Emelie Perland

Classification Systems of Secondary Active Transporters.

Membrane-bound solute carrier (SLC) transporter proteins are vital to the human body, as they sustain homeostasis by moving soluble molecule as nutrients, drugs, and waste across lipid membranes. Of the 430 identified secondary active transporters in humans, 30% are still orphans, and systematic research has been requested to elaborate on their possible involvement in diseases and their potential as drug targets. To enable this, the various classification systems in use must be understood and used correctly. In this review, we describe how various classification systems for human SLCs are constructed, and how they overlap and differ. To facilitate communication between researchers and to avoid ambiguities, everyone must clearly state which classification system they are referring to when writing scientific articles.

Obesity-Linked Homologues TfAP-2 and Twz Establish Meal Frequency in Drosophila melanogaster

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.

The Putative SLC Transporters Mfsd5 and Mfsd11 Are Abundantly Expressed in the Mouse Brain and Have a Potential Role in Energy Homeostasis

Background Solute carriers (SLCs) are membrane bound transporters responsible for the movement of soluble molecules such as amino acids, ions, nucleotides, neurotransmitters and oligopeptides over cellular membranes. At present, there are 395 SLCs identified in humans, where about 40% are still uncharacterized with unknown expression and/or function(s). Here we have studied two uncharacterized atypical SLCs that belong to the Major Facilitator Superfamily Pfam clan, Major facilitator superfamily domain 5 (MFSD5) and Major facilitator superfamily domain 11 (MFSD11). We provide fundamental information about the histology in mice as well as data supporting their disposition to regulate expression levels to keep the energy homeostasis. Results In mice subjected to starvation or high-fat diet, the mRNA expression of Mfsd5 was significantly down-regulated (P<0.001) in food regulatory brain areas whereas Mfsd11 was significantly up-regulated in mice subjected to either starvation (P<0.01) or high-fat diet (P<0.001). qRT-PCR analysis on wild type tissues demonstrated that both Mfsd5 and Mfsd11 have a wide central and peripheral mRNA distribution, and immunohistochemistry was utilized to display the abundant protein expression in the mouse embryo and the adult mouse brain. Both proteins are expressed in excitatory and inhibitory neurons, but not in astrocytes. Conclusions Mfsd5 and Mfsd11 are both affected by altered energy homeostasis, suggesting plausible involvement in the energy regulation. Moreover, the first histological mapping of MFSD5 and MFSD11 shows ubiquitous expression in the periphery and the central nervous system of mice, where the proteins are expressed in excitatory and inhibitory mouse brain neurons.

Characteristics of 29 novel atypical solute carriers of major facilitator superfamily type: evolutionary conservation, predicted structure and neuronal co-expression.

Solute carriers (SLCs) are vital as they are responsible for a major part of the molecular transport over lipid bilayers. At present, there are 430 identified SLCs, of which 28 are called atypical SLCs of major facilitator superfamily (MFS) type. These are MFSD1, 2A, 2B, 3, 4A, 4B, 5, 6, 6 L, 7, 8, 9, 10, 11, 12, 13A, 14A and 14B; SV2A, SV2B and SV2C; SVOP and SVOPL; SPNS1, SPNS2 and SPNS3; and UNC93A and UNC93B1. We studied their fundamental properties, and we also included CLN3, an atypical SLC not yet belonging to any protein family (Pfam) clan, because its involvement in the same neuronal degenerative disorders as MFSD8. With phylogenetic analyses and bioinformatic sequence comparisons, the proteins were divided into 15 families, denoted atypical MFS transporter families (AMTF1-15). Hidden Markov models were used to identify orthologues from human to Drosophila melanogaster and Caenorhabditis elegans. Topology predictions revealed 12 transmembrane segments (for all except CLN3), corresponding to the common MFS structure. With single-cell RNA sequencing and in situ proximity ligation assay on brain cells, co-expressions of several atypical SLCs were identified. Finally, the transcription levels of all genes were analysed in the hypothalamic N25/2 cell line after complete amino acid starvation, showing altered expression levels for several atypical SLCs.

The Novel Membrane-Bound Proteins MFSD1 and MFSD3 are Putative SLC Transporters Affected by Altered Nutrient Intake

Membrane-bound solute carriers (SLCs) are essential as they maintain several physiological functions, such as nutrient uptake, ion transport and waste removal. The SLC family comprise about 400 transporters, and we have identified two new putative family members, major facilitator superfamily domain containing 1 (MFSD1) and 3 (MFSD3). They cluster phylogenetically with SLCs of MFS type, and both proteins are conserved in chordates, while MFSD1 is also found in fruit fly. Based on homology modelling, we predict 12 transmembrane regions, a common feature for MFS transporters. The genes are expressed in abundance in mice, with specific protein staining along the plasma membrane in neurons. Depriving mouse embryonic primary cortex cells of amino acids resulted in upregulation of Mfsd1, whereas Mfsd3 is unaltered. Furthermore, in vivo, Mfsd1 and Mfsd3 are downregulated in anterior brain sections in mice subjected to starvation, while upregulated specifically in brainstem. Mfsd3 is also attenuated in cerebellum after starvation. In mice raised on high-fat diet, Mfsd1 was specifically downregulated in brainstem and hypothalamus, while Mfsd3 was reduced consistently throughout the brain.

Recurrent Sleep Fragmentation Induces Insulin and Neuroprotective Mechanisms in Middle-Aged Flies

Lack of quality sleep increases central nervous system oxidative stress and impairs removal of neurotoxic soluble metabolites from brain parenchyma. During aging poor sleep quality, caused by sleep fragmentation, increases central nervous system cellular stress. Currently, it is not known how organisms offset age-related cytotoxic metabolite increases in order to safeguard neuronal survival. Furthermore, it is not understood how age and sleep fragmentation interact to affect oxidative stress protection pathways. We demonstrate sleep fragmentation increases systems that protect against oxidative damage and neuroprotective endoplasmic reticulum molecular chaperones, as well as neuronal insulin and dopaminergic expression in middle-aged Drosophila males. Interestingly, even after sleep recovery the expression of these genes was still upregulated in middle-aged flies. Finally, sleep fragmentation generates higher levels of reactive oxygen species (ROS) in middle-aged flies and after sleep recovery these levels remain significantly higher than in young flies. The fact that neuroprotective pathways remain upregulated in middle-aged flies beyond sleep fragmentation suggests it might represent a strong stressor for the brain during later life.

Histological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney.

BackgroundMCT14 (SLC16A14) is an orphan member of the monocarboxylate transporter (MCT) family, also known as the SLC16 family of secondary active transmembrane transporters. Available expression data for this transporter is limited, and in this paper we aim to characterize MCT14 with respect to tissue distribution and cellular localization in mouse brain.ResultsUsing qPCR, we found that Slc16a14 mRNA was highly abundant in mouse kidney and moderately in central nervous system, testis, uterus and liver. Using immunohistochemistry and in situ hybridization, we determined that MCT14 was highly expressed in excitatory and inhibitory neurons as well as epithelial cells in the mouse brain. The expression was exclusively localized to the soma of neurons. Furthermore, we showed with our phylogenetic analysis that MCT14 most closely relate to the aromatic amino acid- and thyroid-hormone transporters MCT8 (SLC16A2) and MCT10 (SLC16A10), in addition to the carnitine transporter MCT9 (SLC16A9).ConclusionsWe provide here the first histological mapping of MCT14 in the brain and our data are consistent with the hypothesis that MCT14 is a neuronal aromatic-amino-acid transporter.

The gene expression of the neuronal protein, SLC38A9, changes in mouse brain after in vivo starvation and high-fat diet

SLC38A9 is characterized as a lysosomal component of the amino acid sensing Ragulator-RAG GTPase complex, controlling the mechanistic target of rapamycin complex 1 (mTORC1). Here, immunohistochemistry was used to map SLC38A9 in mouse brain and staining was detected throughout the brain, in cortex, hypothalamus, thalamus, hippocampus, brainstem and cerebellum. More specifically, immunostaining was found in areas known to be involved in amino acid sensing and signaling pathways e.g. piriform cortex and hypothalamus. SLC38A9 immunoreactivity co-localized with both GABAergic and glutamatergic neurons, but not with astrocytes. SLC38A9 play a key role in the mTORC1 pathway, and therefore we performed in vivo starvation and high-fat diet studies, to measure gene expression alterations in specific brain tissues and in larger brain regions. Following starvation, Slc38a9 was upregulated in brainstem and cortex, and in anterior parts of the brain (Bregma 3.2 to -2.1mm). After high-fat diet, Slc38a9 was specifically upregulated in hypothalamus, while overall downregulation was noticed throughout the brain (Bregma 3.2 to -8.6mm).

Putative Membrane-Bound Transporters MFSD14A and MFSD14B Are Neuronal and Affected by Nutrient Availability

Characterization of orphan transporters is of importance due to their involvement in cellular homeostasis but also in pharmacokinetics and pharmacodynamics. The tissue and cellular localization, as well as function, is still unknown for many of the solute carriers belonging to the major facilitator superfamily (MFS) Pfam clan. Here, we have characterized two putative novel transporters MFSD14A (HIAT1) and MFSD14B (HIATL1) in the mouse central nervous system and found protein staining throughout the adult mouse brain. Both transporters localized to neurons and MFSD14A co-localized with the Golgi marker Giantin in primary embryonic cortex cultures, while MFSD14B staining co-localized with an endoplasmic retention marker, KDEL. Based on phylogenetic clustering analyses, we predict both to have organic substrate profiles, and possible involvement in energy homeostasis. Therefore, we monitored gene regulation changes in mouse embryonic primary cultures after amino acid starvations and found both transporters to be upregulated after 3 h of starvation. Interestingly, in mice subjected to 24 h of food starvation, both transporters were downregulated in the hypothalamus, while Mfsd14a was also downregulated in the brainstem. In addition, in mice fed a high fat diet (HFD), upregulation of both transporters was seen in the striatum. Both MFSD14A and MFSD14B were intracellular neuronal membrane-bound proteins, expressed in the Golgi and Endoplasmic reticulum, affected by both starvation and HFD to varying degree in the mouse brain.

Structural prediction of two novel human atypical SLC transporters, MFSD4A and MFSD9, and their neuroanatomical distribution in mice

Out of the 430 known solute carriers (SLC) in humans, 30% are still orphan transporters regarding structure, distribution or function. Approximately one third of all SLCs belong to the evolutionary conserved and functionally diverse Major Facilitator Superfamily (MFS). Here, we studied the orphan proteins, MFSD4A and MFSD9, which are atypical SLCs of MFS type. Hidden Markov Models were used to identify orthologues in several vertebrates, and human MFSD4A and MFSD9 share high sequence identity with their identified orthologues. MFSD4A and MFSD9 also shared more than 20% sequence identity with other phylogenetically related SLC and MFSD proteins, allowing new family clustering. Homology models displayed 12 transmembrane segments for both proteins, which were predicted to fold into a transporter-shaped structure. Furthermore, we analysed the location of MFSD4A and MFSD9 in adult mouse brain using immunohistochemistry, showing abundant neuronal protein staining. As MFSD4A and MFSD9 are plausible transporters expressed in food regulatory brain areas, we monitored transcriptional changes in several mouse brain areas after 24 hours food-deprivation and eight weeks of high-fat diet, showing that both genes were affected by altered food intake in vivo. In conclusion, we propose MFSD4A and MFSD9 to be novel transporters, belonging to disparate SLC families. Both proteins were located to neurons in mouse brain, and their mRNA expression levels were affected by the diet.