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Title
α-Synuclein promotes dilation of the exocytotic fusion pore.
Permalink
https://escholarship.org/uc/item/4zh4z6s8
Journal
Nature neuroscience, 20(5)
ISSN
1097-6256
Authors
Logan, Todd
Bendor, Jacob
Toupin, Chantal
et al.
Publication Date
2017-05-01
DOI
10.1038/nn.4529
Peer reviewed
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University of California
α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore
Todd Logan
1,2,*
, Jacob Bendor
1,*
, Chantal Toupin
1
, Kurt Thorn
3
, and Robert H. Edwards
1,2
1
Departments of Neurology and Physiology, UCSF School of Medicine
2
Graduate Program in Biomedical Sciences, UCSF School of Medicine
3
Department of Biochemistry & Biophysics, UCSF School of Medicine
Summary
The protein α-synuclein has a central role in the pathogenesis of Parkinson’s disease (PD). Similar
to other proteins that accumulate in neurodegenerative disease, however, the function of α-
synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter
release and over-expression inhibits regulated exocytosis, but previous work has failed to identify a
clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin
cells and neurons, we now find that both over-expressed and endogenous synuclein serve to
accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing
pore closure (‘kiss-and-run’). Thus, synuclein exerts dose-dependent effects on dilation of the
exocytotic fusion pore. Remarkably, mutations that cause PD abrogate this property of α-
synuclein without impairing its ability to inhibit exocytosis when over-expressed, indicating a
selective defect in normal function.
Introduction
Despite the established role of multiple proteins in the pathogenesis of neurodegenerative
disease, we know remarkably little about their function. In Parkinson’s disease (PD) as well
as in the related conditions Dementia with Lewy Bodies (DLB) and Multiple System
Atrophy, the peripheral membrane protein α-synuclein accumulates in characteristic
inclusions
1
. Mutations in α-synuclein also produce a dominantly inherited form of PD
2–7
,
demonstrating that the protein has a causative role. Indeed, α-synuclein gene duplication and
particularly triplication produce a severe form of familial PD
8
, implicating the wild type (wt)
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Address correspondence to R.H. Edwards at: Departments of Neurology and Physiology, UCSF School of Medicine, 600 16
th
St.,
GH-N272B, San Francisco, CA 94143, (415) 502-5687 telephone, (415) 502-8644 fax, robert.edwards@ucsf.edu.
*
These authors contributed equally to this work.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Author contributions
R.H.E., T.L. and J.B. designed the research and wrote the manuscript. T.L. and J.B. performed the experiments and analyzed the data,
with assistance from C.T. K.T. provided essential technical assistance with the chromaffin cell imaging experiments.
Competing financial interests
The authors declare no competing financial interests.
HHS Public Access
Author manuscript
Nat Neurosci
. Author manuscript; available in PMC 2017 September 13.
Published in final edited form as:
Nat Neurosci
. 2017 May ; 20(5): 681–689. doi:10.1038/nn.4529.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
protein in disease. Synuclein thus has a central role in PD. However, the normal function of
α-synuclein remains poorly understood.
α-Synuclein normally localizes to the nerve terminal, suggesting a role in neurotransmitter
release
9
. Consistent with this, modest over-expression (insufficient to produce inclusions or
overt toxicity) inhibits the regulated exocytosis of large dense core vesicles (LDCVs) and
synaptic vesicles
10–12
. However, the loss of synuclein has less effect, with minimal or no
increase in glutamate release reported in triple knockout (TKO) mice lacking α-synuclein as
well as closely related β- and γ- isoforms
13, 14
. Knockout mice lacking α- and γ-synuclein
show an increase in evoked dopamine release
15
but the physiological change responsible
remains unknown. Although over-expression inhibits regulated exocytosis, the role of
endogenous α-synuclein has thus remained unknown.
α-Synuclein binds specifically to anionic membranes with high curvature
16–18
, but can also
deform the lipid bilayer. Synuclein aggregates membranes in yeast
19, 20
, tubulates artificial
membranes
in vitro
21
and when over-expressed in mammalian cells, can produce
mitochondrial fragmentation
22, 23
. However, membrane deformation is generally considered
to have an important role in endocytosis rather than exocytosis. The effect of over-expressed
synuclein on exocytosis has thus been difficult to explain on the basis of membrane
curvature-sensing or -promoting properties. Alternatively, synuclein has been suggested to
serve as chaperone for the SNARE complex, but without apparent effect on transmitter
release
14
.
How might membrane deformation by synuclein influence regulated exocytosis? In the
course of exocytosis, synaptic vesicles form a fusion pore that dilates before full collapse
into the plasma membrane. However, the pore can also close as part of a ‘kiss-and-run’
mechanism that immediately regenerates the vesicle
24
. Regulation of membrane curvature
might thus affect behavior of the fusion pore. Since classical transmitters such as glutamate
escape rapidly, postsynaptic recording might not detect a change in fusion pore kinetics. We
have therefore used imaging to monitor directly individual exocytotic events. Single synaptic
vesicle fusion events are difficult to detect by imaging, so we have focused on peptidergic
large dense core vesicles (LDCVs) due to their size (70–200 nm diameter) and relatively
slow release. Adrenal chromaffin cells have been used extensively to study the process of
regulated exocytosis, including release by kiss-and-run
25
, and previous work has indeed
demonstrated the inhibition of LDCV exocytosis by synuclein over-expression in chromaffin
cells
10
. α-Synuclein
+
Lewy pathology also occurs at high frequency in the adrenal gland of
patients with PD and DLB
26
, with effects on catecholamine release into the circulation
27
.
Results
Synuclein over-expression accelerates the kinetics of individual exocytotic events in
chromaffin cells
To understand how synuclein influences release, we first infected primary cultures from the
postnatal mouse adrenal medulla with a lentivirus encoding human α-synuclein. Double
staining for human α-synuclein and the LDCV protein secretogranin II (SgII) confirmed
expression of the human protein within chromaffin cells (Supplementary Fig. 1a). We also
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assessed the expression of endogenous α-synuclein using an antibody that recognizes the
protein from multiple species. Comparison of chromaffin cells from wt and synuclein TKO
mice lacking all synuclein isoforms shows that chromaffin cells express endogenous α-
synuclein, and the lentivirus confers modest over-expression (Supplementary Fig. 1b,c).
To study individual exocytotic events, we used a fusion of brain derived neurotrophic factor
(BDNF) to the ecliptic pHluorin, a modified form of the green fluorescent protein with
enhanced pH sensitivity
28
. Quenched at the low pH of LDCVs, BDNF-pHluorin
fluorescence increases on exposure to the external medium by exocytosis
29
. We monitored
the behavior of BDNF-pHluorin by TIRF microscopy during depolarization with 45 mM K
+
.
Since kiss-and-run may occur more frequently at high external Ca
++30
, we also used 5 mM
external Ca
++
to sample a wider variety of exocytotic events. α-Synuclein over-expression
reduces the number of exocytotic events detected using BDNF-pHluorin (Fig. 1a), as
suggested previously by amperometry
10
. A change in Ca
++
entry cannot account for this
because Ca
++
entry does not significantly differ from wt or TKO (Supplementary Fig. 2).
Individual exocytotic events show a series of characteristic changes due to the over-
expression of α-synuclein. First, synuclein over-expression increases the rate of fluorescence
rise. Although BDNF-pHluorin unquenching occurs rapidly at exocytosis, the fluorescence
of many events increases over more than a single frame
31
and this reflects buffering as well
as dissolution of the LDCV core
32
. Alkalinization or approach to the plasma membrane
might produce similar behavior, but loss of a preloaded dye
33
accompanies or precedes
events with slow rise times (Supplementary Fig. 4a,b). Thus, synuclein over-expression
increases the rate of H
+
loss at exocytosis (Fig. 1b and Supplementary Fig. 3a–c). Second,
after peak fluorescence, the rate of fluorescence decay (due to peptide release) also increases
even when analyzed as means per cell rather than aggregated, individual exocytotic events
(Supplementary Fig. 3d). These results suggest acceleration of the release event by α-
synuclein. However, the fluorescence events fall into at least four distinct classes (Fig. 1c
and Supplementary Fig. 3e). Many events decay immediately to baseline (full decay)
whereas others remain at maximum fluorescence for an interval before full decay (plateau-
decay). In still others, decay is interrupted, suggesting constriction if not closure of the
fusion pore, with or without a plateau preceding the decay (decay-plateau or plateau-decay-
plateau). To determine whether the interruption of fluorescence decay reflects full pore
closure, we quenched residual events using external solution adjusted to pH 5.5 with the
impermeant buffer MES (Supplementary Fig. 4c). All events in the process of decay show
quenching by the acidic buffer (Supplementary Fig. 4d), demonstrating exocytosis and either
full collapse or persistence of a dilated fusion pore rather than movement away from the
plasma membrane. Indeed, the H
+
-ATPase inhibitor bafilomycin does not influence the time
course of events in any genotype (Supplementary Fig. 4e), confirming that loss of
fluorescence indicates the release of peptide, not reacidification. Most of the stable events
(plateau) also show quenching by low external pH, but a fraction do not (Supplementary Fig.
4c,d). Thus, complete closure of the fusion pore occurs only in stable events, but incomplete
closure can still limit the loss of peptide. Consistent with the acceleration of release,
synuclein over-expression increases the proportion of events that undergo full decay (Fig.
1c). Within the group undergoing full decay, however, synuclein over-expression also
increases the rate of decay (Fig. 1d, Supplementary Fig. 3f). Thus, synuclein accelerates
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release independent of effects on pore closure, suggesting a role early in exocytosis to
promote peptide release. Over-expression of synuclein also has no effect on the number of
docked vesicles or their lumenal pH (Supplementary Fig. 5), arguing against a general
disturbance of LDCVs as cause for the change in exocytosis.
Loss of synuclein prolongs the kinetics of release
The difficulty detecting clear effects on transmitter release in knockout mice
11, 13, 14
suggests that α-synuclein over-expression may simply produce toxicity that secondarily
affects release. It was therefore of great interest to determine how the loss of synuclein
influences release kinetics. Consistent with previous work in neurons
11, 13, 14
, chromaffin
cells from synuclein TKO mice show no clear change in event number relative to wt (Fig.
1a). However, analysis of event distribution reveals an increase in the time to peak
fluorescence (Fig. 1b and Supplementary Fig. 3a). Loss of the synucleins modestly reduces
the proportion of events with full decay (Fig. 1c) and also redistributes the decay time
constants to longer values (Fig. 1d). In addition, TKO cells show greatly increased latency to
decay among those events that do not decay immediately (Fig. 1e). (Over-expression does
not affect this parameter presumably because it converts those with a short latency to full
decay.) Thus, loss of synuclein prolongs release, suggesting a similar role for the
endogenous and over-expressed protein in exocytosis. Similar to over-expression, the
synuclein TKO also has no effect on LDCV docking or pH (Supplementary Fig. 5).
Although we used 5 mM external Ca
++
for these experiments because it may promote kiss-
and-run, we also examined BDNF-pHluorin events at the more physiological 2 mM Ca
++
. In
this condition, as at 5 mM Ca
++
, over-expression of human α-synuclein both inhibits the
exocytosis of chromaffin granules and accelerates the loss of BDNF-pHluorin
(Supplementary Fig. 6a,b). The proportion of events again shifts to those with full decay, at
the expense of those with interrupted release, and the time constant for fluorescence decay
shortens (Supplementary Fig. 6c,d). The TKO also shows prolonged decay relative to wt
(Supplementary Fig. 6d). Thus, synuclein has similar effects on release at 2 and 5 mM Ca
++
.
Synuclein inhibits closure of the fusion pore (‘kiss-and-run’)
The effect of synuclein on release could reflect changes in the fusion pore or in the solubility
of dense core vesicle cargo. Indeed, the same LDCVs can release different substances at
different rates
34, 35
, indicating that the properties of the aggregated peptide can influence the
rate of release. It seems unlikely that a cytoplasmic protein such as synuclein would
influence lumenal contents, but to distinguish further between effects on the fusion pore and
on peptide solubility, we used a construct with the pHluorin inserted into a lumenal loop of
the vesicular monoamine transporter VMAT2
36
, a polytopic membrane protein that localizes
to LDCVs. Stimulation of endocrine cells expressing the fusion produces discrete exocytotic
events consistent with LDCVs, and we used bafilomycin for these experiments to prevent
vesicle reacidification. In chromaffin cells, α-synuclein over-expression also inhibits the
exocytosis of VMAT2-pHluorin (Fig. 2b). As a membrane protein, VMAT2-pHluorin cannot
undergo release, and its decay therefore reflects spread within the plasma membrane and
endocytosis, processes limited by the fusion pore
37
. Indeed, we observe events that spread
and others that do not, as well as variation in the time course of fluorescence decay (Fig. 2a).
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