Biology subjects

van Weering, J. R. T.

Publications and source records attributed to van Weering, J. R. T..

7 recordsLinked to original sources

bioRxiv2024

Endosomal sorting protein SNX4 limits synaptic vesicle docking and release

Sorting nexin 4 (SNX4) is an evolutionary conserved organizer of membrane recycling. In neurons, SNX4 accumulates in synapses, but how SNX4 affects synapse function remains unknown. We generated a conditional SNX4 knock-out mouse model and report that SNX4 cKO synapses show enhanced neurotransmission during train stimulation, while the first evoked EPSC was normal. SNX4 depletion did not affect vesicle recycling, basic autophagic flux or the levels and localization of SNARE-protein VAMP2/synaptobrevin-2. However, SNX4 depletion affected synapse ultrastructure: an increase in docked synaptic vesicles at the active zone, while the overall vesicle number was normal, and a decreased active zone length. These effects together lead to a substantially increased density of docked vesicles per release site. In conclusion, SNX4 is a negative regulator of synaptic vesicle docking and release. These findings suggest a role for SNX4 in synaptic vesicle recruitment at the active zone.

cell biology↗

bioRxiv2024

Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis

Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10s established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics and protein synthesis. Gene ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+-pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.

neuroscience↗

bioRxiv2023

Tomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes

Tomosyns are soluble SNARE proteins proposed to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario using a novel mouse model, energy barrier recordings, and single-molecule force measurements. Tomosyn-1/2 deficiency drastically enhanced the probability that synaptic vesicles fuse at synapses, resulting in stronger synapses with faster depression and slower recovery. While wildtype tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Force measurements revealed that tomosyns SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that regions outside the SNARE motif contribute to tomosyns inhibitory function. These results reveal that tomosyns regulate synaptic transmission by preventing SNAP-25 binding to template complexes, increasing the energy barrier for synaptic vesicle fusion, and limiting synaptic depression.

neuroscience↗

bioRxiv2022

SNARE protein tomosyn regulates dense core vesicle composition but not exocytosis in mammalian neurons

Tomosyn is a large, non-canonical SNARE protein proposed to act as a competitive inhibitor of SNARE complex formation in vesicle exocytosis. In the brain, tomosyn inhibits fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model allowing conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l), and an assay that detects DCV exocytosis with single vesicle resolution in primary hippocampal neurons. Surprisingly, loss of both tomosyns did not affect DCV exocytosis but resulted in a strong reduction of intracellular levels of many DCV cargos, most prominently brain-derived neurotrophic factor (BDNF), granin VGF and prohormone convertase PCSK1. Reduced levels of DCV cargos were paralleled by decreased DCV size and impaired mRNA expression of the corresponding genes. We conclude that tomosyns regulate neuropeptide and neurotrophin secretion via control of DCV cargo production, and not at the step of cargo release. Our findings suggest a differential effect of tomosyn on the two main secretory pathways in mammalian neurons and argues against a conserved role of tomosyn as competitive inhibitor of SNARE complex formation.

neuroscience↗

bioRxiv2022

Circulating extracellular vesicles in lung cancer patients are not enriched in tumor-derived DNA fragments as revealed by whole genome sequencing.

Liquid biopsies contain multiple analytes that can be mined to improve the detection and management of cancer. Beyond cell-free DNA (cfDNA), mutations have been detected in DNA associated with extracellular vesicles (EV-DNA). The genome-wide composition and structure of EV-DNA are poorly characterized, and it remains undecided whether circulating EVs are enriched in tumor signal compared to unfractionated cfDNA. Here, using whole genome sequencing from selected lung cancer patients with a high cfDNA tumor content (>5%), we determined that the tumor fraction and heterogeneity are comparable between DNA associated with EVs and matched plasma cfDNA. DNA in EV fractions, obtained with standardized size-exclusion chromatography, are comprised of short [~]150-180 bp fragments and long >1000 bp fragments that are poor in tumor signal. Other fractions only exhibit short fragments with similar tumor DNA content. The composition in bases at the end of EV-DNA fragments, as well as their fragmentation patterns are similar to plasma cfDNA. Mitochondrial DNA is relatively enriched in EV fractions. Our results highlight that cfDNA in plasma is of dual nature, either bound to proteins (including the nucleosome) but also associated to EV. cfDNA associated to small EV (including exosomes) is however not preferentially enriched in tumor signal.

genomics↗

bioRxiv2020

Retrograde transport defects in Munc18-1 null neurons explain abnormal Golgi morphology

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its t-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether these Golgi abnormalities cause defects in the constitutive and regulated secretory pathway that may explain neurodegeneration. Electron microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. Anterograde ER-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking. We conclude that MUNC18-1 deficient neurons have normal anterograde yet reduced retrograde transport to the Golgi. This imbalance in transport routes provides a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons. Significance statementLoss of MUNC18-1 or its t-SNAREs SNAP25 and syntaxin-1 leads to massive, yet unexplained, neurodegeneration. Previous research showed that Golgi abnormalities are the earliest, shared phenotype. Golgi abnormalities are also an early feature in neurodegenerative diseases, such as Alzheimers Disease or Amyotrophic Lateral Sclerosis. This study elucidates the mechanism underlying the Golgi phenotype upon loss of MUNC18-1. By systematically assessing transport routes to and from the Golgi, we show that retrograde endosome-to-Golgi, but not anterograde transport from the Golgi, is disturbed. This imbalance in transport routes provides a plausible explanation for the Golgi phenotype, and may explain the neurodegeneration. The findings in this study contributes to new insights in cellular mechanisms of neurodegeneration.

neuroscience↗

bioRxiv2019

The endosomal protein sorting nexin 4 is a novel synaptic protein

Sorting nexin 4 (SNX4) is an evolutionary conserved protein that mediates recycling from the endosomes back to the plasma membrane in yeast and mammalian cells. SNX4 is expressed in the brain, but its neuronal localization and function have not been addressed. Using a new antibody, endogenous neuronal SNX4 co-localized with both early and recycling endosomes, similar to the reported localization of SNX4 in non-neuronal cells. Neuronal SNX4 was accumulated in synapses, and immuno-electron microscopy revealed that SNX4 was predominantly localized to presynaptic terminals. Acute depletion of neuronal SNX4 using independent shRNAs did not affect the levels of the canonical SNX4-cargo transferrin receptor. Explorative mass spectrometry showed that each SNX4-targetted shRNA resulted in a reproducible and distinct proteome and that synaptic communication-related proteins were downregulated upon expression of the three shRNAs. The identification of SNX4 as a novel synaptic protein indicates a selective demand for SNX4 dependent sorting in synapses.

neuroscience↗