Ling-Gang Wu

Two modes of fusion pore opening revealed by cell-attached recordings at a synapse

Fusion of a vesicle with the cell membrane opens a pore that releases transmitter to the extracellular space. The pore can either dilate fully so that the vesicle collapses completely, or close rapidly to generate ‘kiss-and-run’ fusion. The size of the pore determines the release rate. At synapses, the size of the fusion pore is unclear, ‘kiss-and-run’ remains controversial, and the ability of ‘kiss-and-run’ fusion to generate rapid synaptic currents is questionable. Here, by recording fusion pore kinetics during single vesicle fusion, we found both full collapse and ‘kiss-and-run’ fusion at calyx-type synapses. For full collapse, the initial fusion pore conductance (Gp) was usually >375 pS and increased rapidly at ≥299 pS ms–1. ‘Kiss-and-run’ fusion was seen as a brief capacitance flicker (<2 s) with Gp >288 pS for most flickers, but within 15–288 pS for the remaining flickers. Large Gp (>288 pS) might discharge transmitter rapidly and thereby cause rapid synaptic currents, whereas small Gp might generate slow and small synaptic currents. These results show that ‘kiss-and-run’ fusion occurs at synapses and that it can generate rapid postsynaptic currents, and suggest that various fusion pore sizes help to control the kinetics and amplitude of synaptic currents.

The Origin of Quantal Size Variation: Vesicular Glutamate Concentration Plays a Significant Role

Fusion of a single vesicle induces a quantal response, which is critical in determining synaptic strength. Quantal size varies at most synapses. Its underlying mechanisms are not well understood. Here, we examined five sources of variation: vesicular glutamate concentration ([Glu]v), vesicle volume, ultrafast fusion pore closure, the postsynaptic receptor, and the location between release and the postsynaptic receptor cluster at glutamatergic, calyx of Held synapses. By averaging 2.66 million fusion events from 459 synapses, we resolved the capacitance jump evoked by single vesicle fusion. This capacitance jump, an indicator of vesicle volume, was independent of the amplitude of the miniature EPSC (mEPSC) recorded simultaneously at the same synapses. Thus, vesicle volume is not the main source of mEPSC variation. The capacitance jump was not followed by submillisecond endocytosis, excluding ultrafast endocytosis as a source of variation. Larger mEPSCs were increased to a lesser extent by presynaptic glutamate dialysis, and reduced to a lesser extent by γ-DGG (γ-d-glutamylglycine), a competitive AMPA receptor blocker, suggesting that a higher glutamate concentration in the synaptic cleft contributes to the large size of mEPSCs. Larger mEPSCs were not accompanied by briefer rise times, inconsistent with the prediction by, and thus arguing against, the scenario that larger mEPSCs are caused by a shorter distance between the release site and the postsynaptic receptor cluster. In summary, the different amplitudes of mEPSCs were mainly attributable to release of vesicles having similar volumes, but different glutamate amounts, suggesting that [Glu]v is a main source of quantal size variation.

Calcineurin Is Universally Involved in Vesicle Endocytosis at Neuronal and Nonneuronal Secretory Cells

Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

Voltage-Dependent Calcium Channels at the Plasma Membrane, but Not Vesicular Channels, Couple Exocytosis to Endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.

Senescence impairs direct conversion of human somatic cells to neurons

Recent reports have shown that fibroblasts can be converted to neurons by forced expression of transcription factors. However, the mechanisms underlying this conversion remain unclear. Here, we show that the efficiency of neuronal conversion of embryonic human fibroblasts aged in culture is lower than that in cells in early culture stages. Moreover, depletion of p16(Ink4a) and p19(Arf) involved in the activation of cellular senescence is sufficient to convert human fibroblast and epithelial cells into neurons. The induced neurons express neuron-specific proteins, generate action potentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis shows that the induced neurons have a profile different from fibroblasts and similar to that of control neurons induced by established methods. We further noted that expression of p53 blocks the neuronal conversion, whereas expression of human telomerase reverse transcriptase (hTERT) induces it. Our results indicate that overcoming senescence is a crucial step for neuronal conversion of somatic cells.

Small voltage changes at nerve terminals travel up axons to affect action potential initiation

Nerve terminals are generally considered to be the destination points for electrical signals, which propagate unidirectionally from the soma to nerve terminals. We found that small hyperpolarizations or depolarizations (∼10 mV) generated under physiological conditions in rat nerve terminals backpropagated up the axon (∼400–800 μm) and changed the threshold for initiating action potentials and thus firing patterns. These results suggest a mechanism for information processing in neurons and neuronal circuits.

A Membrane Pool Retrieved via Endocytosis Overshoot at Nerve Terminals: A Study of Its Retrieval Mechanism and Role

Endocytosis overshoot, which retrieves more membrane than vesicles just being exocytosed, occurs at nerve terminals and non-neuronal secretory cells. The mechanism that retrieves the overshoot membrane pool and the role of this pool remain largely unknown. We addressed this issue at the rat calyx of Held nerve terminal with capacitance measurements. We found that every calyx contained an overshoot pool ∼1.8 times the readily releasable pool. Retrieval of this pool required large calcium influx, and was inhibited by blockers of calcium/calmodulin-activated calcineurin and dynamin, suggesting the involvement of calcineurin and dynamin in endocytosis overshoot. Depletion of the overshoot pool slowed down compensatory endocytosis, whereas recovery of the overshoot pool via exocytosis that deposited stranded vesicles to the plasma membrane led to recovery of compensatory endocytosis, suggesting that the overshoot pool enhances endocytosis efficiency. These results suggest that the overshoot pool exists at every nerve terminal, is of limited size arising from vesicles stranded at the plasma membrane, is retrieved via calcium/calmodulin/calcineurin and dynamin signaling pathway, and can enhance endocytosis efficiency. Potential mechanisms for how the endocytosis overshoot pool enhances endocytosis efficiency are discussed.

Conversion of Fibroblasts to Neural Cells by p53 Depletion

SUMMARY Conversion from fibroblasts to neurons has recently been successfully induced. However, the underlying mechanisms are poorly understood. Here, we find that depletion of p53 alone converts fibroblasts into all three major neural lineages. The induced neuronal cells express multiple neuron-specific proteins and generate action potentials and transmitter-receptor-mediated currents. Surprisingly, depletion does not affect the well-known tumorigenic p53 target, p21. Instead, knockdown of p53 upregulates neurogenic transcription factors, which in turn boosts fibroblast-neuron conversion. p53 binds the promoter of the neurogenic transcription factor Neurod2 and regulates its expression during fibroblast-neuron conversion. Furthermore, our method provides a high efficiency of conversion in late-passage fibroblasts. Genome-wide transcriptional analysis shows that the p53-deficiency-induced neurons exhibit an expression profile different from parental fibroblasts and similar to control-induced neurons. The results may help to understand and improve neural conversion mechanisms to develop robust neuron-replacement therapy strategies.

Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic function and plasticity and plays important roles in neuronal development, survival, and brain disorders. Despite such diverse and important roles, how BDNF, or more generally speaking, neurotrophins affect synapses, particularly nerve terminals, remains unclear. By measuring calcium currents and membrane capacitance during depolarization at a large mammalian central nerve terminal, the rat calyx of Held, we report for the first time that BDNF slows down calcium channel activation, including P/Q-type channels, and inhibits exocytosis induced by brief depolarization or single action potentials, inhibits slow and rapid endocytosis, and inhibits vesicle mobilization to the readily releasable pool. These presynaptic mechanisms may contribute to the important roles of BDNF in regulating synapses and neuronal circuits and suggest that regulation of presynaptic calcium channels, exocytosis, and endocytosis are potential mechanisms by which neurotrophins achieve diverse neuronal functions.

Location Matters: Synaptotagmin Helps Place Vesicles Near Calcium Channels

Positioning releasable vesicles near voltage-gated calcium channels may ensure transmitter release upon calcium influx. Disruption of vesicle positioning may underlie short-term synaptic depression. However, how this positioning is achieved is unclear. In this issue of Neuron, Young and Neher find that synaptotagmin 2 helps to align readily releasable vesicles near calcium channels at nerve terminals.