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

Dendritic compartment and neuronal output mode determine pathway-specific long-term potentiation in the piriform cortex

The apical dendrite of layer 2/3 pyramidal cells in the piriform cortex receives two spatially distinct inputs: one projecting onto the distal apical dendrite in sensory layer 1a, the other targeting the proximal apical dendrite in layer 1b. We observe an expression gradient of A-type K(+) channels that weakens the backpropagating action potential-mediated depolarization in layer 1a compared with layer 1b. We find that the pairing of presynaptic and postsynaptic firing leads to significantly smaller Ca(2+) signals in the distal dendritic spines in layer 1a compared with the proximal spines in layer 1b. The consequence is a selective failure to induce long-term potentiation (LTP) in layer 1a, which can be rescued by pharmacological enhancement of action potential backpropagation. In contrast, LTP induction by pairing presynaptic and postsynaptic firing is possible in layer 1b but requires bursting of the postsynaptic cell. This output mode strongly depends on the balance of excitation and inhibition in the piriform cortex. We show, on the single-spine level, how the plasticity of functionally distinct synapses is gated by the intrinsic electrical properties of piriform cortex layer 2 pyramidal cell dendrites and the cellular output mode

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

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

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

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

Titration of syntaxin1 in Mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.

Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms

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