Freja Herborg

Despite their pharmacological importance, multi-pass transmembrane proteins remain among the most challenging targets to study, mainly due to the limited availability of highly specific antibodies. In this work, we describe an optimized fixative based on the dialdehyde glyoxal that enhances the signal intensity and specificity of several antibodies in cells and tissues. Compared with paraformaldehyde, the glyoxal-based formulation is less toxic and improves the preservation of cellular morphology. Using the dopamine (DAT) and serotonin (SERT) transporters as model proteins, we show that glyoxal enables quantitative and specific analysis of transmembrane protein surface dynamics across a broad array of techniques. As proof-of-concept, we combine glyoxal fixation with single-molecule localization microscopy to uncover a previously uncharacterized link between κ-opioid receptor activation and DAT nanoclustering, providing mechanistic insight into their functional interplay. Summarized, our findings establish glyoxal fixation as a versatile and easy-to-implement strategy for probing the native cell biology of transmembrane proteins.

Visual attention enables filtering of irrelevant stimuli and focus on information that is most pertinent to survival. While the locus coeruleus-noradrenaline (LC-NA) system is recognized as crucial in this process, the role of temporally structured NA signaling in visual attentional performance remains unsettled. Here, by using the genetically encoded NA sensor GRAB _NE2h together with fiber photometry in mice, we identify sub-second NA release signatures in the medial prefrontal cortex (mPFC) that correlate with visual attention accuracy in the rodent Continuous Performance Test (rCPT). The dual-peak release signatures were time-locked to correct, rewarded responses, preceded by a ramp-up after stimulus presentation and followed by a decline at reward collection. No such signatures accompanied misses, correct rejections and mistakes. Similar, albeit less pronounced, patterns were observed in the lateral hypothalamus (LH). Activation of the Gq-coupled DREADD (Designer Receptors Exclusively Activated by Designer Drugs) hM3DGq expressed in LC neurons dose-dependently increased tonic NA levels in both mPFC and LH, while simultaneously distorting phasic NA signatures and impairing rCPT performance without affecting locomotor activity. Summarized our data suggest that NA enhances alertness to reward-associated stimuli through precise, dynamic release patterns underscoring its role in modulating attentional performance beyond tonic signaling.

Atypical dopamine transporter (DAT) deficiency syndrome (DTDS) arises from genetic disruption of DAT function and is characterized by early-onset parkinsonism alongside comorbid psychiatric symptoms. However, the underlying pathobiological processes are largely unknown. Here, we present a mouse model of atypical DTDS based on the patient-derived compound heterozygote genotype, DAT-I312F/D421N ^+/+ . DAT-I312F/D421N ^+/+ mice exhibited markedly impaired DAT function, leading to widespread changes in dopamine homeostasis, including elevated extracellular dopamine levels, reduced tyrosine hydroxylase and dopamine D1/D2 receptor expression, and decreased evoked dopamine release, mechanistically linked to enhanced tonic D2 autoreceptor inhibition. Fiber photometry measurements revealed disrupted fast striatal dopamine release dynamics, while confocal imaging showed reduced striatal dopaminergic axon fiber density. These neurochemical changes were accompanied by a psychomotor phenotype characterized by hyperlocomotion, enhanced exploration, and pronounced clasping. Both amphetamine and anticholinergic treatment ameliorated the aberrant hyperactivity. Notably, amphetamine-induced dopamine release was profoundly blunted in ventral striatum but largely preserved in dorsal striatum, implicating region-specific dopamine release dynamics as a determinant of divergent behavioral and pharmacological responses. Summarized, our findings uncover multiscale dopamine dysfunction that links presynaptic DAT impairment to synaptic and circuit-level disruptions, offering insight into atypical DTDS and the co-occurrence of movement and psychiatric features.

Striatal dopamine (DA) release regulates reward-related learning and motivation and is believed to consist of a short-lived phasic and continuous tonic component. Here, we build a large-scale three-dimensional model of extracellular DA dynamics in dorsal (DS) and ventral striatum (VS). The model predicts rapid dynamics in DS with little to no basal DA and slower dynamics in the VS enabling build-up of tonic DA levels. These regional differences do not reflect release-related phenomena but rather differential dopamine transporter (DAT) activity. Interestingly, our simulations posit DAT nanoclustering as a possible regulator of this activity. Receptor binding simulations show that D1 receptor occupancy follows extracellular DA concentration with milliseconds delay, while D2 receptors do not respond to brief pauses in firing but rather integrate DA signal over seconds. Summarised, our model distills recent experimental observations into a computational framework that challenges prevailing paradigms of striatal DA signalling.

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity-dependent internalization, which is orchestrated by adaptor proteins recognizing semispecific motifs within the receptors’ intracellular regions. Here, we describe a complementary trafficking mechanism for G protein–coupled receptors (GPCRs) that is evolutionary conserved and refined. This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a principle of receptor-lipid interactions that may have broad implications for cellular signaling and therapeutic targeting.

Direct reprogramming of somatic cells into induced neurons (iNs) has become an attractive strategy for the generation of patient-specific neurons for disease modeling and regenerative neuroscience. To this end, adult human dermal fibroblasts (hDFs) present one of the most relevant cell sources. However, iNs generated from adult hDFs using two-dimensional cultures are difficult to maintain in vitro and face challenges in survival upon transplantation into the adult brain, thus imposing constraints on biomedical applications of iN technology. Here, we present a platform for direct in vitro reprogramming of adult hDFs inside three-dimensional suspension microcultures (3D-iNs). We show that the 3D environment favors neuronal over fibroblast cellular identity to yield more robust conversion into functional neurons with extended culturing span. The 3D reprogramming approach also provides a platform for fusion into induced assembloids. 3D-iNs can be gently harvested and transplanted into the adult rodent brain to reproducibly generate neuron-rich grafts, thus eliminating a major bottleneck in the direct reprogramming field.

Striatal dopamine (DA) release regulates reward-related learning and motivation and is believed to consist of a short-lived phasic and continuous tonic component. Here, we build a large-scale three-dimensional model of extracellular DA dynamics in dorsal (DS) and ventral striatum (VS). The model predicts rapid dynamics in DS with little-to-no basal DA, and slower dynamics in the VS enabling build-up of tonic DA levels. These regional differences do not reflect release-related phenomena but rather differential dopamine transporter (DAT) activity. Interestingly, our simulations posit DAT nanoclustering as a possible regulator of this activity. Receptor binding simulations show that D1 receptor occupancy follows extracellular DA concentration with milliseconds delay, while D2 receptors do not respond to brief pauses in firing but rather integrate DA signal over seconds. Summarized, our model distills recent experimental observations into a computational framework that challenge prevailing paradigms of striatal DA signalling.

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.

The dopamine transporter (DAT) plays a crucial role in regulating the brain’s dopamine (DA) homeostasis. Atypical DAT deficiency syndrome (DTSD) is a disease characterized by early-onset parkinsonism and comorbid psychiatric symptoms, but the pathobiological processes that link DAT dysfunction to both parkinsonism and psychiatric symptoms are unknown. Here, we present a genetic mouse model of atypical DTDS that expresses two coding DAT variants, DAT-I312F and DAT-D421N, derived from a patient diagnosed with ADHD and parkinsonism. Phenotypic characterization of the mutant mice revealed impaired DAT function and major homeostatic changes including increased ambient extracellular DA levels, decreased evoked DA release, and reduced expression of both tyrosine hydroxylase (TH) and of DA D1/D2 receptors. This was accompanied by diminished striatal dopaminergic axonal density and a psychomotor phenotype characterized by hyperactivity, enhanced exploratory activity, and pronounced clasping. Importantly, both amphetamine and anticholinergic treatment ameliorated aberrant hyperlocomotion in the mice. Summarized, by replicating core aspects of the patient’s phenotype, the mouse model not only provides insights into the mechanisms underlying atypical DTDS but also underlines the broad relevance of DA deficits for understanding the co-morbidity between neuropsychiatric diseases and parkinsonism. ONE SENTENCE SUMMARY: In a new mouse disease model, we explore the behavioral consequences and dopaminergic dysfunction that arise from patient-derived mutations in the dopamine transporter associated with parkinsonism and co-morbid neuropsychiatric disease