Non-synaptic exocytosis enhanced in rat cerebellar granule neurons cultured under survival-promoting conditions

Urakubo, Tomoyoshi; Tominaga-Yoshino, Keiko; Ogura, Akihiko

doi:10.1016/s0168-0102(03)00006-3

Cited by 15 publications

(16 citation statements)

References 11 publications

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“…2 A). These observations are consistent with previous reports that the majority of varicosities containing presynaptic proteins of cultured cerebellar GCs are not apposed to definite postsynaptic structures (Marxen et al, 1999;Urakubo et al, 2003), whereas functional synapses may be formed in a small portion of cultured GC axons as synaptic transmission was detected in cultured GCs (Losi et al, 2002;Lu et al, 2006).…”

Section: Tetramerization Is Required For Glur␦2-ntd To Induce Presynasupporting

confidence: 93%

“…Contacts with postsynaptic sites trigger the stabilization and maturation of synapses. In cultured cerebellar GCs, the majority of varicosities containing presynaptic proteins are not apposed to definite postsynaptic structures (Marxen et al, 1999;Urakubo et al, 2003). Cbln1 is a high-affinity ligand for NRXNs (Uemura et al, 2010;Joo et al, 2011) and is secreted from cerebellar GCs (Bao et al, 2005), suggesting that the interaction between secreted Cbln1 and presynaptic NRXNs takes place before PF-PC synapse formation.…”

Section: Mechanism Of Induction By Glur␦2 Of Pf-pc Synapse Formationmentioning

confidence: 99%

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GluRδ2 Assembles Four Neurexins intoTrans-Synaptic Triad to Trigger Synapse Formation

Lee¹,

Uemura²,

Yoshida³

et al. 2012

J. Neurosci.

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Elucidation of molecular mechanisms of synapse formation is a prerequisite for the understanding of neural wiring, higher brain functions, and mental disorders. The trans-synaptic interaction of postsynaptic glutamate receptor ␦2 (GluR␦2) and presynaptic neurexins (NRXNs) through cerebellin precursor protein 1 (Cbln1) mediates synapse formation in vivo in the cerebellum. Here, we asked how the trans-synaptic triad induces synapse formation. Native GluR␦2 existed as a tetramer in the membrane, whereas the N-terminal domain (NTD) of GluR␦2 formed a stable homodimer. When incubated with cultured mouse cerebellar granule cells (GCs), dimeric GluR␦2-NTD and Cbln1 exerted little effect on the accumulation of punctate immunostaining signals for Bassoon and vesicular glutamate transporter 1 in GC axons. However, tetramerized GluR␦2-NTD stimulated the accumulation of these presynaptic proteins in the axons. Analysis of Cbln1 mutants suggested that the binding sites of GluR␦2 and NRXN1␤ on Cbln1 are differential. Furthermore, there was no competition in the binding to Cbln1 between GluR␦2-NTD and the extracellular domain (ECD) of NRXN1␤. Thus, GluR␦2 and Cbln1 interacted with each other rather independently of Cbln1-NRXN1␤ interaction and vice versa. Gel filtration and isothermal titration calorimetry analyses consistently showed that dimeric GluR␦2-NTD and hexameric Cbln1 assembled in the 1:1 ratio, whereas hexameric Cbln1 and the laminin-neurexin-sex hormone-binding globulin domain of NRXN1␤-ECD assembled in the 1:2 ratio. Thus, the synaptogenic triad is assembled from tetrameric GluR␦2, hexameric Cbln1, and monomeric NRXN in the ratio of 1:2:4. These results suggest that GluR␦2 triggers synapse formation by clustering four NRXNs through triad formation.

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Section: Tetramerization Is Required For Glur␦2-ntd To Induce Presynasupporting

confidence: 93%

Section: Mechanism Of Induction By Glur␦2 Of Pf-pc Synapse Formationmentioning

confidence: 99%

GluRδ2 Assembles Four Neurexins intoTrans-Synaptic Triad to Trigger Synapse Formation

Lee¹,

Uemura²,

Yoshida³

et al. 2012

J. Neurosci.

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“…In these paradigms, exocytosis was examined after cultures were equilibrated in medium containing physiological concentrations of KCl (Harrold et al, 1997;Ward et al, 2000), presumably to promote some recovery of the presynaptic fusion apparatus. Consistent with this, mouse granule neurons recover spontaneous miniature inhibitory postsynaptic potentials and miniature excitatory postsynaptic potentials after overnight equilibration in medium containing 5 mM KCl to replenish vesicles (Mellor et al, 1998;Urakubo et al, 2003), suggesting that particularly dynamic processes are able to revert to a state that resembles granule neurons in vivo when examined over a relatively limited time frame. It is well established, however, that withdrawal of elevated KCl from neuroadapted cultures is itself a potent trigger of neuronal death and is used extensively as a model of apoptosis (Miller and Johnson, 1996;Watson et al, 1998;Contestabile, 2002).…”

Section: Monaco and Vallanosupporting

confidence: 57%

“…Almost without exception, however, cultures are grown in media containing elevated KCl (or NMDA) because it enhances long-term survival compared with those grown in physiological media (Gallo et al, 1987). This is despite mounting evidence that continually depolarized cultures undergo numerous adaptations, including perturbed patterns of AMPA (Condorelli et al, 1993), NMDA (Vallano et al, 1996), and GABA (Mellor et al, 1998) receptor subunit expression; abnormal induction of inositol 1,4,5-trisphosphate receptor (Choi et al, 2004); calpain-mediated down-regulation of Ca 2ϩ /calmodulin-dependent protein kinase type IV (Tremper-Wells and Vallano, 2005), a net decrease in synaptic vesicles (Urakubo et al, 2003); suppression of spontaneous synaptic activity and action potentials (Mellor et al, 1998); reduced VDCC currents; and increased susceptibility to death upon activity reduction (Moulder et al, 2003). On the other hand, a significant proportion of granule neurons grown in medium containing 5 mM KCl require both tetrodotoxin-sensitive Na ϩ currents and active NMDA receptors for their survival (Fig.…”

Section: Discussionmentioning

confidence: 99%

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Monaco

Vallano²

2005

Molecular Pharmacology

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Cerebellar granule neurons are highly susceptible to injury in vivo and in vitro, and primary cultures are widely used to characterize relevant receptors and signaling pathways. However, there are problems associated with their use. In particular, cultures are typically grown in medium supplemented with elevated KCl levels because it improves survival, but accumulating evidence indicates that this causes profound neuroadaptations. For example, growth in elevated KCl levels renders neurons electrically silent. Thus, they cannot be used to examine excitotoxicity of synaptic origins. On the other hand, cultures grown in physiological medium are rarely studied because a proportion undergoes apoptosis. Herein, we provide evidence that mature neurons cultured in physiological KCl develop spontaneous action potentials that support survival through N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms. Furthermore, the cdk inhibitor roscovitine enhances the coupling between tetrodotoxin-sensitive action potentials and P/Q-type voltage-dependent calcium channels (VDCCs), thereby converting this survival program to excitotoxicity of synaptic origin. Therefore, roscovitine-triggered necrosis requires spontaneous Na ϩ -based action potentials (tetrodotoxin inhibits, (Ϯ)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (-agatoxin-IVA and -conotoxin-MVIIC inhibit, but not -conotoxin-GVIA), intact vesicle fusion processes (tetanus toxin inhibits), and transmitter-filled vesicles (concanamycin and bafilomycin inhibit). From a postsynaptic standpoint, roscovitine-mediated excitotoxicity requires the functionally linked activation of ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/ KA) and NMDA receptors, which is consistent with evidence that activated AMPA/KA receptors relieve the voltage-dependent Mg 2ϩ block of NMDA receptors, resulting in excitotoxic Ca 2ϩ influx. In the end, NMDA receptor-linked pathways transduce excitotoxicity. On the other hand, L-type VDCC blockers are not protective. Further characterization of this new model is expected to provide important insights about excitotoxicity of synaptic origins and about roscovitine as a selective modulator of this process.Glutamate-mediated excitotoxicity is extensively studied in cell cultures and animals to model key events underlying neuronal loss in human neurodegeneration (Aarts and Tymianski, 2004). Numerous experimental paradigms support the theory that sustained NMDA receptor activation leads to toxic intracellular Ca 2ϩ deregulation and death by necrosis and/or apoptosis. Therefore, NMDA receptor antagonists afford protection and provide a rationale for their use in clinical trials in humans. Unfortunately, these trials were not correspondingly successful, prompting investigators to re-evaluate the predictive value of existing models and stimulating renewed efforts to identify novel therapeutic targets.The "source-specificity hypothesis" of excitotoxicity posits that discrete Ca 2ϩ microdomains, as opposed to alteration...

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“…Varicosities (VRs) are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrates and vertebrates, for example, Helix neurons (Cibelli et al, 1996;Fiumara et al, 2001); Aplysia neurons (Hatada et al, 2000;Grabham et al, 2005); fish retinal explants (Koenig et al, 1985); rat cortical neurons (Morgenthaler et al, 2003); rat granule cells (Urakubo et al, 2003); pyramidal neurons (Umeda et al, 2005); and hippocampal neurons (Krueger et al, 2003; Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11068-006-9007-7 Sankaranarayanan et al, 2003). In culture, VRs can be formed by neurons that have not made contact with postsynaptic cells, as well as by neurons that form en passant synapses where they are often referred to as presynaptic boutons (Hatada et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

et al. 2007

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Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane "sleeve", split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities' organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as "ready to go" prefabricated presynaptic terminals. We suggest that the varicosities' motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse.

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Non-synaptic exocytosis enhanced in rat cerebellar granule neurons cultured under survival-promoting conditions

Cited by 15 publications

References 11 publications

GluRδ2 Assembles Four Neurexins intoTrans-Synaptic Triad to Trigger Synapse Formation

GluRδ2 Assembles Four Neurexins intoTrans-Synaptic Triad to Trigger Synapse Formation

Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

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