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Arf6 regulates the cycling and the readily releasable pool of synaptic vesicles at hippocampal synapse.

Tagliatti E, Fadda M, Falace A, Benfenati F, Fassio A - Elife (2016)

Bottom Line: Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs.The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting.We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.

View Article: PubMed Central - PubMed

Affiliation: Center of Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, Italy.

ABSTRACT
Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.

No MeSH data available.


Related in: MedlinePlus

SypHy expression at Arf6-silenced synapses and effect of Arf6 shRNA#2.DOI:http://dx.doi.org/10.7554/eLife.10116.013
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fig5s2: SypHy expression at Arf6-silenced synapses and effect of Arf6 shRNA#2.DOI:http://dx.doi.org/10.7554/eLife.10116.013

Mentions: To couple the synaptic ultrastructure with functional analysis of SV trafficking, we employed SynaptophysinpHluorin (Syphy), a fluorescent probe exquisitely designed to monitor the SV exo-endocytosis cycle at single synapses (Miesenböck et al., 1998; Fassio et al., 2011). Arf6-shRNA (or the mismatched control) together with the RFP reporter, and SypHy were transiently co-transfected in rat hippocampal neurons in order to record from a silenced neuron embedded in a network of non-silenced ones (Figure 5—figure supplement 1, Figure 5—figure supplement 2). Considering the emerging role of Arf6 in AMPA receptor trafficking (Scholz et al., 2010; Myers et al., 2012; Oku and Huganir, 2013; Zheng et al., 2015), this approach allows ruling out the contribution of postsynaptic effects on the observed phenotype. Experiments were performed at 17–18 DIV (3–4 days after transfection) and RFP/SypHy-positive axonal processes were selected for stimulation and analysis (Figure 5A). When the amount of surface-expressed SypHy was evaluated, we observed a significant increase of extracellularly exposed SypHy at Arf6-silenced synapses compared with control synapses, suggestive of a global impairment of SV cycling (Figure 5—figure supplement 2). Neurons were first stimulated with 40 action potentials (APs) at 20 Hz, a protocol widely employed to measure the readily releasable pool (RRP) of SVs and, therefore, suitable to reveal if the increased docked SVs observed by EM were functional or represented mistargeted/unprimed SVs, not competent for fusion. Both the shRNA #1 (Figure 5B,C) and the shRNA #2 (Figure 5—figure supplement 2) significantly increased the peak fluorescence at the end of the 2 s. Stimulation without affecting the kinetics of fluorescence return to basal level at the end of the stimulation, which describe ‘post-stimulus’ endocytosis. To reveal whether the increased peak fluorescence was due to higher exocytosis or impairment of ‘during-stimulus’ endocytosis we employed the H+ATPase inhibitor Bafilomycin. This drug prevents reacidification of internalized SVs and allows monitoring net exocytosis. The peak fluorescence was equally increased when neurons were stimulated in the presence of Bafilomycin, suggesting that the observed peak fluorescence increase was indeed attributable to an increased number of exocytosed SVs. (Figure 5D). Next, to exclude the involvement of shRNA-mediated off-target effects, we performed rescue experiments. When hippocampal neurons were triple transfected with SypHy, Arf6 shRNA#1 and a V5-tagged Arf6 rat variant resistant to shRNA#1 silencing (Arf6-res, Figure 5—figure supplement 1) the peak fluorescence at the end of the 40APs@20Hz stimulation returned to the control level (Figure 5E). These data demonstrate that increased docked SVs in Arf6-depleted synapses are indeed functional releasable SVs and that Arf6 silencing causes an alteration in the process of neurotransmission in the absence of any effects on the kinetics of RRP endocytosis.10.7554/eLife.10116.011Figure 5.Increased readily releasable pool at Arf6-deficient synapses.


Arf6 regulates the cycling and the readily releasable pool of synaptic vesicles at hippocampal synapse.

Tagliatti E, Fadda M, Falace A, Benfenati F, Fassio A - Elife (2016)

SypHy expression at Arf6-silenced synapses and effect of Arf6 shRNA#2.DOI:http://dx.doi.org/10.7554/eLife.10116.013
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764570&req=5

fig5s2: SypHy expression at Arf6-silenced synapses and effect of Arf6 shRNA#2.DOI:http://dx.doi.org/10.7554/eLife.10116.013
Mentions: To couple the synaptic ultrastructure with functional analysis of SV trafficking, we employed SynaptophysinpHluorin (Syphy), a fluorescent probe exquisitely designed to monitor the SV exo-endocytosis cycle at single synapses (Miesenböck et al., 1998; Fassio et al., 2011). Arf6-shRNA (or the mismatched control) together with the RFP reporter, and SypHy were transiently co-transfected in rat hippocampal neurons in order to record from a silenced neuron embedded in a network of non-silenced ones (Figure 5—figure supplement 1, Figure 5—figure supplement 2). Considering the emerging role of Arf6 in AMPA receptor trafficking (Scholz et al., 2010; Myers et al., 2012; Oku and Huganir, 2013; Zheng et al., 2015), this approach allows ruling out the contribution of postsynaptic effects on the observed phenotype. Experiments were performed at 17–18 DIV (3–4 days after transfection) and RFP/SypHy-positive axonal processes were selected for stimulation and analysis (Figure 5A). When the amount of surface-expressed SypHy was evaluated, we observed a significant increase of extracellularly exposed SypHy at Arf6-silenced synapses compared with control synapses, suggestive of a global impairment of SV cycling (Figure 5—figure supplement 2). Neurons were first stimulated with 40 action potentials (APs) at 20 Hz, a protocol widely employed to measure the readily releasable pool (RRP) of SVs and, therefore, suitable to reveal if the increased docked SVs observed by EM were functional or represented mistargeted/unprimed SVs, not competent for fusion. Both the shRNA #1 (Figure 5B,C) and the shRNA #2 (Figure 5—figure supplement 2) significantly increased the peak fluorescence at the end of the 2 s. Stimulation without affecting the kinetics of fluorescence return to basal level at the end of the stimulation, which describe ‘post-stimulus’ endocytosis. To reveal whether the increased peak fluorescence was due to higher exocytosis or impairment of ‘during-stimulus’ endocytosis we employed the H+ATPase inhibitor Bafilomycin. This drug prevents reacidification of internalized SVs and allows monitoring net exocytosis. The peak fluorescence was equally increased when neurons were stimulated in the presence of Bafilomycin, suggesting that the observed peak fluorescence increase was indeed attributable to an increased number of exocytosed SVs. (Figure 5D). Next, to exclude the involvement of shRNA-mediated off-target effects, we performed rescue experiments. When hippocampal neurons were triple transfected with SypHy, Arf6 shRNA#1 and a V5-tagged Arf6 rat variant resistant to shRNA#1 silencing (Arf6-res, Figure 5—figure supplement 1) the peak fluorescence at the end of the 40APs@20Hz stimulation returned to the control level (Figure 5E). These data demonstrate that increased docked SVs in Arf6-depleted synapses are indeed functional releasable SVs and that Arf6 silencing causes an alteration in the process of neurotransmission in the absence of any effects on the kinetics of RRP endocytosis.10.7554/eLife.10116.011Figure 5.Increased readily releasable pool at Arf6-deficient synapses.

Bottom Line: Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs.The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting.We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.

View Article: PubMed Central - PubMed

Affiliation: Center of Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, Italy.

ABSTRACT
Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.

No MeSH data available.


Related in: MedlinePlus