Biology subjects

Martinez-Marmol, R.

Publications and source records attributed to Martinez-Marmol, R..

7 recordsLinked to original sources

bioRxiv2023

VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming

SNARE-mediated secretory vesicle (SV) exocytosis underpins neuronal communication. Munc18-1 orchestrates SNARE complex formation by controlling the opening of syntaxin-1A. How the SV-plasma membrane interface becomes fusion-competent at the nanoscale level is poorly understood. Here, we propose that the interaction of Munc18-1 with VAMP2 during vesicular docking triggers nanoscale re-organization which renders the SV-plasma membrane interface fusion-competent. We identified and mutated key residues in Munc18-1 domain 3a (A297 and T304) hypothesised to impair its interaction with VAMP2. Munc18-1A297H, and to a lesser extent Munc18-1T304H, constrained SVs on the plasma membrane and reduced stimulated secretion, under re-expression conditions in Munc18-1/2 double knockout neurosecretory cells. Moreover, the de-clustering of Munc18-1 in response to activity was lost for both mutants. The interaction of VAMP2 with the Munc18-1 domain 3a therefore controls the re-organization of the nanoscale environment of the docked SV-plasma membrane interface, fostering syntaxin-1A opening and Munc18-1 release to ensure that SNARE assembly only occurs within the confinement of docked vesicles.

neuroscience↗

bioRxiv2022

Surface SV2A-Syt1 nanoclusters act as a sequestration hub that limits dynamin-1 recruitment and targeting to recycling synaptic vesicles

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been postulated as a pre-assembly mechanism for endocytosis - ensuring high-fidelity retrieval. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through interaction of its C2B domain with SV2A, that are sensitive to mutations in this domain (Syt1K326A/K328A) and knocking down SV2A. SV2A co-cluster with Syt1 and blocking SV2As cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced plasma membrane recruitment of key endocytic protein dynamin-1, leading to accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. SV2A-Syt1 surface nanoclusters therefore negatively regulate the rate of their own re-entry into recycling SVs by controlling the recruitment of the endocytic machinery.

neuroscience↗

bioRxiv2021

SV2A-Syt1 interaction controls surface nanoclustering and access to recycling synaptic vesicles

Following exocytosis, the recapture of vesicular proteins stranded at the plasma membrane in recycling synaptic vesicles (SVs) is essential to sustain neurotransmission. Nanoclustering is emerging as a mechanism through which proteins may be pre-assembled prior to endocytosis, to ensure high fidelity of retrieval for subsequent rounds of vesicle fusion. Here, we used single molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A). Syt1 forms surface nanoclusters through interaction of its C2B domain (K326/K328) with SV2A, as demonstrated by mutating Syt1 (K326A/K328A) and knocking down endogenous SV2A. Blocking cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Impaired nanoclustering of Syt1 and SV2A leads to accelerated endocytosis of Syt1, altered intracellular sorting and decreased trafficking of Syt1 to a Rab5-positive endocytic pathway. We conclude that the interaction between SV2A and Syt1 locks both molecules into surface nanoclusters, controlling their entry into recycling SVs.

neuroscience↗

bioRxiv2021

The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion

Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells1. During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood2-4. Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.

neuroscience↗

bioRxiv2021

Syntaxin-1 is necessary for UNC5/Netrin-1-dependent macropinocytosis and chemorepulsion

Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by Uncoordinated locomotion 5 (UNC5) receptors located on external granule layer (EGL) tracts. Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5 receptors form a protein complex with the t-SNARE syntaxin-1 (Stx1). By combining botulinum neurotoxins, a shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin-1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.

neuroscience↗

bioRxiv2020

Frontotemporal dementia mutant tau (P301L) locks Fyn in an open, active conformation conducive to nanoclustering

Fyn is a Src kinase that controls critical signalling cascades and its postsynaptic enrichment underpins synaptotoxicity in Alzheimers disease (AD) and frontotemporal dementia (FTLD-tau). Previously, we found that pathogenic FTLD tau mutant (P301L) expression promotes aberrant trapping of Fyn in nanoclusters within hippocampal dendrites via an unknown mechanism (Padmanabhan et al., 2019). Here, we imaged Fyn-mEos2 using single particle tracking photoactivated localization microscopy (sptPALM) to demonstrate that nanoclustering of Fyn in hippocampal dendrites is promoted by Fyns open, primed conformation. Disrupting the auto-inhibitory, closed conformation of Fyn through phospho-inhibition, and perturbation of Fyns SH3 domain increases, Fyns nanoscale trapping. However, inhibition of Fyns catalytic domain has no impact on its mobility. Tau-P301L promotes Fyn lateral trapping via Fyn opening and ensuing increased catalytic activation. Pathogenic tau may therefore drive synaptotoxicity by locking Fyn in an open, catalytically active conformation, leading to postsynaptic entrapment and aberrant signalling cascades.

neuroscience↗

bioRxiv2020

Hericerin derivatives from Hericium erinaceus exert BDNF-like neurotrophic activity in central hippocampal neurons and enhance memory

The traditional medicinal mushroom Hericium erinaceus has long been known for enhancing the peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. It was also reported to protect against ageing-dependent cognitive decline in wildtype and in Alzheimers disease mouse models suggesting a yet to be defined action on neurons of the central nervous system. Here, we purified and identified biologically active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom together with its hydrophobic derivative hericene A, were highly potent in inducing extensive axon outgrowth and neurite branching in the absence of serum demonstrating high neurotropic activity. NDPIH also induced enlarged growth cones suggestive of a brain-derived neurotrophic factor (BDNF)-like activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA12 prevented NDPIH-induced neurotrophic activity providing evidence that NDPIH acts via TrkB receptors to mediate its neurotrophic effect in central neurons. Finally, in vivo treatment with H. erinaceus crude extract and hericene A significantly increased BDNF and downstream pathway and enhanced learning and memory in the novel object recognition memory test. Our results suggest that hericene A can promote BDNF-like activity in neurons in vitro and in vivo thereby enhancing recognition memory.

neuroscience↗