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

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

Textiloma: a case of foreign body mimicking a spinal mass

Items such as cotton or gauze pads can be mistakenly left behind during operations. Such foreign materials (called textilomas or gossypibomas) cause foreign body reaction in the surrounding tissue. The complications caused by these foreign bodies are well known, but cases are rarely published because of medico-legal implications. Some textilomas cause infection or abscess formation in the early stage, whereas others remain clinically silent for many years. Here, we describe a case of textiloma in which the patient presented with low-back pain 4 years after lumbar discectomy. Imaging revealed an abcess-like mass in the lumbar epidural space

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.Peer reviewe

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. Deleting OSH1-3 in 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