Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses

Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2012; 109(8):E515-23. DOI: 10.1073/pnas.1121626109
Source: PubMed


Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.

Download full-text


Available from: Mirko Messa, Jul 02, 2014
  • Source
    • "These findings suggest that the additional action potentials delivered during TBS were not sufficient to alter the vesicular composition of boutons and that the decrease in vesicles with LTP involved NMDA-receptor activation. Synaptic vesicles in axonal boutons are recycled via endocytosis in order to sustain release (Murthy and De, 2003; Ryan, 2006; Haucke et al., 2011; Lou et al., 2012). Such a tight coupling between release and endocytosis suggests that anatomical evidence of endocytosis could serve to mark which boutons were undergoing presynaptic activity at the time of fixation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 min after the induction of LTP; however, by 2 hr many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with three-dimensional reconstructions from serial section electron microscopy at 30 min and 2 hr after induction of LTP by theta-burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hr, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 µm). Docked vesicles were reduced at 30 min and then returned to control levels by 2 hr following induction of LTP. By 2 hr there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin-mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin-coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 min and 2 hr after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 min but markedly reduced by 2 hr after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on NMDA receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Jun 2013 · The Journal of Comparative Neurology
  • Source
    • "However, other reports indicate there are reasons that may explain them. Dynasore affects a number of dynamin isoforms, disabling not only dynamin 1, dynamin 2 and Drp1, the mitochondrial dynamin (Macia et al., 2006), but also potentially dynamin 3 (Lou et al., 2012). It is also possible that Dynasore is targeting another unidentified GTPase required at the early stages of invagination (Nankoe and Sever, 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking remain poorly understood. We assessed the role of clathrin, adaptor complexes, lipid rafts and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNAi-mediated depletion of caveolin 1, 2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant negative mutant not only disrupted clathrin-regulated pathways but also blocked PM to TGN internalization of ATP7A. Depletion of the μ subunits of either AP-2 or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Importantly, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and wash-out, suggesting forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane associated factors including clathrin, AP-2, AP-1 and Rab22 are regulators of ATP7A trafficking.
    Full-text · Article · Apr 2013 · Molecular biology of the cell
  • [Show abstract] [Hide abstract]
    ABSTRACT: Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization.
    No preview · Article · Jul 2012 · Cold Spring Harbor perspectives in biology
Show more