PhD defence: Jonatan Fullerton Støier

Department of Neuroscience

4 July 2022, 14:00-17:00, Henrik Dam Auditorium

Assessment Committee
Professor Claus Juul Løland (Chairperson)
Professor Harald Sitte
Assistant professor Jana Haase

Insight into Monoamine Transporter Dysfunction: Hard Drugs, Sprinkle Cells and Depression

Abstract: The monoamines – dopamine, serotonin and noradrenaline – are neurotransmitters serving a critical role in modulating complex behaviors and emotions. When released from a neuron, the monoamines are signaling by activation of specific receptors.  Major regulators of this signaling are the monoamine transporters, which are responsible for the reuptake of monoamines into neurons. Dysfunctional regulations of the monoamines are seen in many psychiatric, neurological and somatic disorders, including substance use disorders. Thus, monoamine transporters serve as an important target for many therapeutic drugs and drugs of abuse, and several coding variants of the monoamine transporters have been linked to disease.

In Paper 1, we developed a new method to detect dopamine in vitro and ex vivo by generating stable cell lines with inducible expression of fluorescent dopamine sensors, which we term “sniffer cells”. We performed proof-of-principle experiments illustrating the usage of the sniffer cells for measuring endogenous dopamine released from cultured neurons and striatal slices, and for quantifying the dopamine content in striatal tissue preparations. Additionally, we used the sniffer cell to carry out a high-throughput study of dopamine transporter kinetics and pharmacology by co-transfecting the sniffer cells with the dopamine transporter. Doing so, we were able to investigate changes in dopamine transporter function upon treatment with drugs and from genetic insults to the transporter. Lastly, as the list of fluorescent dopamine sensors is expansive we did a head-to-head comparison of the different sensors’ properties under the same experimental conditions as guidance for others to select an appropriate sensor for their experiments. Importantly, the framework of generating stable cell lines expressing dopamine sensors presented in Paper 1 can be expanded for fluorescent sensors for other biomolecules for which the list is rapidly growing.

In Paper 2, we used the sniffer cells developed in Paper 1 to study the Ca2+-dependence of amphetamine-induced dopamine efflux in primary cultured midbrain neurons. By combining the sniffer cells with the targeted expression of a Ca2+-sensor in dopaminergic neurons, we were able to measure dopamine release and cytosolic Ca2+ level, simultaneously. In contrary to what has been published on the matter, we found no dependence of Ca2+ on the ability of amphetamine to cause dopamine efflux. Neither increasing or chelating cytosolic Ca2+ nor blocking relevant Ca2+ channels had any effect on amphetamine-induced dopamine efflux. Additionally, we did not find the efflux to rely on the activity of kinases activated by Ca2+, Ca2+/calmodulin-dependent protein kinase IIα and protein kinase C β, which has previously been shown to be implicated in the action of amphetamine. However, we found the action of amphetamine to be highly dependent on the tandem action of the vesicular monoamine transporter 2 and the dopamine transporter.

In Paper 3, we identified two different coding variants of the serotonin transporter in a cohort of treatment-resistant patients with affective disorders. The two variants, SERT-N217S and SERT-A500T, were found to be enriched in the cohort when comparing the allele frequency among the patients with that of either a large exome sequencing database or a healthy ethnicity-matched control population. Interestingly, by functional characterization of the two variants, we found both to be loss-of-function variants, making them the first loss-of-function variants of the serotonin transporter to be linked to disease. Furthermore, the SERT-N217S variant was found to act dominant-negative on wild-type serotonin transporter when co-expressed together. As the SERT-N217S variant leads to the loss of one of the two N-glycosylation sites present in the transporter, we found the missense mutation caused defective folding of the protein, which we could rescue using pharmacochaperoning.

Overall, the work presented in this thesis provides new tools to study monoamine transporter function in vitro, which we used to gain mechanistic insight into amphetamine-induced dopamine efflux. The work also expands the spectrum of disease-associated monoamine transporter variants to include two loss-of-function coding variants of the serotonin transporter linked to treatment-resistant affective disorders.