The vesicular monoamine transporter 1 (VMAT1) is essential for storage of monoamines, such as epinephrine and serotonin, in secretory vesicles of neuroendocrine cells. Recently the VMAT1 protein was detected in human and mouse brain, and mutations of the VMAT1 gene at single DNA nucleotides (single nucleotide polymorphisms or SNPs) were associated with schizophrenia. In this study, Chinese hamster ovarian cells were stably transfected with either human VMAT1 DNA (GenBank: #NM_003053.1 or DNA with the Thr4Pro SNP, which results in a threonine to proline change in amino acid number 4 of the VMAT1 protein. Western blot analysis revealed that cells with the SNP produced immunoreactive human VMAT1 proteins of altered molecular size, suggesting that SNP Thr4Pro modifies either folding or processing of the VMAT1 protein. This finding is the first evidence for biochemical consequences of a mutation in the human VMAT1 gene.

In der vorliegenden Arbeit wurde die Regulation der Aktivität der vesikulären Monoamintransporter VMAT1 und VMAT2 durch heterotrimere G-Proteine untersucht. In der humanen neuroendokrinen Zellinie BON werden VMAT1 und VMAT2 exprimiert. Sie colokalisieren in diesen Zellen mit der a-Untereinheit des heterotrimeren G-Proteins Go2 vorwiegend auf großen elektronendichten Vesikeln, den LDCVs. Die Aktivität beider Transporter unterliegt einer Regulation durch Gao2. Nach Aktivierung des G-Proteins kommt es zu einer Hemmung der vesikulären Monoaminaufnahme. Die Aktivität von VMAT2 wird dabei empfindlicher reguliert als die Aktivität von VMAT1. In Primärkulturen von Rapheneuronen der Ratte wird VMAT2 als neuronale Variante des Transporters exprimiert. VMAT2 lokalisiert in diesen Neuronen überwiegend auf kleinen synaptischen Vesikeln, den SSVs. Hier kommt es zu einer Colokalisation mit Gao2 auf diesem Vesikeltyp. Auch in Rapheneuronen wird die Aktivität von VMAT2 durch diese G-Protein Untereinheit gehemmt. Elektronenmikroskopische Befunde belegen die Lokalisation von VMAT2 und Gao2 auf SSVs von serotonergen Axonterminalen im präfrontalen Cortex der Ratte. An einer Präparation synaptischer Vesikel aus diesem Gehirnbereich konnte ebenfalls eine Hemmung der Transportaktivität von VMAT2 durch Gao2 nachgewiesen werden. Auch in Thrombozyten der Maus unterliegt die vesikuläre Serotoninaufnahme einer Hemmung durch ein heterotrimeres G-Protein. In chronisch entleerten Vesikeln aus Mäusen, in denen das Gen für die periphere Tryptophanhydroxylase deletionsmutiert vorlag, konnte zunächst keine Hemmung der Serotoninaufnahme durch heterotrimere G-Proteine beobachtet werden. Nach Vorbeladung der Vesikel mit Serotonin war dies jedoch der Fall. Die Aktivierung des G-Proteins wird somit sehr wahrscheinlich über den Füllungszustand der Vesikel gesteuert. / In this study we investigated the regulation of the activity of the vesicular monoamine transporters VMAT1 and VMAT2 by heterotrimeric G-proteins. In the human neuroendocrine cell line BON both transporters are expressed. They colocalize in these cells with the a-subunit of the heterotrimeric G-protein Go2 predominantely on Large Dense Core Vesicles (LDCVs). The activity of both VMAT1 and VMAT2 is regulated by Gao2. G-protein activation results in a down-regulation of vesicular monoamine uptake. VMAT2 appears to be more sensitive towards the observed G-protein regulation than VMAT1. Serotonergic raphe neurons in primary culture express VMAT2 as the neuronal form of the transporter. In these neurons VMAT2 predominantely localizes to Small Synaptic Vesicles (SSVs). Here, VMAT2 colocalizes with Gao2 on SSVs. In these neurons Gao2-dependent down-regulation of VMAT2 activity was observed, too. Immunoelectron microscopic analysis confirmed a localization of VMAT2 and Gao2 on SSVs from serotonergic terminals in the rat prefrontal cortex. In addition, Gao2-dependent regulation of VMAT2 activity could also be demonstrated when using a crude synaptic vesicle preparation of this brain area. Even in platelets obtained from mice the vesicular serotonin uptake is down-regulated by heterotrimeric G-proteins. In serotonin-depleted platelets from peripheral tryptophane-hydroxylase knockout mice no G-protein-dependent down-regulation of monoamine uptake was observed. After preincubation of the platelets with serotonin, the G-protein regulation was restored. Therefore, the vesicular transmitter content appears to be a likely factor of G-protein activation in platelets.

Abstract FUNCTIONAL AND BIOCHEMICAL CONSEQUENCES OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE HUMAN VESICULAR MONOAMINE TRANSPORTER 1 GENE (SLC18A1) By Sally Gamal Shukry, B.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Advisor: Jennifer K. Stewart Associate Professor and Graduate Director, Department of Biology Single nucleotide polymorphisms (SNP) in the human VMAT1 gene (SLC18A1) have been associated with schizophrenia in three different populations: Han Chinese, Western European and Japanese. Effects of these mutations on transport function of the hVMAT1 protein have not been reported. The goal of this study was to investigate functional and biochemical differences in human VMAT1 proteins with a threonine or proline at amino acid position 4 (Thr4Pro) and a serine or threonine at position 98 (Ser98Thr). COS1 cells were transfected with variant SNPs coding for 4Thr/98Ser, 4Pro/98Ser, or 4Thr98Thr. Western blotting demonstrated robust over expression of the genes and no differences in electrophoretic mobility of the proteins. Maximal transport of serotonin by the VMAT1 protein with 4Pro/98Ser was less than that of the 4Thr/98Ser or the 4Thr/98Thr. Response of the 4Pro/Ser98 to the VMAT inhibitor reserpine was lower than that of the 4Thr/98Thr. These findings suggest mechanisms for human VMAT1 links to schizophrenia.

Schizophrenia is a debilitating mental disorder that causes a large economic burden and is prevalent across all cultures and countries around the world. Although both environmental factors and genetics are known to play an important role in the etiology of schizophrenia, the exact role of genetics and its interaction with environmental factors in an individual’s predisposition to develop schizophrenia is poorly understood. Schizophrenia is characterized by symptoms that include positive symptoms (e.g. delusions, hallucinations, disorganized thinking and speech), negative symptoms (e.g. avolition, anhedonia, depressive-like behavior), and cognitive dysfunctions (e.g. executive functioning deficits in learning and memory, attention, and vigilance). Genomic screening has identified polymorphisms of the vesicular monoamine transporter 1 (VMAT1) gene (SLC18A1) that are associated with schizophrenia and bipolar disorder. The current study represents the first extensive phenotyping of both young and aged mice in which the VMAT1 gene (SLC18A1) has been deleted. The results demonstrated behavioral effects of deleting the VMAT1 gene that may relate to aspects of schizophrenic-like behavioral changes in this model. Specifically, young VMAT1 knockout mice displayed significant deficits in sensorimotor gating in the prepulse inhibition (PPI) task and in the acquisition of operant learning in the autoshaping task. When exposed to a mild stressor (24 hours of food deprivation), young VMAT1 knockout mice displayed a significant reduction in locomotor activity that was not evident under free-feeding conditions. Thus, young VMAT1 knockout mice showed deficits in tasks that model positive symptoms and cognitive deficits seen in schizophrenia; however, they did not display differences in behaviors related to models of the negative symptoms of schizophrenia or deficits in tasks designed to measure motor skills. While less extensive phenotyping was conducted in aged VMAT1 knockout mice, there were no significant deficits evident in any of the assays conducted in older animals. These findings demonstrated that deletion of the VMAT1 gene has behavioral effects that appear to be mediated by changes in brain monoamine function and changes in response to stressors (i.e. food deprivation) that may reflect changes in adrenal gland monoamine function.