34 (PMP34),

21 encoded by SLC25A17, a member of the mitochondrial transporter family [114]. PMP34 functions as an antiporter for several free cofactors such as acetyl-CoA from the cytosol in to the peroxisome lumen [115]. Lysosomes are vesicles specialized in macromolecular degradation. They vary in size, shape and quantity in different cells, but all contain around 50 enzymes that co-ordinate the degradation of intra- and extracellular material [116]. The SLC17 family has two members expressed in lysosomes: sialin and VNUT. Sialin has dual functions: it is expressed in lysosomes as an H+- coupled transporter of sialic acid, and it is responsible for storage and subsequent exocytosis of aspartate and glutamate in vesicles, as mentioned previously [86, 117, 118]. Likewise, VNUT has dual functions and acts as a vesicular nucleotide transporter [89]) and lysosomal ATP-transporter [119]. Two members of the SLC15 family of proton-dependent oligopeptide transporters are found in lysosomes: SLC15A3 [120, 121] and SLC15A4 [121, 122]. They are both symporters of di- and tripeptides as well as histidine and are involved in the pathogen sensing of endolysosomal complexes [122]. In the SLC38 family (System A and System N sodium-coupled neutral amino acid transporter family, or SNAT), SNAT9, encoded by SLC38A9 is expressed in lysosomes and is an integral part of the lysosomal amino acid- sensing machinery that activates the mTORC1 pathway [123, 124]. Members from the proton-coupled amino acid transporter family SLC36 [125] are also involved in the mTORC1 pathway. PAT1, encoded by SLC36A1, is expressed in neurons and epithelial cells lining the intestine, predominantly in the lysosomes but also in the plasma membrane [126, 127]. In lysosomes, it functions as a proton-dependent symporter of neutral amino acids, and is responsible for the efflux of amino acids following protein degradation from the acidic lysosomal lumen to the cytosol. The lysosomal V- ATPase assists in this symport by creating an H+-gradient [128] with a high concentration of H+ in the lumen, which is used by PAT1 to drive the efflux of amino acids. In the plasma membrane, PAT1 mediates the uptake of extracellular amino acids with the same symport mechanism. Mouse PAT4, encoded by Slc36a4 also exhibits neuronal and epithelial expression (Paper III) [129]. It is a high- affinity/low-capacity transporter of neutral amino acids, but unlike PAT1, the transport is proton-independent and electroneutral [130]. In mouse, PAT4 expression is abundant in brain, is mainly intracellular, and is partially co-localized with lysosomal markers [129]. Recent reports have localized PAT4 on and adjacent to the trans- Golgi network [131]. In addition to the transporters described above, a number of potential lysosomal transporters have been identified, including additional members of the SLC38 family [132], but their definite localization and function needs further investigation. Some of these SLC transporters have an established expression in the plasma membrane, pointing towards possible differential expression and multiple functions of these proteins, a recurrent theme

22 amongst vesicular transporters [133]. The physiological importance of lysosomes is reflected in the many serious diseases that result from an improper degradation of macromolecules. Lysosomal storage diseases are a large group of about 50 rare, inherited diseases that result from lysosomes not functioning properly [134]. Studying the lysosome and the lysosomal transport proteins in normal cells and during pathological conditions will contribute to our understanding of their functional role in cell physiology.

SLCs and amino acid sensing: the mTOR and GCN pathways There are two major signaling pathways for amino acid sensing in mammalian cells: the mechanistic target of rapamycin (mTOR) and the general non- derepressible 2 (GCN2) pathway. These pathways provide a link between nutrient sensing and the regulation of major metabolic responses in the cell.

The mTOR pathway The mTOR pathway integrates an array of intra- and extracellular signals to regulate basic cellular processes, like growth and proliferation, by counter- balancing anabolic and catabolic processes [135-137]. The key protein in this pathway is TOR, an evolutionary conserved serine/threonine kinase part of the phospho-inositide 3-kinase (PI3K)-related kinase family [138, 139]. TOR nucleates two functionally and structurally distinct large multiprotein complexes; in mammals, these are termed the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [140, 141]. Each complex phosphorylates a different subset of effectors to meet the varying energy demands of the cell. Relatively little is known about the effectors of mTORC2 and the underlying mechanisms by which it functions, while mTORC1 is better characterized [142]. mTORC2 regulates cytoskeletal organization and is associated with cell survival and metabolism [143, 144]. mTORC1 is involved in cell growth and proliferation, primarily by promoting anabolic and limiting catabolic processes [142] in response to growth hormones, energy status, oxygen levels, stress, and availability of amino acids [136, 137, 145-147] . The complexes also differ in their sensitivity to rapamycin, the immunosuppressant known to target TOR [148-151]. Although the molecular compositions of mTORC1 and mTORC2 are distinct, they share a number of common subunits: TOR, the DEP-domain- containing mTOR-interacting protein (Deptor), mammalian lethal with Sec13 protein 8 (mLST8), Tel two interacting protein (Tt1) and telomere maintenance 2 (Tel2) [152-154]. mTORC1 also contains proline-rich AKT substrate 40 kDa (PRAS40) and regulatory-associated protein of mTOR

23 (Raptor) [155, 156], whereas mTORC2 comprises rapamycin-insensitive companion of mTOR (Rictor), protein observed with Rictor-1 (Protor-1) and mammalian stress-activated protein kinase interacting protein (mSIN1) [150, 157-159]. Little is known about the activation of mTORC2, but activation of mTORC1 occurs via multiple routes. The presence of amino acids signals nutrient availability and is a potent stimulus for mTORC1 to trigger anabolic processes like protein synthesis [160, 161]. In the current view, amino acids activate proteins in the Ras-related GTP-binding protein (Rag) family of small GTPases, more specifically RagA-D [162]. The Rag proteins form heterodimers of RagA or B with RagC or D . In their inactive form, the Ra- gA/B component is GDP-bound and the RagC/D component GTP-bound (RagA/B*GDP-RagC/D*GTP). Amino acids activate the Rag proteins so that the RagA/B-component becomes bound to GTP and the RagC/D to GDP (RagA/B*GTP-RagC/D*GDP). This activation occurs when the amino acid sensor leucyl-tRNA synthetase (LRS) binds leucine. Leucine-bound LRS acts as a GTPase for RagD and thus activates the Rag complex [163]. The activated Rag complex binds Raptor and recruits mTORC1 to the surface of the lysosome by docking to a protein complex termed Ragulator [162, 164] assisted by a V-ATPase [165]. The mTORC1 complex then binds to the activated form of Ras homolog enriched in brain (Rheb). Rheb is activated in turn, i.e. it becomes GTP-bound instead of GDP-bound, by a complex termed the tuberous sclerosis complex (TC) that consists of tuberous sclerosis 1 and 2 (TSC1 and TSC2). TC acts as a GTPase-activating protein for Rheb. Active TC thus inactivates Rheb and is negative regulator of mTORC1 activity [166] (Fig. 3). Amino acids are an important exception to the intra- and extracellular factors regulating mTORC1 through the TSC [167]. The activated mTORC1 phosphorylates a set of effectors, of which the ribosomal S6 kinase (S6K1), elF4E-binding protein 1 (4E-BP) and UNC-51-like kinase 1 (ULK1) are best characterized [168, 169]. S6K1 has substrates that are upstream of mTORC and acts on PI3K pathway, and is therefore part of the negative feedback loop that helps regulate mTORC1 activity [170]. The lysosome is the main site of action for mTORC1; however, the lysosome does more than just serve a platform for mTORC1 activation. The lysosome and mTORC1 seem to be interconnected and modulate each other’s activity and function [171]. As the endpoint of many catabolic processes, the lysosome offers insights about the metabolic state of the cell[171]. Amino acid levels, and hence amino acid transporters together with the V-ATPases in the lysosome [128], are therefore key components of the mTOR pathway.

24 LEUCINE GLUTAMINE GLUTAMINE LEUCINE AAs H+ AAs

B0AT2 ASCT2 LAT1/CD98 PAT1 PAT4

LEUCINE GLUTAMINE GLUTAMINE LEUCINE AAs H+ AAs

EAAs

LRS Protein S6K S6 mTOR PRAS40 mLST8 synthesis TC and cell Rags mTORC1inactive 4E-BP eIF4E growth DEPTOR RAPTOR

mTORPRAS40 mLST8 RAPAMYCIN GTP GTP mTORC1active AAs PAT4 Rheb Rheb DEPTOR RAPTOR ARGININE + RagulatorRags H SNAT9 PAT1 AAs ARGININE H+ + H + H LYSOSOME V-ATPase Figure 3. A simplified illustration of the amino-acid mediated activation of mTORC1 in the mTOR pathway. Extracellular amino acids like leucine, a potent activator of mTORC1 are transported into the cell by amino acid transporters. These amino acids act on Leucyl t-RNA synthetases (LRS) which in turn act asGTPases for Rag dimers and activate them The active Rag proteins bind to the Raptor subunit of mTORC1 and help translocate it to the lysosome by docking to Ragulator, where it also binds the activated for av Rheb. Rheb is kept active by inhibition of its sup- pressor TC by amino acids. Amino acid transporters in the lysosome like SNAT9, PAT1 and PAT4 work together with a V-ATPase regulate mTORC1 activation by sensing the intralysosomal amino acid concentration and thus acts transceptors.The outcome of mTORC1 activation is an increased cell proliferation and growth. Credit to Somersault18:24 (www.somersault1824.com) for figure components, shared under a creative commons license (CC-BY-NC-SA 4.0).

Several SLC families are implicated in the regulation of mTORC1. Leucine, arginine and other essential amino acids initiate protein synthesis and cell growth, mediated by mTORC1 [172]. Several transporters co-operate in maintaining sufficient leucine levels upstream of the mTOR pathway. The neutral amino acid transporter Alanine/serine/cysteine transporter 2 (ASCT2) encoded by SLC1A5 is a high-affinity symporter for Na+ and glutamine [173] and indirectly activates mTORC1. The glutamine transported into the cell by ASCT2 is used by the antiporter dimer, L-type amino acid transporter 1 (LAT1)/CD98 encoded by SLC7A5/SLC32A2. It mediates the inward transport of leucine, coupled to the outward transport of glutamine, making them tertiary active transporters [174-176]. The glutamine symporter SNAT2 (SLC38A8) mediates Na+-coupled concentrative glutamine uptake

25 and contributes to the glutamine pool. B0AT2 (SLC6A15) also provides the cell with extracellular leucine, affecting the mTOR pathway (Paper I) [177- 179]. Furthermore, the arginine transporter SNAT9 was recently identified as a lysosomal amino-acid sensor associated with the Rag GTPase-Ragulator complex [123, 124]. SNAT3 is another putative indirect activator of mTORC1, similar to SNAT2 and SLC1A5 [180-182]. The lysosomal transporter PAT1 is a proton-coupled antiporter mediating the outward transport of neutral amino acids and works together with V-ATPases that pump the protons back in to the lysosome [49, 128]. PAT1 colocalizes with RagC and is involved in mTORC1 translocation to lysosomes in response to amino acids [49]. Knockdown studies of the Drosophila PAT ortholog, and PAT1 and PAT4 in cell lines, suggests that PAT1 and PAT4 work together in amino acid activation of mTORC1 [183, 184]. Furthermore, upregulation of PAT4 drives rapamycin-resistant mTORC1-mediated cell proliferation by interacting with Rab1a, a small GTPase linking mTORC1 with Rheb, on the Golgi, and is associated with colorectal cancer [131, 172]. Due to its central role in cell proliferation, cancer is one of the major pathological conditions implicated in a defective mTOR regulation [136, 137, 185].

The GCN2 pathway The other principal pathway for regulating cellular function by amino acid sensing is the GCN2 pathway, also known as the amino-acid response element (AARE) pathway. GCN2 is a conserved serine/threonine kinase that senses low levels of intracellular amino acids by the direct binding of accu- mulated uncharged tRNA [186, 187], which gives rise to a conformational change and a subsequent activation [188, 189]. The activated GCN2 in turn phosphorylates and inactivates the eukaryotic initiation factor 2α (eIF2α). Under normal circumstances, eIF2α initiates protein translation by forming a ternary complex with GTP and the Met-tRNA. This complex binds to the 40S subunit of the ribosome and forms the 43S pre-initiation complex [190] Thus, phosphorylated eIF2α strongly inhibits the initiation of translation, leading to a reduced global protein synthesis. The activating transcription factor 4 (ATF4) is upregulated by several transcription factors in response to the inhibited protein synthesis [191, 192], and it binds to AAREs in genes associated with the biosynthesis, retention and scavenging of amino acids [16]. The asparagine synthetase (Asns) and CCAAT/enhancer binding protein homologous protein (Chop) were among the first genes in which the AAREs [193] were identified, and these studies were the first insight into the molecular mechanisms involved in the control of gene expression by amino acid starvation. The elements in question are short DNA sequences that function as enhancers in an orientation- and position independent manner, and confer regulated transcription to a heterologous promoter [194].

26 Members of the SLC7 family of cationic amino-acid transporters exhibit an altered cellular transcriptional response to nutritional stress via the GCN2 pathway. Cationic amino acid transporter 1 (CAT-1, encoded by SLC7A1) transports arginine and lysine and is upregulated during amino acid starvation [195]. Both components of the LAT1/CD98 glutamine/leucine antiporter dimer hold AAREs [196], as well as xCT (SLC7A11), the cystine/glutamate antiporter [197]. ASCT1 and ASCT2 (SLC1A4 and SLC1A5 respectively) from the SLC1 high-affinity glutamate and neutral amino acid transporter family contain AAREs and have an inducible gene expression [198, 199]. Moreover, an AARE element has been identified in the first intron of the glutamine transporter SNAT2. ATF4 binds to this element to activate transcription and reduce degradation [194, 200]. In addition to this regulation of translational and transcriptional activity by the GCN2, the changes in amino acid availability (reflected by the binding of the amino acids to their respective transporters) can also be converted to an intracellular signaling response. The term transceptor has been adopted for transport proteins with this feature, i.e. transporters with receptor quali- ties [201, 202]. The intrinsic ability of a protein to control upregulation or degradation of the protein itself, based on the levels of available substrates, is a characteristic feature for this group of transporters [203]. This is well- documented for SNAT2, in studies where amino acid withdrawal leads to an increase in System A activity and an increase in expression and surface localization of SNAT2. The latter is a response known as adaptive regulation [204, 205]. PAT1, and related Drosophila transporters path and CG1139, influence growth and cell proliferation by mechanisms independent of the actual transport of substrates across the plasma membrane, and possible downstream targets of the substrate [184]. Rather, it seems like the limited transport abilities, and hence saturation of these transporters, can be a constitutive signal to regulate mTOR activity [49, 206]. PAT2 is suggested to share transceptor characteristics [183], as does PAT4 [206, 207]. Interesting- ly, all suggested transceptors across different phylogenies belong to APC clan of transporters, which indicates that the innate sensing and signaling functions of these transporters may have been conserved during evolution [208].

The monocarboxylate transporter family The SLC16 family is a large transporter family, currently consisting of 14 members [209]. It is also denoted as the monocarboxylate transporter family (MCT), after the preferred substrates of the first identifiedmembers. The first indication that monocarboxylates were transported across membranes, rather than being subject to passive diffusion, was the finding that the uptake of

27 monocarboxylates could be inhibited by α-cyano-4-hydroxycinnamate (CHC) [210]. Subsequent characterization of monocarboxylate transport led to the identification of the first proton-linked monocarboxylate transporter, termed MCT1 [211, 212]. Homology cloning as well as searches of expressed sequence tag (EST) and human genome projects have expanded this family to now comprise the current number of members. The nomenclature for members within this family is inconsistent; gene names were named se- quentially SLC16A1-14 as their cDNA became available, while the MCTs were numbered in the order in which they were functionally characterized. Furthermore, only four members have been shown to transport monocarboxylates, MCT1-4, while other substrates of the family include thyroid hormones and aromatic amino acids. All MCTs are predicted to have the typical 12 transmembrane (TM) helices, with intracellular C- and N- terminals, and a large cytosolic loop between TM 6 and 7 [213]. A summary of aliases and substrates can be found in Table 1.

Table 1.Gene- and protein name, aliases and predominant substrates of SLC16 gene family members. Adapted from [214]. Human gene name Protein name Aliases Predominant substrates SLC16A1 MCT1 MOT1 Lactate, pyruvate, ketone bodies SLC16A2 MCT8 MOT8, XPCT, T2, rT3, T4, T4 MCT7 SLC16A3 MCT4 MOT3, MCT3 Lactate, ketone bodies SLC16A4 MCT5 MOT5, MCT4 Orphan

SLC16A5 MCT6 MOT6, MCT5 Bumetanide, probei- cid, nateglinide SLC16A6 MCT7 MOT7, MCT6 Orphan

SLC16A7 MCT2 MOT2 Pyruvate, lactate, ketone bodies SLC16A8 MCT3 MOT3, REMP Lactate

SLC16A9 MCT9 MOT9 Orphan

SLC16A10 TAT1, MCT10 MOT Aromatic amino acids SLC16A11 MCT11 MOT11 Orphan

SLC16A12 MCT12 MOT12 Orphan

SLC16A13 MCT13 MOT13 Orphan

SLC16A14 MCT14 MOT14 Orphan

28 Transport of monocarboxylates Monocarboxylates such as lactate, pyruvate and ketone bodies are key biomolecules in the metabolism of carbohydrates, fats and amino acids, all of which require rapid transport across membranes. MCTs 1-4 mediate this transport and are thus essential for metabolism. The transport of monocarboxylates is proton-linked, and the direction of net transport is dependent on the concentration gradient of both protons and substrate. The isoforms of MCTs differ in their tissue expression profile and their relative affinities for their substrates. Taken together, this relates to the particular role of a transporter in a given tissue, although the same function can be performed by several MCTs [209]. MCT1 (SLC16A1) is found in almost all tissues and is responsible for stereoselective, unidirectional transport of mainly L-lactate, but also pyruvate and ketone bodies, such as β- hydroxybuturat and acetoacetate [211, 215]. MCT2 (SLC16A7) is also a monocarboxylate transporter, but with a more specific substrate profile, and with higher affinity for its substrates. The tissue expression in humans is relatively low, but it is present in many tissues [216, 217]. MCT2 expression in the brain is largely postsynaptic , where it is believed to be responsible for the uptake of lactate used as respiratory fuel [218]. Interestingly, when MCT2 is co-expressed with MCT1 in a tissue, they differ in cellular localization, suggesting they play distinct functional roles within that tissue, further related to their differences in substrate affinity [213, 215]. Relatively little is known about MCT3 (SLC16A8), but it has been shown to transport lactic acid out of the retina and its expression is confined to the retinal pigment- and the choroid plexus epithelium [219, 220]. MCT4 (SLC16A3), on the other hand, is widely expressed, especially in glycolytic tissue such as astrocytes, white blood cells and white skeletal muscle. It is therefore believed to be mainly involved in the efflux of lactic acid derived from glycolysis [213, 215, 221]. All monocarboxylate transporting MCTs demonstrate a broad specificity for short-chain monocarboxylates and a strong stereoselectivity for the L- enantiomer [214, 222]. L-Lactate is the predominant substrate, and MCT1-4 are mainly [215] responsible for the efflux of lactic acid as a product of glycolysis. Glycolysis is commonly anaerobic, in which case MCT1 is the primary transporter. However, in certain cell types, the glycolysis is aerobic, and these cells exhibit high MCT4 levels rather than MCT1. MCT4 is also upregulated during hypoxia, a condition in which all cells rely on glycolysis [215, 223]. In contrast, some cells use lactic acid as a substrate for lipogenesis and gluconeogenesis. The liver, kidney and adipose tissues are responsible for these processes, and they express either MCT1 or MCT2 [222, 224]. An interesting example of MCTs working together to regulate monocarboxylate transport is the shuttling of lactic acid in a tissue, between cells that produce it and cells that use it. Astrocytes produce lactic acid by

29 glycolysis, which is then transported out by MCT4 or MCT1, to later be taken up by MCT1 or MCT2 into neurons for oxidation as respiratory fuel [218]. Other SLCs involved in the transport of monocarboxylates are PAT1 [225], OATP2A1 (SLC21A1) [226], and two sodium-coupled monocarboxylate transporters (SMCT): SMCT1 (SLC5A8) and SMCT2 (SLC5A12) [227, 228]. MCT1-4 and MCT6 (SLC16A5) are also implicated with the transport of a number of drugs, including alproic acid, salicylate, bumetanide, nateglinide, simvastatin and atorvastatin [215, 229]. Expression of MCTs at the blood- brain barrier makes them important in drug disposition and delivery [230]. Furthermore, MCTs are being used to optimize drug transport; the novel Gabapentin prodrug, XP13512, has been designed so that it can be taken up by MCT1 through intestinal epithelia when administered orally [231].

Transport of aromatic amino acids MCT10 or T-type amino acid transporter (TAT1, SLC16A10) is expressed in the kidney, liver, skeletal muscle heart, placenta, and basolateral epithelium of the intestine [232] [213]. It is a facilitative efflux transporter of aromatic amino acids (phenylalanine, tryptophan, tyrosine) as well as L-dopa and the thyroid hormones T3 and T4 [233, 234]. MCT10 can also control the neutral amino acid efflux via the L-type amino acid transporter (LAT1)/CD98 exchanger dimer by recycling its aromatic influx substrates [235]. The localization of MCT10 together with its efflux properties suggests that it is a part of an amino-acid reabsorption system in epithelia of the small intestine and proximal kidney tubule [232]. Other transporters of aromatic amino acids are PAT4, a high affinity tryptophan transporter [130], and LAT1/CD98, a low- affinity L-dopa transporter [236].

Transport of thyroid hormones

The thyroid gland secretes two hormones: the prohormone T4 (3,3*5,5*- tetraiodothyronine) and the biologically active T3 (3,3',5-triiodothyronine). Thyroid hormones act on virtually every cell by binding nuclear receptors and altering the expression of a wide array of genes. They regulate the metabolism of fat, carbohydrates and protein, and are essential for the proper development of the CNS and somatic tissue. Thyroid hormones act by binding their receptors, which in turn function as transcription factors that mediate changes in gene transcription. T4 exerts minimal activity but has a longer half-life and can be converted to T3 in most tissues by intracellular deiodinating enzymes. In order for the receptor binding and conversion to occur, the thyroid hormones must first enter the cell [237, 238]. It was previously thought that they did so by passive diffusion. However, after the identification of specific thyroid hormone transporters located in the plasma

30 membrane, it is increasingly clear that there is a regulated influx and efflux of thyroid hormones. The first thyroid hormone transporter, termed MCT8 (SLC16A2), was identified in 1994 [239], but its function was not determined until a decade later, when it was shown to be a specific and active transporter with a strong ligand specificity towards iodothyronines including T3 and T4 [240, 241]. MCT8 is expressed in a wide range of tissues, and has been localized to liver, kidney, pituitary and thyroid, and to various regions in mouse brain involved in thyroid signaling [241-243]. The transport of thyroid hormones is facilitative and bidirectional, as is the thyroid hormone transport exhibited by MCT10 mentioned previously [241, 244]. Mutations in MCT8 can lead to severe diseases such as Allen-Herndon- Dudley syndrome, manifested by marked mental retardation and endocrine abnormalities in affected male subjects [245], and Pelizaeus-Merzbacher- like disease with similar clinical features [246]. These conditions further underline the importance of thyroid hormone transporters in thyroid hormone homeostasis. A number of transporters belonging to the organic anion-transporting (OATP, SLC0) family transport thyroid hormones, but OATC1P1 (SLC01B1) displays a more high-affinity and narrow substrate profile than the other members [247]. Furthermore, the LAT1/CD98 heterodimer exchanger transports thyroid hormones [248], although the physiological relevance for these transporters remains unknown.

Orphan transporters The remaining transporters in the MCT family remain largely unexplored with respect to both tissue expression and function. MCT6 has unknown endogenous substrates but has been shown to transport carnitine and the antidiabetic drug nateglinide. It has been localized to the human intestine [229, 249]. MCT12 has also been identified as a carnitine transporter, localized to the eye, brain, heart, kidney and muscle, and together with MCT11, in rat skeletal muscle [250, 251]. MCT9 has been implicated in renal overload in gout patients, and variations of the SLC16A9 gene have been found in chronic hepatitis B patients treated with peginterferon and adefovir [252, 253]. MCT5, MCT7, MCT13 and MCT14 (paper IV) have been localized to cattle gastrointestinal tracts, rat pancreas and bovine adrenal gland [254- 257]. Much remains to be elucidated about the expression profile and function of these transporters. .

31 Aims

Determination of distribution pattern and function of SLCs is essential for elucidation of their implications in global pathways and neuronal circuits. The phylogenetic relationship between transporters within an individual SLC family can provide clues on the function of a novel transporter, as similarities in sequence can translate into similarities in function. The aim of this thesis was to decipher functional characteristics of solute carrier transporters, in order to obtain a better understanding of their physiological role. For this purpose, we used two separate transgenic mouse lines. Furthermore, we aimed at providing basal characteristics for novel solute carriers with respect to phylogeny and expression profile on a regional- and cellular level.

The specific aims were:

Paper I The aim of this study was to investigate the role of Slc6a15 (B0AT2) leucine transporter in mediating the anorexigenic effect of leucine on food intake, as well as in the activation of the mTOR pathway. We also aimed at studying the B0AT2 expression in the CNS.

Paper II Here, we aimed at studying the effects of postnatal forebrain-specific deletion of Slcz1 on locomotor activity and object-based memory function as well as determine its cellular localization in the brain.

32 Paper III This study aimed at a determining the overall tissue expression of PAT4 as well as a detailed investigation of the cellular and subcellular localization of PAT4 in the CNS.

Paper IV Herein we sought to perform a detailed histological analysis of the MCT14 transporter in the mouse brain, determine the tissue expression profile and cellular localization. Furthermore, we aimed to analyze the phylogenetic relationship between members in the MCT family.

33 Methodological considerations: mouse genetics

The experimental procedures in this thesis are described in detail in the in- cluded papers. Herein, I focus on generation of the two transgenic mouse lines used in paper I and II.

Linking polymorphisms of SLCs to Mendelian diseases through genome- wide association studies is an example of an efficient top-down research strategy, that starts with a phenotype of interest and uses genetic analysis to identify candidate genes. However, when deciphering the contribution of individual SLCs to neuronal networks and pathways, the bottom-up strategy is advantageous, where a genotype is used to study a phenotypical outcome. A revolutionizing tool in this strategy is the use of genetically modified mice, where pronuclear injection of new genetic information into the mouse genome allows for obtainment of spatial and temporal control of specific genes in the mouse genome, and thereby analysis of their contribution to cellular pathways and neuronal circuits. Commonly, a gene is knocked out, either by a conventional or a conditional approach. In the conventional approach, a null mutation is introduced into the target allele so that the altered gene will be translated into a nun-functional protein, if translated at all. However, major drawbacks with this method are the risk of embryonic le- thality, prevention of the study of gene function later in life, and compensa- tory up- or downregulations of other systems. A conditional knockout strategy using the Cre-LoxP system on the other hand, allows for genes to be knocked out in a controlled manner, and can be used for region- or time- specific gene deletion. The Cre-LoxP system utilizes the recombination properties of the P1 bacteriophage enzyme Cre (Cyclization recombinase) and its 34 bp long recognition sites, called LoxP. They are introduced by homologous recombination so that the gene of interest is flanked by LoxP or "floxed". Mice that are homologous for the floxed allele are then bred with mice expressing Cre, resulting in a recombination at the LoxP sites and subsequent excision of the floxed region. The Cre expression is driven by a specific promoter with the required spatial and temporal expression, thereby restricting the recombination and subsequent deletion to specific areas and to specific time points (Fig. 4) [258-260].

Floxed mouse Tissue-specific-Cre mouse X

LoxP LoxP GENE X Promoter Cre

LoxP LoxP GENE X

Non-Cre expressing cells

LoxP

Tissue-specific deletion of Gene X

Figure 4. Application of Cre-LoxP technology. Mice carrying the floxed target allele are crossed with mice carrying a tissue- and time specific Cre. Recombination at the LoxP sites allows for excision of the floxed gene segment, resulting in a non- functional gene. Credit to Somersault18:24 (www.somersault1824.com) for figure components, shared under a creative commons license (CC-BY-NC-SA 4.0).

So called reporter mouse lines can be used for visualization of Cre activity. This is achieved by targeted insertion of a construct containing the promoter, followed by a floxed-Stop cassette-controlled fluorescent marker gene. The stop cassette is excised in the presence of Cre, allowing expression and subsequent detection of the reporter. The TdTomato reporter line used in paper IV in this thesis contains the strong artificial CAG promoter, placed at the ROSA26 locus for constitutive and ubiquitous gene expression [261]. Upon Cre-mediated excision of the Stop cassette, the promoter drives the expression of the fluorescent TdTomato in all Cre-expressing cells (Fig. 5).

35 TdTomato Reporter mouse Tissue-specific-Cre mouse X

Rosa26 locus LoxP LoxP CAG STOP TdTomato Promoter Cre

LoxP LoxP CAG STOP TdTomato Non-Cre expressing cells

LoxP CAG TdTomato

Tissue-specific expression of TdTomato

Figure 5. Cre activity can be monitored with a reporter mouse line. Here, the expression of the fluorescent red protein TdTomato is inhibited by a floxed Stop-cassette. Crossing reporter mice carrying this construct with a Cre-expressing mouse line results in the excision of the Stop cassette and subsequent expression of TdTomato in all Cre-expressing cells. Credit to Somersault18:24 (www.somersault1824.com) for figure components, shared under a creative commons license (CC-BY-NC-SA 4.0).

The full Slc6a15 knockout mice were generated by Drgonova et al in 2007 [178] with a conventional method. In short, homologous recombination in ES cells was used to inactivate exon 3 in the Slc6a15 gene, known to contain the initiation codon and the TM domain for substrate binding [262] (Fig. 6). ES cells that were correctly recombined were selected by using the antibiotic neomycin and the antiviral drug ganciclovir, identified by polymerase chain reaction (PCR) and confirmed with Southern Blot. The ES cells were next injected into a blastocyst which in turn were inserted into a pseudopregnant mouse and mated with C57BL/6 resulting in chimeric transgenic mice. The heterozygous progeny were intercrossed to obtain KO mice. The knocking of Slc6a15 was later confirmed with qPCR.

36 Slc6a15 locus 3 4 5

Targeting construct Neo TK

Recombined Neo 4 5 allele Figure 6. Disruption of exon 3 in the Slc6a15 gene using homologous recombination with a targeting construct containing a neomycin cassette for positive selection, and Herpes Simplex Virus thymidine kinase for negative selection of successfully recombined ES cells. Figure redrawn from [178].

The generation of the Slcz1floxed mice has been described in detail elsewhere [263], but in short; homologous recombination was used to replace part of the Slcz1 gene. A targeting construct containing floxed exons 3, 4 and 5 of Slcz1, followed by a neomycin casette flanked by so called Frt sites; Frt sites are the yeast equivalent of LoxP sites, recognized by the flippase recombinase, also known as Flp (Fig. 7), were recombined with the wildtype allele. Neomycin-selected positive ES cells were injected into a blastocyst and inserted into a pseudopregnant mouse to generate chimeric mice. The chimeric mice were bred with C57BL/6 mice to produce heterozygous progeny, that were crossed to produce homozygous Slcz1f/f mice. The neomycin selection cassette was removed by crossing the Slcz1f/f mice to Deleter-FlpE mice [264].

Slcz1 locus 3 4 5

LoxP LoxP Frt Frt Targeting 3 4 5 construct Neo

LoxP Frt Frt LoxP Recombined 3 4 5 Neo allele

Del-FlpE

LoxP Frt LoxP Flipped 3 4 5 allele

Figure 7. Gene targeting strategy for generation of Slcz1f/f mice. Homologous recombination is used to flank exon 3, 4 and 5 of the Slcz1 gene, as well as for the introduction of neomycin casette flanked by Frt sites, resulting in a recombined, floxed allele. The neomycin cassette is removed by crossing mice carrying the recombined allele with Del-FlpE mice, resulting in a flipped Slcz1f/f allele. Figure redrawn from [263].

37 Results and discussion

Paper I In this paper we examine the role of B0AT2, a neuronal transporter of essential branched chain amino acids, in the regulation of food intake To characterize the tissue expression of B0AT2, we screened the mouse brain for Slc6a15 mRNA and B0AT2 protein expression. qPCR on a tissue panel showed widespread expression of Slc6a15 in the CNS, while expression in peripheral tissue was low or non-detectable. We used in situ hybridization to further determine Slc6a15 mRNA localization in the brain and found Slc6a15 mRNA in striatum, cortex, hippocampus, amygdala, pons, as well as several regions in hypothalamus implicated in the regulation of food intake, such as supraoptic nucleus, ventromedial hypothalamic nucleus, the anterior hypothalamic area, basolateral amygdala, arcuate nucleus and locus coerulous . We generated a polyclonal B0AT2 antibody to verify the deletion of Slc6a15 in the KO mice using non-fluorescent immunohistochemistry. Furthermore, the antibody was used to determine in which cell types B0AT2 is expressed. Using fluorescent immunohistochemistry, we showed that B0AT2 expression is mainly neuronal, including many GABAergic neurons as well as motor neurons in the spinal cord. Moreover, we saw expression in astrocytes, and in epithelial cells of the choroid plexus. Interestingly, the astrocytical expression was confined to periventricular regions. We also noted a colocalization of B0AT2 with diazepam binding inhibitor (DBI) in epithelial cells in choroid plexus, and in astrocytes close to the ventricles. DBI, also known as the Acyl-CoA-binding protein, is expressed in hypothalamus and circumventricular organs, especially in astroglial and ependymal cells [265]. It is implicated in Acyl-CoA transfer and thus the metabolism of fatty acids [266]. The colocalization with DBI combined with the hypothalamic and periventricular expression of B0AT2 suggests it is involved in the uptake of circulating amino acids from the bloodstream, as well as in the sensing of amino acid levels. B0AT2 is an amino acid transporter of essential branched-chain amino acids, with a high affinity for leucine [177, 267]. Leucine is known to signal amino acid availability and is a potent activator of the mTOR pathway. We showed that wildtype mice that were administered dietary leucine for 48 hours exhibited a downregulation of Slc6a15 and Mtor, the gene coding for the mTOR kinase, as well downstream factors Rps6 and Eif4e. This is possi-

38 bly a negative feedback mechanism due to prolonged exposure to dietary leucine. To investigate the role of B0AT2 in mediating the effects of leucine in the central nervous system, we used Slc6a15 KO mice. We measured food intake after intraperitoneal injections of leucine, and found a lower reduction of food intake in Slc6a15 KO mice compared with wildtype controls. We also investigated the neuronal activation of areas related to food intake following leucine injections, and found a lower neuronal activity in the ventromedial hypothalamic nucleus in Slc6a15 KO mice in comparison with controls. The altered effect of leucine in Slc6a15 KO suggests that it is involved in mediating hypothalamic sensing of leucine availability.

Paper II Here, we study the function of SLCZ1, the mouse ortholog to human SLC18B1, a putative transporter of the polyamines spermidine and spermine [100]. We generated conditional Slcz1 KO mice (cKO) by crossing Slcz1f/f with a Cre line specific for CaMKIIα-expressing neurons in order to obtain a late- onset deletion of Slcz1 in the forebrain. The production of the Slcz1f/f has been described elsewhere [263]. To monitor Cre activity, mice were bred with a TdTomato reporter line. We generated a custom-made polyclonal antibody directed against SLCZ1, and used it to ensure deletion of Slcz1 with fluorescent immunohistochemistry together with visualization of the TdTomato in mouse brain sections. We also used the SLCZ1 antibody in immunohistochemical assays to determine the localization of SLCZ1 to CaMKIIα-expressing neurons but not astrocytes. Furthermore, the expression of SLCZ1 was shown to be intracellular, not overlapping with the plasmalemmal marker WGA. The deletion of Slcz1 was driven by the CaMKIIα promoter, and the deletion was thus restricted so subpopulations of the amygdala, cortex and hippocampus. To assess general activity in these mice, the Slcz1 cKO mice were pharmacologically challenged with the psychostimulant amphetamine and the sedative diazepam, whereafter their total locomotive activity was assessed in automated activity cages, so called locoboxes. The cKO mice showed a gender-independent reduction in response to diazepam with higher total levels of activity, whereas no difference was measured in the response to amphetamine. This led us to study the neuronal activity in areas subject to Slcz1 deletion after diazepam injections. The cKO mice exhibited an increased neuronal activity in the retrosplenial cortex and hippocampus but not in amygdala after diazepam injections, in both male and females. Moreover, we studied the recognition memory of the cKO mice in the novel-object recognition task, finding an impaired short-term memory in both male and female cKO, whereas the long-term memory remained unaffected. Recogni-

39 tion memory formation is believed to be hippocampus-independent, and linked to cortical regions such as the perirhinal cortex [268, 269]. The deletion of Slcz1 in the superficial layers of cortex may account for this effect. Slcz1 cKO mice also exhibited a reduced digging behavior compared to controls. The digging behavior is in part dependent on hippocampal function, and there has also been reports in overlap of neuronal circuitry involved in locomotion and digging behavior [270]. Deletion of Slcz1 in hippocampus may account for the altered digging behavior. As SLCZ1 is a putative spermidine transporter, we conclude that our findings regarding phenotype of Slcz1 cKO mice may be accounted for by disruption of the spermidine transport in affected forebrain regions. The underlying mechanisms are however largely unknown and need further investigation.

Paper III In this paper, we performed a histological analysis of the PAT4, a transporter of large, neutral amino acids [130]. We performed extensive mapping of Slc36a4 mRNA in mouse brain by in situ hybridization, and showed widespread distribution. Slc36a4 mRNA was localized to the hippocampus, piriform cortex and throughout various structures in the hypothalamus, like the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, anterior cortical amygdaloid- and suprachiasmatic nucleus. We verified the specificity of a commercially available polyclonal antibody directed against PAT4, and used fluorescent and non-fluorescent immunohistochemistry to determine the expression profile. We found widespread PAT immunoreactivity in the hippocampus, cortex, piriform cortex and large parts of the hypothalamus, including the ventromedial and dorsomedial hypothalamus and arcuate nucleus. The fluorescent immunohistochemistry determined the cellular localization of PAT4 to the soma of both inhibitory and excitatory neurons, as well as in epithelial cells lining the ventricles in mouse brain. The subcellular localization of PAT4 was investigated by immunocytochemistry on hypothalamic cells, showing a intracellular localization of PAT4, as well as a partial overlap with lysosomal markers. PAT4 is an amino acid transporter and has been implicated in the activation of mTORC1, which is in agreement with the cellular and subcellular localization in our findings. We also determined the phylogenetic relationship between the SLC36 gene family and the SLC32 and SLC38 families, and showed that PAT4 is the evolutionary oldest member of the SLC36 family.

40 Paper IV The focus of this paper is the expression profile of orphan transporter MCT14. Slc16a14 mRNA was highly expressed in the kidney, while moderate levels where detected in the CNS, uterus, testis and liver as determined by qPCR on a panel of mouse tissues, while levels in other tissues were low. The presence of Slc16a14 mRNA in the CNS was confirmed by in situ hybridization, finding mRNA throughout a variety of brain regions, such as the piriform cortex, hippocampus and hypothalamus. Non-fluorescent immunohistochemistry with a verified commercially available polyclonal antibody showed that the MCT14 expression followed the same pattern as the mRNA, with immunoreactivity in retrosplenial and piriform cortex, hippocampus and hypothalamus. The cellular localization of MCT14 was determined with fluorescent immunohistochemistry, revealing immunoreactivity in the soma of both excitatory and inhibitory neurons, and in epithelial cells. From the immunohistochemistry we also concluded that MCT14 was absent in astrocytes, dendrites and pre-synaptic vesicles. Immunocytochemistry on immor- talized, rat adrenal gland cells indicated both plasmalemmal and intracellular expression of MCT14, while it did not overlap with synaptic vesicles. The phylogenetic analysis revealed that Slc16a14 is more closely related to Slc16a2, Slc16a10 and Slc16a9 than other members within the same family. MCT8 corresponding to Slc16a2 is a transporter of thyroid hormones, and MCT10, encoded by Slc16a10, transports aromatic amino acids. The similarities between MCT14 and MCT8 and 10 and 9 with respect to phylogeny as well as functional characteristics such as lack of proton-binding domain suggests a similar function for MCT14.

41 Closing remarks: conclusions and perspectives

The aim of this thesis was to perform a histological and functional characterization of both known and novel solute carrier transporters expressed in the central nervous system. We determined the expression profile in both central and peripheral tissues, and regional and cellular localization in the brain. We also used transgenic mouse models for functional studies of behavior and food intake.

Paper I Our findings in this study further contribute to the hypothesis that B0AT2 is involved in the sensing and transporting of amino acids and subsequent activation of the mTOR pathway. B0AT2 is highly expressed in the mouse central nervous system, and we found it to be localized to neurons as well as epithelial cells and astrocytes close to the ventricles, indicating that it can be involved in the transport of leucine from the bloodstream into the brain. We used the Slc6a15 knockout mice to investigate the anorexigenic effect of leucine, finding a decreased reduction of food intake in the knockout mice after intraperitoneal injections of leucine. Central intracerebroventricular injections of leucine into the third ventricle has shown a stronger decrease of food intake. By circumventing the blood brain barrier, leucine can affect the hypothalamic signaling in a more direct way. We would like to apply this route of administration of leucine to the Slc6a15 knockout mice, to investigate if the anorexigenic effects of leucine are altered. Furthermore, B0AT2 has been associated with depression, and it would be of further interest to evaluate the Slc6a15 mice in models for depression-like behavior, such as the Porsolt forced swim test.

Paper II In this paper, we studied the phenotypical outcome of postnatal forebrain- specific deletion of Slcz1, the mouse ortholog to putative spermidine transporter SLC18B1. By using a region-specific CaMKIIα-Cre line with a late onset, we generated Slcz1conditional knockout mice. We assessed general activity by pharmacologically challenging the mice with the psychostimulant

42 amphetamine and the sedative diazepam, and found gender-independent differences in the conditional mice response to the sedative. This led us to investigate the neuronal activity upon diazepam injections in areas where Slcz1 had been deleted, and found an increased activity in hippocampus and cortex, but not amygdala, in the conditional knockout mice. It would be interesting to explore the mechanisms behind the blunted response to diazepam, and to analyze other aspects of locomotion in these mice. Furthermore, we found differences in short-term memory, specifically in recognition memory formation, in the conditional mice compared with the controls. We would like to explore this behavioral phenotype more completely through cognitive testing, as additional behavioral deficiencies may contribute to the memory impairments. This type of memory formation is believed to be hippocampus-independent, so we would like to test investigate other aspects of memory formation in these conditional mice. The marble burying test revealed alterations in the digging behavior, which is connected to repetitive behavior in mice and often implicated in models of autism spectrum disorders. We would like to explore other repetitive behaviors in these mice. In addition to the behavioral tests, we would like to further investigate the exact cellular and subcellular localization of SLCZ1. The human ortholog is a putative transporter of polyamines in synthetic liposomes, and we would like to express this transporter in Xenopus Laevis oocytes to explore the substrate specificity of SLCZ1. In a parallel project, this transporter has been subject to a full knockout using a PGK-driven Cre, leading to early and uniform gene deletion in mice. Results from those mice are in the pipeline.

Paper III In paper III, we performed a detailed characterization of PAT4, finding it to be widely distributed in the brain, in both inhibitory and excitatory neurons and epithelial cells lining the ventricles. We also found PAT4 to be present on the plasma membrane as well as intracellularly, and partially colocalizing with lysosomal markers. PAT4 is a transporter of neutral amino acids like tryptophan and proline, and is believed to be a part of the intricate mTORC1-activating machinery. Our findings of the cellular and subcellular localization of PAT4 are in good agreement with this. Recent reports have found that PAT4-mediated mTORC1 activation through amino acid sensing occurs on the Golgi apparatus, although the underlying mechanisms are not fully elucidated [131]. In the light of these findings, further studies on the subcellular localization of PAT4 are needed, to determine if its interaction with the mTORC1 is restricted to the Golgi apparatus or if it acts on the lysosome as well. One approach to this examine the interaction between PAT4

43 and PAT1, organelle-specific markers as well as with other components of the mTOR pathway using a proximity ligation assay (PLA). In our phylogenetic analysis, we found PAT4 to be the oldest member of the conserved SLC36 family, most likely predating animals. This taken together with its widespread distribution suggests an important role for this transporter, conserved in other species. Therefore, we are also in the process of characterizing a closely related Drosophila ortholog. By using different driver lines in the Gal4/UAS system, we are in the process of knocking it out in neuronal subpopulations as well as on a whole-body level, in order to further investigate the role of PAT4 in the mTOR pathway.

Paper IV We performed the first detailed histological characterization of MCT14 in mouse brain in paper IV, and determined it to have plasmalemmal and intracellular expression in both excitatory and inhibitory neurons as well as epithelial cells. MCT14 is an orphan transporter from the monocarboxylate transporter family. Besides the four monocarboxylate transporters, this family also contains transporters of thyroid hormones and amino acids, as well as a number of uncharacterized transporters, both with respect to expression- and substrate profile. Our phylogenetic analysis revealed that Slc16a14 clus- ters with Slc16a2, Slc16a8 and Slc16a9. The endogenous substrate for the MCT9 protein encoded by the latter is still unknown. Slc16a2 and Slc16a10, encoding MCT8 and MCT10 respectively, are known transporters of thyroid hormones and aromatic amino acids. We analyzed Slc16a14 in both central and peripheral tissues, finding moderate levels of Slc16a14 in the central nervous system, liver, tests and uterus, and high levels in the kidney. Taken together, the expression profile and phylogenetic analysis suggests MCT14 is similar to MCT8 and MCT10 in substrate profile, which needs to be confirmed through uptake assays. Expression of the transporter in Xenopus Laevis oocytes is an advantageous model for this purpose. We are also in the process of characterizing two other transporters in the same family, MCT11 and MCT13, both orphan transporters with an unknown expression profile.

44 Acknowledgments

At last, a few lines to express my appreciation towards everyone who has helped me along the way (also, the only part of this book most of you will read) - I couldn't have done any of this without you.

First, I'd like to extend my gratitude towards my main supervisor Robert Fredriksson. Thank you for creating a warm, inspirational work atmosphere, and for always taking the time to answer my many, many questions. I've learnt a lot from working with you, and thank you for helping me develop as a researcher. Thank you to my co-supervisor Helgi Schiöth for creating such an exciting environment to conduct research in, and for pushing me to finally get my driver's license. I would also like to thank Karin Nordström for intro- ducing me to the Neuroscience department. There has been many fantastic people in Helgilab throughout the years - thank you all for making it so much fun to go to work! A special thanks to: Maria, my first supervisor turned colleague, Sofie - for always being friendly and helpful (and fast!). To all my fellow PhD student (most of you have finished and are missed): Smitha, Markus, Mathias, Johan, Jonathan, Anders, Linda, Josefine, Olga, Anica, Emil, Philip, Arun, Wei, Frida R, Sarah, Hao, Nathalie, Marcus and Misty - thank you all for sharing this adventure with me! Thank you also to Galina, Jessica, Lyle, Christian, Mike and Madeleine for great company and for always being helpful. Thank you Pleunie for being a great friend outside of work, we miss you already! Thank you to all project students and all co-authors for all their hard work, especially Elin, Johan, Luca, Atieh, Yinan, Amanda, Vanni and Frida L! Thank you and sorry to the many many students that helped me with the gazillion PCRs... To all my friends in the department - thank you Bejan for always being so nice and helpful (we made it!), Kasia, Sharn, Fatima, Casey, Emma, Bo, Daniel, and Xesus to name a few; you have all been so much fun to be around. Greta, thank you for being so supportive and for all the fun lunches! To my current colleagues and friends in the Robertlab/Helgilab mashup - oh how sad I'll be to leave you. Karin, I'm really glad you joined our little group, finally someone I can talk (or harass) mouse genetics with! You've been a great colleague and friend, and thank you for helping me even before I know I need help. Emelie, you are a great fellow lab-mother, colleague and most importantly, a sweet friend whom I cherish. Emilia, your enthusiasm is

45 contagious and it has been so much fun working with you, wish it could have been longer and under less sleep-deprived conditions. Mikaela, thank you for all your help in the lab, and for always being so friendly and easy to work with. Sonchita, you're such a bubbly, inspirational person, and a lot of fun to be around. Thank you also to Noura, Rekha and Vasiliki for being such great co-workers, I'm going to miss you! Thank you to everyone at the Pharmaceutical Biosciences department for being so friendly and welcoming (and organized!), especially Lisa and Lova.

Mitt högst älskade BLAJ-gäng - vilken fantastisk skara människor ni är. Bland det bästa jag gjort i livet är omge mig med er. Peppande, inspirerande, roliga och smarta, och det är mycket tack vare er den här boken blev till. Linnea, din pepp och hjälp har tagit mig igenom de tyngsta av dagar, du borde säljas på burk. Du inspirerar mig till att njuta mer av livets glädjeäm- nen och har alltid sån koll på allt. Emelie, du ger mig alltid nya (smarta) perspektiv, och är så otroligt hjälpsam. Tack för all choklad! Shima, tack för att du höll ett öga på mig efter det där första (men inte sista!) tidningsomsla- get. Beundrar din entusiasm inför livet och din peppighet. Srebrenka, du inspirerar mig på många sätt, och din pepp har varit ovärderlig. Sofie, ditt lugn och din härliga personlighet lättar upp vilken grå doktorand-dag som helst. Världens bästa Dvärgkollektiv: min första tid i Uppsala var oförglömlig tack vare er. Episka torsdagsmiddagar, för-, huvud- och efterfester, och yo- gaagenter. Det var så självklart att vi skulle bli kompisar för livet. Henrik, tack för att du välkomnade mig in i kaoset, och för att du lät mig tvångsad- optera ca hela din klass. Bodil, du förgyller dagligen mitt liv på så många plan! Du e min kompis! Tack för Daimtårtan. Tack till mitt älskade "Göteborgsbästisgäng" som jag saknar precis hela tiden: ni är underbara! Jag är tacksam för att ni finns i mitt liv och är så fina vänner trots att jag är dålig på att svara i telefon och bor i andra änden av Sverige. Hanna - du har varit ett sånt otroligt stöd för mig på alla vis i livet, kommer hålla hårt i dig och hoppas kunna återgälda dig en vacker dag. Neda, min stöttepelare och bästis, tack för allt. Azadeh, du är världens mest godhjärtade människa, är så tacksam för dig! Hana, du och jag är great suc- cess, alltid. Omtänksamma Raghda, den roligaste jag vet. Ala, så klok och kreativ, saknar dig! Anahita, vi träffas för sällan men det är som om ingen tid har passerat mellan gångerna. Sayeh, är så glad att jag lärt känna dig, tack för att du är så omtänksam och rolig, och en sån fin vän på alla vis. Vida, Sofie Bäcklin, Sofia och Mar- tin - tack för att ni är så fina, stöttande vänner som jag önskar jag träffade oftare. Till min familj: kan inte nog beskriva tacksamheten jag känner till er. Ni har alltid varit den bästa hejaklacken, inspirationen och tryggheten. Älskar er! Mamanjoon - min hjälte. Beundrar dig så otroligt mycket för allt du har

46 tagit dig för i livet, och du bara fortsätter att imponera på mig. Du har alltid gjort allt i din makt för att ge mig ett så bra liv som möjligt, och för det är jag dig evigt tacksam. Du är en fantastisk mamma och mormor. Solli, du kan ca allt, och du känner mig som ingen annan (på gott och ont), och jag är så lyckligt lottad som har dig som bror. Du ger mig alltid perspektiv på min tillvaro och är en klippa på alla sätt. Snart kommer läkemedlet! Sepid: tack för att du är en syster, en bästis, en förebild och någon som alltid håller min hand genom alla stadier av livet. Din tilltro till mig har gjort att jag aldrig tvekar att ta mig an nya utmaningar, och det var du som peppade mig att börja den här banan, på vilken vi har följts åt. Mina syskonbarn Gabriel, Lucas och Liv - jag älskar er och saknar er hela tiden. Till min älskade ex- tramamma/moster Mojgan - Jag har alltid känt mig hemma i Uppsala för att jag har haft dig, Arash och Sara. Kan inte tacka dig nog för all hjälp. Tack till Monica och Hans, Magnus och Maggie, Martin och Åsa med familj, som till min stora glädje nu också är min familj, för att ni har hejat, peppat och avlastat. Till Olle, den bästa människan jag vet, och mitt största fan (känslan är ömsesidig). Varje dag får jag nypa mig själv för att försäkra mig om att du är på riktigt. Jag är så tacksam för dig, och vårt liv tillsammans. Tack för att du gör mig så lycklig, och för att du är ett orubbligt stöd i livets alla skeenden. Till mina barn Nora och Love; jag älskar er mest av allt! (men om det inte hade varit för er hade jag blivit klar för två år sen).

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1215 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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