Biology subjects

Herborg, F.

Publications and source records attributed to Herborg, F..

9 recordsLinked to original sources

bioRxiv2024

Modelling determinants of region-specific dopamine dynamics in the striatum

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.

neuroscience↗

bioRxiv2024

Membrane curvature association of amphipathic helix 8 drives constitutive GPCR endocytosis

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 semi-specific motifs within the receptors intracellular regions. Here we describe a complementary and evolutionary conserved and refined trafficking mechanism for G-protein coupled receptors (GPCR). 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 new principle of receptor-lipid interactions that may have broader implications for cellular signaling and therapeutic targeting. One-Sentence SummaryReceptor proteins navigate cellular membranes by interacting with their curvature using an evolutionary conserved mechanism that relies on amphipathic helices and complements direct coupling to the endocytic protein machinery.

cell biology↗

bioRxiv2023

Injectable 3D microcultures enable intracerebral transplantation of mature neurons directly reprogrammed from patient fibroblasts

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 (2D) cultures poorly survive transplantation into the adult brain in part due to the need for enzymatic or mechanical cellular dissociation before transplantation. Three-dimensional (3D) culturing methodologies have the potential to overcome these issues but have largely been unexplored for the purposes of direct neuronal reprogramming. Here we report a strategy for direct in vitro reprogramming of adult hDFs inside suspension 3D microculture arrays into induced DA neurospheroids (iDANoids). We show that iDANoids express neuronal and DA markers and are capable of firing mature action potentials and releasing dopamine. Importantly, they can be gently harvested and transplanted into the brain of a Parkinsons disease rat model to reproducibly generate functionally integrated neuron-rich grafts. The 3D culturing approach presented here thus eliminates a major bottleneck in direct neuronal reprogramming field and, due to its simplicity and versatility, could readily be adapted as a culturing platform used for a broad range of transplantation studies as well as disease modeling.

neuroscience↗

bioRxiv2022

Behavioral encoding across timescales by region-specificdopamine dynamics

The dorsal (DS) and ventral striatum (VS) receive dopaminergic projections that control motor functions and reward-related behavior. It remains poorly understood how dopamine release dynamics across different temporal scales in these regions are coupled to behavioral outcomes. Here, we employ the dopamine sensor dLight1.3b together with multi-region fiber photometry and machine learning-based analysis to decode dopamine dynamics across striatum during self-paced exploratory behavior in mice. Our data show a striking coordination of rapidly fluctuating signal in the DS, carrying information across dopamine levels, with a slower signal in the VS, consisting mainly of slow-paced transients. Importantly, these release dynamics correlated with discrete behavioral motifs, such as turns, running and grooming on a subsecond-to-minutes time scale. Disruption of dopamine dynamics with cocaine caused randomization of action selection sequencing and disturbance of DS-VS coordination. The data suggest that distinct dopamine dynamics of DS and VS jointly encode behavioral sequences during unconstrained activity with DS modulating the stringing together of actions and VS the signal to initiate and sustain the selected action. Significance StatementNew genetically encoded dopamine sensors offer unprecedented temporal resolution for measurement of dopamine release dynamics across different brain regions over extended periods. In this study, we use the dopamine sensor dLight1.3b to decipher the role of dopamine release dynamics in the dorsal (DS) and ventral striatum (VS) of mice during simple, self-paced exploratory behavior. By AI-based splitting of behavioral kinematics into individual motifs, we link differential but highly cooperative dopamine release dynamics of DS and VS with movements on a subsecond-to-minutes time scales. In addition to coupling region-specific dopamine dynamics to behavioral sequences, our study demonstrates the strength of a machine learning-based data analysis pipeline that can be readily applied to other neurotransmitters for which genetically encoded biosensors are available.

neuroscience↗

bioRxiv2022

Structural basis of organic cation transporter-3 inhibition

Organic cation transporters (OCTs) facilitate the translocation of catecholamines and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 [A] resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.

pharmacology and toxicology↗

bioRxiv2022

Relative enrichment - a density-based colocalization measure for single-molecule localization microscopy

Dual-color single-molecule localization microscopy (SMLM) provides unprecedented possibilities for detailed studies of colocalization of different molecular species in a cell. However, the informational richness of the data is not fully exploited by current analysis tools that often reduce colocalization to a single value. Here, we describe a new tool specifically designed for determination of co-localization in both 2D and 3D from SMLM data. The approach uses a novel function that describes the relative enrichment of one molecular species on the density distribution of a reference species. The function reframes the question of colocalization by providing a density-context relevant to multiple biological questions. Moreover, the function visualize enrichment (i.e. colocalization) directly in the images for easy interpretation. We demonstrate the approachs functionality on both simulated data and cultured neurons, and compare it to current alternative measures. The method is available in a Python function for easy and parameter-free implementation.

cell biology↗

bioRxiv2021

Multimodal Detection of Dopamine by Sniffer Cells Expressing Genetically Encoded Fluorescence Sensors

Dopamine serves an important role in supporting both locomotor control and higher brain functions such as motivation and learning. Dopaminergic dysfunction is implicated in an equally multidimensional spectrum of neurological and neuropsychiatric diseases. Extracellular dopamine levels are known to be tightly controlled by presynaptic dopamine transporters (DAT), which is also a main target of psychostimulants. Still, detailed data on dopamine dynamics in space and time is needed to fully understand how dopamine signals are encoded and translated into cellular and behavioral responses, and to uncover the pathological effects of dopamine-related diseases. The recently developed genetically encoded fluorescent dopamine sensors enable unprecedented monitoring of dopamine dynamics and have changed the field of in vivo dopamine recording. However, the potential of these sensors to be used for in vitro and ex vivo assays remains unexplored. Here, we demonstrate a generalizable blueprint for making "sniffer" dopamine cells for multimodal detection of dopamine in vitro and ex vivo. We generated sniffer cell lines with inducible expression of six different dopamine sensors and performed a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we show how the sniffer cells can be applied to measure release of endogenous dopamine from cultured neurons and striatal slices, and for determining total dopamine content in striatal tissue. Furthermore, we use the sniffer cells to quantify DAT-mediated dopamine uptake, and AMPH-induced and constitutive dopamine efflux as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cells framework can readily be applied to other transmitter systems for which the list of genetically encoded fluorescent sensors is rapidly growing.

neuroscience↗

bioRxiv2020

A dominant-negative variant in the dopamine transporter PDZ-binding motif is linked to parkinsonism and neuropsychiatric disease

Dopaminergic dysfunction is central to movement disorders and mental diseases. The dopamine transporter (DAT) is essential for the regulation of extracellular dopamine but the genetic and mechanistic link between DAT function and dopamine-related pathologies remains elusive. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we identify a novel coding DAT variant, DAT-K619N, in a patient with early-onset parkinsonism and comorbid neuropsychiatric disease and in 22 individuals from exome-sequenced samples of neuropsychiatric patients. The variant localizes to the critical C-terminal PDZ-binding motif of DAT and causes reduced uptake capacity, decreased surface expression, and accelerated turnover of DAT in vitro. In vivo, we demonstrate that expression of DAT-K619N in mice and dropsophila imposes impairments in dopamine transmission with accompanying changes in dopamine-directed behaviors. Importantly, both cellular studies and viral overexpression of DAT-K619N in mice show that DAT-K619N has a dominant-negative effect which collectively implies that a single dominant-negative genetic DAT variant can confer risk for neuropsychiatric disease and neurodegenerative early-onset parkinsonism.

neuroscience↗

bioRxiv2020

Dopaminergic neurons establish a distinctive axonal arbor with a majority of non-synaptic terminals

Chemical neurotransmission in the brain typically occurs through synapses, which are structurally and functionally defined as sites of close apposition between an axon terminal and a postsynaptic domain. Ultrastructural examinations of axon terminals established by monoamine neurons in the brain often failed to identify a similar tight pre- and postsynaptic coupling, giving rise to the concept of "diffuse" or "volume" transmission. Whether this results from intrinsic properties of such modulatory neurons remains undefined. Using an efficient co-culture model, we find that dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in exocytosis such as synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to be active. Finally, we found that the presynaptic protein Nrxn-1SS4- and the postsynaptic protein NL-1AB, two major components involved in excitatory synapse formation, play a critical role in the formation of synapses by dopamine neurons. Taken together, our findings support the idea that dopamine neurons in the brain are endowed with a distinctive developmental program that leads them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and GABAergic neurons involved in fast point-to-point signaling. SIGNIFICANCE STATEMENTMidbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a particularly large number of release sites, most of which not adopting a classical synaptic structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding suggests that, NL-1A+B and Nrxn-1SS4-, play a critical role in the formation of synapses by DA neurons.

neuroscience↗