Natali L. Chanaday

Synaptobrevin‐2 (Syb2) is a SNARE protein essential for neurotransmitter release and communication in the nervous system. We previously showed that Syb2 is also exchanged among neurons via extracellular vesicles (EVs). Host neurons rapidly integrate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release. However, the endocytic mechanism by which neurons incorporate Syb2‐containing EVs is unknown. Here, we use a fusion of Syb2 with the pH‐sensitive GFP (Syb2‐pHluorin) to track the incorporation of Syb2‐containing EVs into rat hippocampal and cortical neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2‐containing EVs are endocytosed via a Dynamin‐dependent pathway, largely independently of macropinocytosis. The same endocytic route is used in the somatodendritic and axonal compartment and it occurs very rapidly, with the majority of Syb2‐pHluorin residing in internal acidic organelles at 30 min after EV addition, including synaptic vesicles at synapses. Leveraging this finding, we used EVs to sparsely deliver Syb2‐pHluorin to synapses and track the fusion and endocytosis of single synaptic vesicles. The results indicate that Syb2‐pHluorin‐positive synaptic vesicles are endocytosed with either ultrafast (< 1 s) or fast (~1–3 s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. Our findings expand our understanding of the mechanisms EVs use to enter neurons and open the door for future applications of EVs as vehicles to deliver fluorescent molecules in a neuron‐specific, targeted manner. image

Glycosphingolipid glycosyltransferases (GGTs) can organize as multienzyme complexes localized along the Golgi complex. However, the influence of the relative presence of GGTs on the localization of their clients is unclear. Here, we determine that expression of certain full-length GGTs increases the levels of Golgi phosphoprotein 3 (GOLPH3), an adaptor oncoprotein involved in Golgi trafficking and organization. Furthermore, we demonstrate that expression of the N-terminal domain of GGTs, which lacks the catalytic domain, is sufficient to achieve this regulation on GOLPH3 in a cell type-dependent manner. We also identify the N-terminal domain of β4GalT-VI GGT as an inhibitor of GOLPH3 expression and thus a potential therapeutic application, since GOLPH3 overexpression is associated with progression and poor prognosis of multiple tumor types. Our data further suggest that the cytoplasmic tail of β4GalT-VI N-terminal domain interferes with the ability of GOLPH3 to interact with phosphatidylinositol 4-phosphate, which consequently reduces the levels of GOLPH3, thereby impairing its function in the acquisition of mesenchymal features.

Synaptobrevin-2 (Syb2) is an essential SNARE protein for neurotransmitter release and communication in the nervous system. We previously showed that Syb2 is also exchanged among neurons via extracellular vesicles (EVs). Host neurons can rapidly incorporate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release, however the endocytic mechanism of Syb2-containing EVs is unknown. Here, we use a fusion of Syb2 with the pH-sensitive GFP (Syb2-pHluorin) to track the incorporation of Syb2-containing EVs into neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2-containing EVs are endocytosed via Clathrin- and Dynamin-independent pathways. Moreover, Syb2-containing EVs are directly uptaken by axons and are rapidly incorporated into functional synaptic vesicles. These Syb2-pHluorin positive synaptic vesicles are endocytosed with either ultrafast (<1s) or fast (∼1-3s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. This work introduces a novel application of EVs as vehicles to deliver fluorescent molecules in a neuron-specific, targeted manner to investigate protein transport mechanisms without overexpression artifacts. Our findings expand our understanding of the mechanisms EVs use to enter neurons.

SUMMARY: RNAs move through the extracellular space to transmit information between cells, including mammalian neurons, yet how specific RNAs are channeled into these extracellular routes is unknown. Using genome-wide CRISPR screening, proteomics, and high-sensitivity transcriptomics in a neuronal model system, we identify domesticated retroviral proteins and RNA-modifying enzymes that regulate RNA loading into and transportation via extracellular vesicles. We show that the pseudouridine synthase PUS1 is a key determinant of RNA trafficking, and that its catalytic product in RNA, pseudouridine, is enriched in extracellular RNAs from transformed and primary neurons. Furthermore, the presence of pseudouridine on select RNAs is both necessary and sufficient for their extracellular export. Finally, we show that myosin light chain 6 (MYL6) is a pseudouridine-binding protein required for secretion of synthetic and endogenous RNAs. These findings reveal a biochemical code linking chemical RNA modification to extracellular transport, and establish a framework to study the function of extracellular RNAs in the nervous system and beyond.

Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons down to negligible levels. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Recycling of synaptic vesicles during spontaneous or low levels of evoked activity were largely impervious to dynamin depletion, while retrieval of synaptic vesicle components at higher levels of activity was partially arrested. These results suggest the existence of balancing dynamin‐independent mechanisms for synaptic vesicle recycling at central synapses. Classical dynamin‐dependent mechanisms are not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion, but they become more relevant for membrane retrieval during intense, sustained neuronal activity. image Key points: Loss of dynamin 2 does not impair synaptic transmission. Loss of all three dynamin isoforms mostly affects evoked neurotransmission. Excitatory synapse function is more susceptible to dynamin loss. Spontaneous neurotransmission is only mildly affected by loss of dynamins. Single synaptic vesicle endocytosis is largely dynamin independent.

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release, however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release. In non-neuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of non-photobleached probes within 10-seconds. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

The nature of the mechanism coupling fusion and endocytosis of synaptic vesicles has been long debated. Here, Chanaday and Kavalali show that the SNARE synaptobrevin-2 couples fast synchronous, single synaptic vesicle release to a Ca²⁺-dependent fast mode of retrieval.