Disruptive mutations in the serotonin transporter associate serotonin dysfunction with treatment-resistant affective disorder.

Affective or mood disorders are a leading cause of disability worldwide. The serotonergic system has been heavily implicated in the complex etiology and serves as a therapeutic target. The serotonin transporter (SERT) is a major regulator of serotonin neurotransmission, yet the disease-relevance of impaired SERT function remains unknown. Here, we present the first identification and functional characterization of disruptive coding SERT variants found in patients with psychiatric diseases. In a unique cohort of 144 patients characterized by treatment-resistant chronic affective disorders with a lifetime history of electroconvulsive therapy, we identified two previously uncharacterized coding SERT variants: SERT-N217S and SERT-A500T. Both variants were significantly enriched in the patient cohort compared to GnomAD (SERT-N217S: OR = 151, P = 0.0001 and SERT-A500T: OR = 1348, P = 0.0022) and ethnicity-matched healthy controls (SERT-N217S: OR ≥ 17.7, P ≤ 0.013 and SERT-A500T: OR = ∞, P = 0.029). Functional investigations revealed that the mutations exert distinct perturbations to SERT function, but their overall effects converge on a partial loss-of-function molecular phenotype. Thus, the SERT-A500T variant compromises the catalytic activity, while SERT-N217S disrupts proper glycosylation of SERT with a resulting dominant-negative trafficking deficiency. Moreover, we demonstrate that the trafficking deficiency of SERT-N217S is amenable to pharmacochaperoning by noribogaine. Collectively, our findings describe the first disease-associated loss-of-function SERT variants and implicate serotonergic disturbances arising from SERT dysfunction as a risk factor for chronic affective disorders.

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2.

Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.

Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors.

Dopamine supports locomotor control and higher brain functions such as motivation and learning. Consistently, dopaminergic dysfunction is involved in a spectrum of neurological and neuropsychiatric diseases. Detailed data on dopamine dynamics is needed to understand how dopamine signals translate into cellular and behavioral responses, and to uncover pathological disturbances in dopamine-related diseases. Genetically encoded fluorescent dopamine sensors have recently enabled unprecedented monitoring of dopamine dynamics in vivo. However, these sensors' utility for in vitro and ex vivo assays remains unexplored. Here, we present a blueprint for making dopamine sniffer cells for multimodal dopamine detection. We generated sniffer cell lines with inducible expression of seven different dopamine sensors and perform a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we apply the sniffer cells to record endogenous dopamine release from cultured neurons and striatal slices, and for determining tissue dopamine content. Furthermore, we use the sniffer cells to measure dopamine uptake and release via the dopamine transporter as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cell framework can readily be applied to the growing list of genetically encoded fluorescent neurotransmitter sensors.

α-Synuclein-induced dysregulation of neuronal activity contributes to murine dopamine neuron vulnerability.

Pathophysiological damages and loss of function of dopamine neurons precede their demise and contribute to the early phases of Parkinson's disease. The presence of aberrant intracellular pathological inclusions of the protein α-synuclein within ventral midbrain dopaminergic neurons is one of the cardinal features of Parkinson's disease. We employed molecular biology, electrophysiology, and live-cell imaging to investigate how excessive α-synuclein expression alters multiple characteristics of dopaminergic neuronal dynamics and dopamine transmission in cultured dopamine neurons conditionally expressing GCaMP6f. We found that overexpression of α-synuclein in mouse (male and female) dopaminergic neurons altered neuronal firing properties, calcium dynamics, dopamine release, protein expression, and morphology. Moreover, prolonged exposure to the D2 receptor agonist, quinpirole, rescues many of the alterations induced by α-synuclein overexpression. These studies demonstrate that α-synuclein dysregulation of neuronal activity contributes to the vulnerability of dopaminergic neurons and that modulation of D2 receptor activity can ameliorate the pathophysiology. These findings provide mechanistic insights into the insidious changes in dopaminergic neuronal activity and neuronal loss that characterize Parkinson's disease progression with significant therapeutic implications.