Anja W. Fjorback

Sort1, Encoded by the Cardiovascular Risk Locus 1p13.3, Is a Regulator of Hepatic Lipoprotein Export

Recent genome-wide association studies (GWAS) have revealed strong association of hypercholesterolemia and myocardial infarction with SNPs on human chromosome 1p13.3. This locus covers three genes: SORT1, CELSR2, and PSRC1. We demonstrate that sortilin, encoded by SORT1, is an intracellular sorting receptor for apolipoprotein (apo) B100. It interacts with apoB100 in the Golgi and facilitates the formation and hepatic export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol. Absence of sortilin in gene-targeted mice reduces secretion of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3.

Membrane Glycoprotein M6B Interacts with the Human Serotonin Transporter

The serotonin transporter (SERT) belongs to a family of sodium- and chloride-dependent neurotransmitter transporters that are responsible for the active re-uptake of the neurotransmitter serotonin from the synapse. In the present study, using the yeast two-hybrid system, we identified the membrane glycoprotein M6B as a binding partner of SERT. This interaction was further verified by co-immunoprecipitation and glutathione-S-transferase pull-down assays. M6B belongs to a proteolipid protein family, which is expressed in neurons and in oligodendrocytes in the brain. The knowledge of the biological function of this protein family is sparse, but their expression in most brain regions have led to the hypothesis that they are involved in cellular housekeeping functions such as membrane trafficking and cell-to-cell communication. The co-expression of SERT with M6B results in a significant decrease in SERT-mediated serotonin uptake caused by a down-regulation of SERT surface expression. Furthermore, we find, using confocal microscopy, that M6B co-localizes with SERT when transiently expressed in HEK-MSR-293 cells and when endogenously expressed in RN46A cells. Taken together, our data suggest that M6B regulates the serotonin uptake by affecting cellular trafficking of the serotonin transporter.

Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy

The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy.

SorLA is a molecular link for retromer-dependent sorting of the Amyloid precursor protein.

Deficiency in the retromer sorting pathway is known to be associated with the onset of Alzheimer disease (AD), and has been suggested to involve regulation of Amyloid precursor protein (APP) trafficking. Absence of the APP sorting receptor sorLA is also associated to AD, as amyloidogenic processing of APP is increased due to missorting. Reduced activity of either retromer or sorLA thus both lead to enhanced amyloidogenic APP processing, and these pathways are therefore important factors for understanding the development of AD. It is therefore key to outline the neuronal APP trafficking in order to determine the mechanisms that influence AD onset.

Modulation of the dopamine transporter by interaction with Secretory Carrier Membrane Protein 2.

The monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) facilitate the homeostatic balance of neurotransmitters in the synaptic cleft and thus, play a fundamental role in regulating neuronal activity. Despite the importance of these monoamine transporters in controlling brain function, only relatively little information is available regarding the cellular and molecular regulation of these proteins. The monoamine transporters have been found to associate with a number of different proteins that regulate the function and subcellular localization of the transporters. We recently reported a functional interaction between SERT and the Secretory Carrier Membrane Protein 2 (SCAMP2). Here, we demonstrate that SCAMP2 also plays a role in the functional regulation of DAT. DAT and SCAMP2 interaction is here verified by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) microscopy. Moreover, co-expression of DAT and SCAMP2 results in a decrease in DAT-mediated dopamine uptake caused by reduced levels of DAT molecules on the cell surface. Our finding that SCAMP2 interacts with and regulates the subcellular distribution of both DAT and SERT suggests that interaction with SCAMP2 may constitute an important mechanism for coordinating cell surface expression of monoamine transporters.

Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.

P25α / TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity

Parkinson’s disease is characterized by preferential degeneration of the dopamine‐producing neurons of the brain stem substantia nigra. Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro‐oxidative cytosolic dopamine. The microtubule‐stabilizing protein p25α accumulates in dopaminergic neurons in Parkinson’s disease. We hypothesized that p25α modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity. Here we show that co‐expression of the dopamine transporter with p25α in HEK‐293‐MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter. No direct interaction between p25α and the dopamine transporter was demonstrated, but they co‐fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25α peptides to brain vesicles was demonstrated. Truncations of the p25α peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding. Co‐expression of p25α and the dopamine transporter in HEK‐293‐MSR cells sensitized them to the toxicity of extracellular dopamine. Neuronal expression of p25α thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter.