Biology subjects

Lycas, M. D.

Publications and source records attributed to Lycas, M. D..

6 recordsLinked to original sources

bioRxiv2021

Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. Summarized, the data reveal a mechanism for rapid alterations in nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.

neuroscience↗

bioRxiv2021

Disrupted PDZ-domain binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis and reward motivation

The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors different from those seen in other genetic DAT mouse models. DA levels in striatum are reduced to [~]45% of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics.

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

Coding variants identified in diabetic patients alter PICK1 BAR domain function in insulin granule biogenesis

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that shape negatively charged curved lipid membranes during membrane remodeling processes. The BAR domain proteins ICA69, PICK1 and arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify four coding variants in the PICK1 gene from a Danish whole-exome screening of diabetic patients, that all involve change of positively charged residues in the PICK1 BAR domain. All four coding variants failed to rescue the insulin content in INS-1E cells upon KD of endogenous PICK1. Moreover, two variants showed dominant negative properties. Interestingly, in vitro assays addressing the BAR domain function suggest that the coding variants compromised membrane binding capacity but increased capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin from ISGs during the maturation process and that the coding variants may cause premature budding possibly explaining their dominant negative function.

cell biology↗

bioRxiv2020

Rab2 drives axonal transport of dense core vesicles and lysosomal organelles

Long range fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles and presynaptic components is critical for maintaining the functionality of neurons. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 has previously been shown to mediate DCV biogenesis and endosome-lysosome fusion. Here we use the Drosophila model system to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2 is also critical for DCV exit from cell bodies into axons. Our results uncover the mechanisms responsible for axonal transport of DCVs and reveal surprising parallels between the regulation of DCVs and lysosomal motility.

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↗