BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines.

Synaptic structural plasticity, the expansion of dendritic spines in response to synaptic stimulation, is essential for experience-dependent plasticity and is driven by branched actin polymerization. The WAVE regulatory complex (WRC) is confined to nanodomains at the postsynaptic membrane where it catalyzes actin polymerization. As the netrin/RGM receptor Neogenin is a critical regulator of the WRC, its nanoscale organization may be an important determinant of WRC nanoarchitecture and function. Using super-resolution microscopy, we reveal that Neogenin is highly organized on the spine membrane at the nanoscale level. We show that Neogenin binding to the WRC promotes co-clustering into nanodomains in response to brain-derived neurotrophic factor (BDNF), indicating that nanoclustering occurs in response to synaptic stimulation. Disruption of Neogenin/WRC binding not only prevents BDNF-mediated actin remodeling but also inhibits BDNF-induced calcium signaling. We conclude that the assembly of Neogenin/WRC nanodomains is a prerequisite for BDNF-mediated structural and synaptic plasticity.

SV2A controls the surface nanoclustering and endocytic recruitment of Syt1 during synaptic vesicle recycling.

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 proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. 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 the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs.

PMP22 duplication dysregulates lipid homeostasis and plasma membrane organization in developing human Schwann cells.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.

Actomyosin-II protects axons from degeneration induced by mild mechanical stress.

Whether, to what extent, and how the axons in the central nervous system (CNS) can withstand sudden mechanical impacts remain unclear. By using a microfluidic device to apply controlled transverse mechanical stress to axons, we determined the stress levels that most axons can withstand and explored their instant responses at nanoscale resolution. We found mild stress triggers a highly reversible, rapid axon beading response, driven by actomyosin-II-dependent dynamic diameter modulations. This mechanism contributes to hindering the long-range spread of stress-induced Ca2+ elevations into non-stressed neuronal regions. Through pharmacological and molecular manipulations in vitro, we found that actomyosin-II inactivation diminishes the reversible beading process, fostering progressive Ca2+ spreading and thereby increasing acute axonal degeneration in stressed axons. Conversely, upregulating actomyosin-II activity prevents the progression of initial injury, protecting stressed axons from acute degeneration both in vitro and in vivo. Our study unveils the periodic actomyosin-II in axon shafts cortex as a novel protective mechanism, shielding neurons from detrimental effects caused by mechanical stress.

Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters.

Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.

Synapsin 2a tetramerisation selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles.

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Location, location, location: Protein kinase nanoclustering for optimised signalling output.

Protein kinases (PKs) are proteins at the core of cellular signalling and are thereby responsible for most cellular physiological processes and their regulations. As for all intracellular proteins, PKs are subjected to Brownian thermal energy that tends to homogenise their distribution throughout the volume of the cell. To access their substrates and perform their critical functions, PK localisation is therefore tightly regulated in space and time, relying upon a range of clustering mechanisms. These include post-translational modifications, protein-protein and protein-lipid interactions, as well as liquid-liquid phase separation, allowing spatial restriction and ultimately regulating access to their substrates. In this review, we will focus on key mechanisms mediating PK nanoclustering in physiological and pathophysiological processes. We propose that PK nanoclusters act as a cellular quantal unit of signalling output capable of integration and regulation in space and time. We will specifically outline the various super-resolution microscopy approaches currently used to elucidate the composition and mechanisms driving PK nanoscale clustering and explore the pathological consequences of altered kinase clustering in the context of neurodegenerative disorders, inflammation, and cancer.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

Phospholipase Modulation of Synaptic Membrane Landscape: Driving Force Behind Memory Formation?

The synapse is the communication unit of the brain, linking billions of neurons through trillions of synaptic connections. The lipid landscape of the synaptic membrane underpins neurotransmitter release through the exocytic fusion of neurotransmitter-containing vesicles, endocytic recycling of these synaptic vesicles, and the postsynaptic response following binding of the neurotransmitter to specialized receptors. How the connected brain can learn and acquire memories through synaptic plasticity is unresolved. Phospholipases, and especially the phospholipase A1 isoform DDHD2, have recently been shown to play a critical role in memory acquisition through the generation of saturated free fatty acids such as myristic and palmitic acids. This emerging synaptic plasticity pathway suggests that phospholipases cannot only respond to synaptic activity by altering the phospholipid landscape but also contribute to the establishment of long-term memories in our brain.

SARS-CoV-2 omicron BA.5 and XBB variants have increased neurotropic potential over BA.1 in K18-hACE2 mice and human brain organoids.

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential.

Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles.

Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.

Fear extinction is regulated by the activity of long noncoding RNAs at the synapse.

Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.

Functional Roles of UNC-13/Munc13 and UNC-18/Munc18 in Neurotransmission.

Neurotransmitters are released from synaptic and secretory vesicles following calcium-triggered fusion with the plasma membrane. These exocytotic events are driven by assembly of a ternary SNARE complex between the vesicle SNARE synaptobrevin and the plasma membrane-associated SNAREs syntaxin and SNAP-25. Proteins that affect SNARE complex assembly are therefore important regulators of synaptic strength. In this chapter, we review our current understanding of the roles played by two SNARE interacting proteins: UNC-13/Munc13 and UNC-18/Munc18. We discuss results from both invertebrate and vertebrate model systems, highlighting recent advances, focusing on the current consensus on molecular mechanisms of action and nanoscale organization, and pointing out some unresolved aspects of their functions.

Lipids and Secretory Vesicle Exocytosis.

In recent years, the number of studies implicating lipids in the regulation of synaptic vesicle exocytosis has risen considerably. It has become increasingly clear that lipids such as phosphoinositides, lysophospholipids, cholesterol, arachidonic acid and myristic acid play critical regulatory roles in the processes leading up to exocytosis. Lipids may affect membrane fusion reactions by altering the physical properties of the membrane, recruiting key regulatory proteins, concentrating proteins into exocytic "hotspots" or by modulating protein functions allosterically. Discrete changes in phosphoinositides concentration are involved in multiple trafficking events including exocytosis and endocytosis. Lipid-modifying enzymes such as the DDHD2 isoform of phospholipase A1 were recently shown to contribute to memory acquisition via dynamic modifications of the brain lipid landscape. Considering the increasing reports on neurodegenerative disorders associated with aberrant intracellular trafficking, an improved understanding of the control of lipid pathways is physiologically and clinically significant and will afford unique insights into mechanisms and therapeutic methods for neurodegenerative diseases. Consequently, this chapter will discuss the different classes of lipids, phospholipase enzymes, the evidence linking them to synaptic neurotransmitter release and how they act to regulate key steps in the multi-step process leading to neuronal communication and memory acquisition.

Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing.

Single-molecule localization microscopy techniques are emerging as vital tools to unravel the nanoscale world of living cells by understanding the spatiotemporal organization of protein clusters at the nanometer scale. Current analyses define spatial nanoclusters based on detections but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in video games to detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to determine the overlap of the bounding boxes of individual molecular trajectories to establish membership in nanoclusters. Extending the spatial indexing into the time dimension allows the resolution of spatial nanoclusters into multiple spatiotemporal clusters. Using spatiotemporal indexing, we found that syntaxin1a and Munc18-1 molecules transiently cluster in hotspots, offering insights into the dynamics of neuroexocytosis. Nanoscale spatiotemporal indexing clustering (NASTIC) has been implemented as a free and open-source Python graphic user interface.

SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity.

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca2+ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation.

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.