BDNF Expression in Cortical GABAergic Interneurons

Brain-derived neurotrophic factor (BDNF) is a major neuronal growth factor that is widely expressed in the central nervous system. It is synthesized as a glycosylated precursor protein, (pro)BDNF and post-translationally converted to the mature form, (m)BDNF. BDNF is known to be produced and secreted by cortical glutamatergic principal cells (PCs); however, it remains a question whether it can also be synthesized by other neuron types, in particular, GABAergic interneurons (INs). Therefore, we utilized immunocytochemical labeling and reverse transcription quantitative PCR (RT-qPCR) to investigate the cellular distribution of proBDNF and its RNA in glutamatergic and GABAergic neurons of the mouse cortex. Immunofluorescence labeling revealed that mBDNF, as well as proBDNF, localized to both the neuronal populations in the hippocampus. The precursor proBDNF protein showed a perinuclear distribution pattern, overlapping with the rough endoplasmic reticulum (ER), the site of protein synthesis. RT-qPCR of samples obtained using laser capture microdissection (LCM) or fluorescence-activated cell sorting (FACS) of hippocampal and cortical neurons further demonstrated the abundance of BDNF transcripts in both glutamatergic and GABAergic cells. Thus, our data provide compelling evidence that BDNF can be synthesized by both principal cells and INs of the cortex

The lipid transporter ORP2 regulates synaptic neurotransmitter release via two distinct mechanisms

Funding Information: We thank Marisa Brockmann and Gülcin Vardar for initial help with SynGCamp6f imaging and electrophysiology, respectively. We thank Katja Pötschke, Bettina Brokowski, Heike Lerch, Nadine Albrecht-Koepke, and Berit Söhl-Kielczynski for expert technical assistance and the Viral Core Facility of the Charité – Universitätsmedizin Berlin for lentivirus and AAV production. We thank the Core Facility for Electron Microscopy of the Charité for their support with the electron microscope. This study was supported by the Charité Universitätsmedizin Berlin (M.W.-B., J.K., T.T., C.R.), the German Research Council via a Reinhart Koselleck project (C.R.), the Lydia Rabinowitsch-Förderung (M.W.-B.), the Academy of Finland (grant 3222647 to V.M.O.), and the Sigrid Jusélius Foundation (V.M.O.). Publisher Copyright: © 2022 The AuthorsCholesterol is crucial for neuronal synaptic transmission, assisting in the molecular and structural organization of lipid rafts, ion channels, and exocytic proteins. Although cholesterol absence was shown to result in impaired neurotransmission, how cholesterol locally traffics and its route of action are still under debate. Here, we characterized the lipid transfer protein ORP2 in murine hippocampal neurons. We show that ORP2 preferentially localizes to the presynapse. Loss of ORP2 reduces presynaptic cholesterol levels by 50%, coinciding with a profoundly reduced release probability, enhanced facilitation, and impaired presynaptic calcium influx. In addition, ORP2 plays a cholesterol-transport-independent role in regulating vesicle priming and spontaneous release, likely by competing with Munc18-1 in syntaxin1A binding. To conclude, we identified a dual function of ORP2 as a physiological modulator of the synaptic cholesterol content and a regulator of neuronal exocytosis.Peer reviewe

Differential pH Dynamics in Synaptic Vesicles From Intact Glutamatergic and GABAergic Synapses

Synaptic transmission requires the presynaptic release of neurotransmitter from synaptic vesicles (SVs) onto the postsynaptic neuron. Vesicular neurotransmitter transporter proteins, which use a V-ATPase-generated proton gradient, play a crucial role in packaging neurotransmitter into SVs. Recent work has revealed different proton dynamics in SVs expressing the vesicular glutamate transporter (VGLUT) or the vesicular GABA transporter (VGAT) proteins. At the whole synapse level, this results in different steady-state pH and different reacidification dynamics during SV recycling (Egashira et al., 2016). In isolated SVs, the presence of VGAT causes a higher steady state pH, which is correlated with a faster proton efflux rate (Farsi et al., 2016). To address whether proton efflux from GABAergic and glutamatergic SVs in intact synapses differs, we applied a glutamatergic- or GABAergic neuron-specific expression strategy (Chang et al., 2014) to express a genetically encoded pH sensor (synaptophysin pHluorin; SypHy) and/or light-activated proton pump (pHoenix; (Rost et al., 2015). We confirm, with SypHy post-stimulation fluorescence dynamics, that the pH profile of recycling GABAergic SVs differs from that of recycling glutamatergic SVs (Egashira et al., 2016). Using light-activation of pHoenix in pH-neutral vesicles, we investigated the pH dynamics of actively filling vesicles, and could show that proton efflux from GABAergic SVs is indeed initially faster than glutamatergic SVs in intact synapses. Finally, we compared the filling rate of empty glutamatergic and GABAergic vesicles using pHoenix as a proton source, and find a slightly faster filling of glutamatergic vs. GABAergic SVs

ORP/Osh mediate cross-talk between ER-plasma membrane contact site components and plasma membrane SNAREs

OSBP-homologous proteins (ORPs, Oshp) are lipid binding/transfer proteins. Several ORP/Oshp localize to membrane contacts between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate lipid transfer or regulate lipid-modifying enzymes. A common way in which they target contacts is by binding to the ER proteins, VAP/Scs2p, while the second membrane is targeted by other interactions with lipids or proteins. We have studied the cross-talk of secretory SNARE proteins and their regulators with ORP/Oshp and VAPA/Scs2p at ER-plasma membrane contact sites in yeast and murine primary neurons. We show that Oshp-Scs2p interactions depend on intact secretory SNARE proteins, especially Sec9p. SNAP-25/Sec9p directly interact with ORP/Osh proteins and their disruption destabilized the ORP/Osh proteins, associated with dysfunction of VAPA/Scs2p. DeletingOSH1-3in yeast or knocking down ORP2 in primary neurons reduced the oligomerization of VAPA/Scs2p and affected their multiple interactions with SNAREs. These observations reveal a novel cross-talk between the machineries of ER-plasma membrane contact sites and those driving exocytosis.Peer reviewe

Autaptic cultures of human induced neurons as a versatile platform for studying synaptic function and neuronal morphology

Recently developed technology to differentiate induced pluripotent stem cells (iPSCs) into human induced neurons (iNs) provides an exciting opportunity to study the function of human neurons. However, functional characterisations of iNs have been hampered by the reliance on mass culturing protocols which do not allow assessment of synaptic release characteristics and neuronal morphology at the individual cell level with quantitative precision. Here, we have developed for the first time a protocol to generate autaptic cultures of iPSC-derived iNs. We show that our method efficiently generates mature, autaptic iNs with robust spontaneous and action potential-driven synaptic transmission. The synaptic responses are sensitive to modulation by metabotropic receptor agonists as well as potentiation by acute phorbol ester application. Finally, we demonstrate loss of evoked and spontaneous release by Unc13A knockdown. This culture system provides a versatile platform allowing for quantitative and integrative assessment of morphophysiological and molecular parameters underlying human synaptic transmission

The RGS gene loco is essential for male reproductive system differentiation in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>The <it>loco </it>gene encodes several different isoforms of a regulator of G-protein signalling. These different isoforms of LOCO are part of a pathway enabling cells to respond to external signals. LOCO is known to be required at various developmental stages including neuroblast division, glial cell formation and oogenesis. Less is known about LOCO and its involvement in male development therefore to gain further insight into the role of LOCO in development we carried out a genetic screen and analysed males with reduced fertility.</p> <p>Results</p> <p>We identified a number of lethal <it>loco </it>mutants and four semi-lethal lines, which generate males with reduced fertility. We have identified a fifth <it>loco </it>transcript and show that it is differentially expressed in developing pupae. We have characterised the expression pattern of all <it>loco </it>transcripts during pupal development in the adult testes, both in wild type and <it>loco </it>mutant strains. In addition we also show that there are various G-protein α subunits expressed in the testis all of which may be potential binding partners of LOCO.</p> <p>Conclusion</p> <p>We propose that the male sterility in the new <it>loco </it>mutants result from a failure of accurate morphogenesis of the adult reproductive system during metamorphosis, we propose that this is due to a loss of expression of <it>loco c3</it>. Thus, we conclude that specific isoforms of <it>loco </it>are required for the differentiation of the male gonad and genital disc.</p

Layer 1 of somatosensory cortex: an important site for input to a tiny cortical compartment

Neocortical layer 1 has been proposed to be at the center for top-down and bottom-up integration. It is a locus for interactions between long-range inputs, layer 1 interneurons, and apical tuft dendrites of pyramidal neurons. While input to layer 1 has been studied intensively, the level and effect of input to this layer has still not been completely characterized. Here we examined the input to layer 1 of mouse somatosensory cortex with retrograde tracing and optogenetics. Our assays reveal that local input to layer 1 is predominantly from layers 2/3 and 5 pyramidal neurons and interneurons, and that subtypes of local layers 5 and 6b neurons project to layer 1 with different probabilities. Long-range input from sensory-motor cortices to layer 1 of somatosensory cortex arose predominantly from layers 2/3 neurons. Our optogenetic experiments showed that intra-telencephalic layer 5 pyramidal neurons drive layer 1 interneurons but have no effect locally on layer 5 apical tuft dendrites. Dual retrograde tracing revealed that a fraction of local and long-range neurons was both presynaptic to layer 5 neurons and projected to layer 1. Our work highlights the prominent role of local inputs to layer 1 and shows the potential for complex interactions between long-range and local inputs, which are both in position to modify the output of somatosensory cortex.Peer Reviewe

Distinct localization of SNAP47 protein in GABAergic and glutamatergic neurons in the mouse and the rat hippocampus

Synaptosomal-associated protein of 47 kDa (SNAP47) isoform is an atypical member of the SNAP family, which does not contribute directly to exocytosis and synaptic vesicle (SV) recycling. Initial characterization of SNAP47 revealed a widespread expression in nervous tissue, but little is known about its cellular and subcellular localization in hippocampal neurons. Therefore, in the present study we applied multiple-immunofluorescence labeling, immuno- electron microscopy and in situ hybridization (ISH) and analyzed the localization of SNAP47 in pre- and postsynaptic compartments of glutamatergic and GABAergic neurons in the mouse and rat hippocampus. While the immunofluorescence signal for SNAP47 showed a widespread distribution in both mouse and rat, the labeling pattern was complementary in the two species: in the mouse the immunolabeling was higher over the CA3 stratum radiatum, oriens and cell body layer. In contrast, in the rat the labeling was stronger over the CA1 neuropil and in the CA3 stratum lucidum. Furthermore, in the mouse high somatic labeling for SNAP47 was observed in GABAergic interneurons (INs). On the contrary, in the rat, while most INs were positive, they blended in with the high neuropil labeling. ISH confirmed the high expression of SNAP47 RNA in INs in the mouse. Co-staining for SNAP47 and pre- and postsynaptic markers in the rat revealed a strong co-localization postsynaptically with PSD95 in dendritic spines of pyramidal cells and, to a lesser extent, presynaptically, with ZnT3 and vesicular glutamate transporter 1 (VGLUT1) in glutamatergic terminals such as mossy fiber (MF) boutons. Ultrastructural analysis confirmed the pre- and postsynaptic localization at glutamatergic synapses. Furthermore, in the mouse hippocampus SNAP47 was found to be localized at low levels to dendritic shafts and axon terminals of putative INs forming symmetric synapses, indicating that this protein could be trafficked to both post- and presynaptic sites in both major cell types. These results reveal divergent localization of SNAP47 protein in mouse and rat hippocampus indicating species- and cell type-specific differences. SNAP47 is likely to be involved in unique fusion machinery which is distinct from the one involved in presynaptic neurotransmitter release. Nonetheless, our data suggest that SNAP47 may be involved not only postsynaptic, but also in presynaptic function

Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming

The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex

Investigation of the physiological role of PRG-1 by generating and phenotyping of PRG-1 deficient mouse models

Die Informationsübertragung an Synapsen ist essentiell für neuronale Prozesse im Nervensystem. In dieser Arbeit wurde gezeigt, dass das gehirnspezifische, neuronale Protein Plasticity-Related-Gene-1 (PRG-1) die exzitatorische Informationsübertragung an Synapsen moduliert. PRG-1 ist ein integrales Membranprotein und zeigt deutliche Homologien zu Lipid Phosphat Phosphatasen (LPP). So besitzt PRG-1 die charakteristischen LPP Merkmale von drei konservierten Domänen, welche es den LPPs erlauben, „bioaktive“ Lipidphosphate wie Phosphatidat (PA), Lysophosphatidat (LPA) oder Sphingosin-1-Phosphat (S1P) enzymatisch zu dephosphorylieren. Solche Lipidphosphate sind Schlüsselfaktoren in Rezeptor vermittelten Signalkaskaden (z.B. über LPA-Rezeptoren) und sind daher an einer Vielzahl von zellulären Prozessen beteiligt. Deshalb wird angenommen, dass LPPs mittels dieses Phosphatase Motivs als negative Regulatoren solcher rezeptorvermittelten Signaltransduktionen fungieren. Im Gegensatz zu den LPPs besitzt jedoch PRG-1 ein unvollständiges Phosphatase Motiv, so dass es nicht diesen Mechanismus der Dephosphorylierung durchführen kann. Trotzdem konnte in früheren Arbeiten in einer neuronalen Zelllinie, die mit einem PRG-1-GFP Fusionskonstrukt transfiziert wurde und LPA ausgesetzt wurde, eine erhöhte Konzentration an LPA Degradationsprodukten sowie Schutz vor LPA-induzierter Neuritenretraktion beobachtet werden. Um die physiologische Rolle von PRG-1 und dessen Einfluss auf Lipidphosphat- vermittelte Signaltransduktion genauer zu untersuchen, wurde in dieser Arbeit eine konstitutive und eine konditionelle Knockout (KO) Maus für PRG-1 generiert. Unter Verwendung einer eingefügten Reporterkassette innerhalb des konstitutiven KO Deletionskonstruktes konnte im Hippocampus eine spezifische Expression von PRG-1 in glutamatergen, Neuronen gezeigt werden. Bei heterozygoten Verpaarungen wurde schließlich beobachtet, dass ca. 50 % der PRG-1 defizienten Nachkommen an den Folgen konvulsiver Anfälle innerhalb der ersten 3 bis 4 Wochen starben. Durch eine Kooperation mit der Arbeitsgruppe von Prof. Dietmar Schmitz konnten diese juvenilen Anfälle durch in-vivo EEG Aufnahmen dargestellt werden. Weiterhin zeigten Kainat Applikationen an adulten heterozygoten und homozogoten PRG-1-KO Tieren eine erhöhte Anfallsbereitschaft dieser Tiere. Elektrophysiologische Messungen in Kooperation mit der AG Schmitz zeigten eine spezifische erhöhte Erregbarkeit der exzitatorisch glutamatergen, hippocampalen CA1 Neurone. Trotz dieser Veränderungen waren die zelluläre Morphologie in Bezug auf die Entwicklung der Neurone bzw. Interneurone, die Expression der wichtigsten synaptischen Marker und die elektrophysiologischen intrinsischer Eigenschaften der Zellen unverändert. Durch die Klonierung von Expressionsvektoren für PRG-1 bzw. Cre- Rekombinase konnte mittels in utero Elektroporationstechniken PRG-1 spezifisch in einzelnen hippocampalen CA1 Neuronen von PRG-1 defizienten Tiere exprimiert bzw. in den generierten, konditionellen PRG-1-cKO Mäusen deletiert werden. Die elektrophysiologische Analyse der elektroporierten und nicht-elektroporierten Zellen zeigte, dass rein postsynaptisches PRG-1 die Präsynapse retrograd beeinflussen muss. Um diesen Einfluss von PRG-1 im Hinblick auf die LPA Signaltransduktionsvorgänge zu untersuchen, wurden Aufnahmeexperimente mit fluoreszenzmarkierten PA Analoga an primären Neuronen von Wildtyp und PRG-1-KO Mäusen durchgeführt. Es zeigte sich, das KO Neurone eine geringere Fähigkeit besitzen, diese Lipidphosphate intrazellulär zu akkumulieren, so dass angenommen wird, dass PRG-1 die extrazelluläre Konzentration an Lipidphosphaten im synaptischen Spalt regulieren könnte. Schließlich zeigte 1. die in utero Elektroporation (IUE) eines PRG-1 Konstruktes, welches an einer essentiellen Aminosäure innerhalb des Phosphatase Motivs mutiert wurde, und 2. die Verpaarung mit einer LPA2 Rezeptor KO Maus, dass die physiologische Funktion von PRG-1 durch dessen Einfluss auf eine Lipidphosphat vermittelte Signaltransduktion an der Synapse definiert sein müsste. Unter Berücksichtigung der Tatsache, dass der LPA2 Rezeptor präsynaptisch, und PRG-1 postsynaptisch spezifisch auf exzitatorischen Synapsen glutamaterger Neurone lokalisiert wurde, beschreibt diese Arbeit einen neuen möglichen Regulationsmechanismus der exzitatorischen Signalübertragung, der über PRG-1 vermittelten wird.Synaptic transmission is the essential feature of neuronal information processing in the nervous system. This thesis reveals that the brain-specific, neuronal protein Plasticity-Related-Gene-1 (PRG-1) modulates synaptic transmission specifically at excitatory synapses. PRG-1 is an integral membrane protein and shows homologies to lipid phosphate phosphatases (LPP). PRG-1 contains the characteristic LPP features of three conserved extracellular domains which enables the LPPs to dephosphorylate bioactive lipid phosphates like phosphatidate (PA), lysophosphatidate (LPA) or sphingosine-1-phosphate (S1P). These bioactive lipid phosphates are key factors in initiating receptor-directed signalling cascades (e.g. via LPA receptors) and are therefore involved in diverse cellular processes. Therefore it has been proposed that LPPs may act as negative regulators of these signalling. In contrast to the LPPs PRG 1 lacks critical amino acids within the conserverd domains which imply that PRG-1 is not able to dephosphorylate LPA using the same mechanism which has been proposed for the LPPs. However, in earlier studies it has been shown, that the neuronal cell line N1E-115, transfected with a PRG-1-GFP fusion construct and exposed to LPA, exhibits increased extracellular LPA dephosphorylation products and thereby protection against LPA induced neurite collapse. In order to elucidate the physiological role of PRG-1 and its influence in lipid phosphate mediated signal transduction a constitutive and a conditional PRG-1 knockout (KO) mouse were generated. Thereby a specific expression of PRG-1 has been shown in glutamatergic neurons of the hippocampus by using an introduced reporter construct within the constitutive deletion construct. Furthermore it was observed that during heterozygous breeding approximately 50 % of PRG-1 deficient litters died due to the consequences of spontaneous seizures within the first three postnatal weeks. By cooperation with the group of Prof. Dietmar Schmitz these juvenile seizures could be monitored by in-vivo EEG recordings. Furthermore kainat applications demonstrated in adult heterozygous and homozygous PRG-1 KO animals a reduced threshold to develop seizures. Electrophysiological measurement in cooperation with the group of Prof. Dietmar Schmitz revealed a specific increase of excitability of hippocampal glutamatergic CA1 neurons. Despite these results, the cellular morphology regarding the development of neurons and interneurons, the expression of important synaptic markers and the electrophysiological intrinsic properties of the cells were unchanged. By generating expression vectors for PRG-1 and Cre-recombinase and utilizing in-utero electroporation techniques PRG-1 could be expressed specifically in single CA1 neurons of PRG-1 deficient animals respectively could be deleted in the generated conditional PRG-1-cKO mice. The electrophysiological analysis of electroporated and non-electroporated cells revealed that postsynaptic PRG 1 influences the presynapse. To further elucidate the impact of PRG-1 regarding LPA signal transduction uptake experiments with fluorescent labelled PA analogs has been performed on WT and PRG-1-KO primary neurons. Thereby it was revealed that KO neurons possess a reduced capability to accumulate lipid phosphates within the cell which led to the hypothesis that PRG-1 can regulate the extracellular concentration of lipid phosphate within the synaptic cleft. Finally, by in-utero electroporation of a PRG-1 expression construct which has been mutated at an essential amino acid within the conserved domain and by breeding of the PRG-1-KO mice with the LPA2 receptor KO mice it has been shown, that the physiological function of PRG-1 may be defined by its influence in lipid phosphate mediated signal transduction at the synapse. Under consideration of the fact that the LPA2 receptor is localised presynaptically and PRG-1 is localised postsynaptically at excitatory synapses of glutamatergic neurons this thesis describes a new possible mechanism of regulating excitatory transmission mediated by PRG-1