The results show that over-expression of αsyn inhibits neurotransmitter release. Optical imaging of transfected neurons and electrophysiologic recording from the brain slices of transgenic mice both indicate a defect in release, consistent with the presynaptic location of αsyn. Using the same experimental paradigm, we find little or no difference between wild type and αsyn knockout. Relative to the KO, increased expression of αsyn thus appears to have a much greater effect on synaptic vesicle exocytosis.
Considering the minimal effect of the knockout, why does synuclein over-expression have a dramatic effect on transmitter release? First, over-expression could result in toxicity due to the gain of an abnormal function such as the impaired membrane trafficking observed in yeast, with indirect effects on transmitter release (Outeiro and Lindquist, 2003
; Willingham et al., 2003
; Cooper et al., 2006
; Soper et al., 2008
). However, we find no change in the level of VGLUT1-pHluorin reporter used for optical imaging of the synaptic vesicle cycle, excluding a major defect in the early secretory pathway. In addition, we deliberately over-expressed αsyn in the range predicted for patients with a duplication or triplication of the gene, reducing the likelihood of toxicity due to massive accumulation. The similar effects of αsyn over-expression in transgenic mice also exclude a role for the toxicity of acute transfection, and support the physiological significance of these observations for synaptic transmission in vivo
. In addition, we did not detect any inclusions by immunofluorescence or oligomers by western analysis. The ability of a point mutation (A30P) to block the inhibition of synaptic vesicle exocytosis by αsyn further supports the specificity of the effect by wild type. The lack of change in total synaptic vesicle pool size, endocytosis and the kinetics of exocytosis also argue against non-specific toxicity.
Second, functional redundancy with βsyn and γsyn may account for the minimal phenotype of the αsyn KO. Indeed, the three isoforms are very similar in sequence, overlap in distribution, and double KO mice lacking γsyn as well as αsyn show a substantial increase in dopamine release not observed in single knockout mice (Senior et al., 2008
). Consistent with potential redundancy, we now find that over-expression of βsyn also inhibits transmitter release. However, the minimal effect of the α/βsyn double KO on synaptic vesicle exocytosis and synaptic transmission (Chandra et al., 2004
) makes redundancy unlikely to account for the lack of detectable effect in the αsyn single KO. Since βsyn has been suggested to protect against the aggregation of αsyn (Hashimoto et al., 2001
; Uversky et al., 2002
; Park and Lansbury, 2003
), does not produce toxicity and is not associated with PD, the ability of βsyn to inhibit transmitter release argues further against a role for toxicity.
Third, the roughly linear inhibition of transmitter release suggests that the effect of the KO may simply be difficult to detect. Based on the response to over-expression 2-3-fold above endogenous, the effect of losing synuclein is in fact predicted to be small, and possibly within the noise associated with these measurements. Synuclein may thus have an important role at baseline. However, the effects of over-expression also suggest that the endogenous protein may have a particularly important role when up-regulated. Indeed, synuclein up-regulates under a variety of conditions (Vila et al., 2000
; Quilty et al., 2006
) including sporadic PD (Chiba-Falek et al., 2006
), and our observations predict dramatic effects on synaptic transmission.
The hydrophilic C-terminus of αsyn has been suggested to interact with a number of proteins (Payton et al., 2004
; McFarland et al., 2008
), and to undergo phosphorylation at multiple sites (Okochi et al., 2000
; Ellis et al., 2001
; Fujiwara et al., 2002
; Chen and Feany, 2005
). However, we find that deletion of the C-terminus does not affect the ability of αsyn to inhibit synaptic vesicle exocytosis, excluding a requirement for the C-terminus in this function of αsyn, although it might still have a regulatory role.
Consistent with a crucial role for the N-terminal membrane-binding domain, the PD-associated A30P mutation abolishes the inhibition of transmitter release by αsyn. Since this mutation prevents the membrane association of αsyn in a number of experimental systems (Jo et al., 2002
; Outeiro and Lindquist, 2003
; Kubo et al., 2005
), and prevents the synaptic enrichment of αsyn in neurons (Fortin et al., 2004
), a high concentration of the protein at these structures appears essential for the effect on transmitter release. In adrenal medullary cells, the A30P mutant still inhibits the exocytosis of chromaffin granules (Larsen et al., 2006
), but this may reflect the compact round shape of these cells that does not require specific targeting of synuclein to the release site. The A30P may thus retain some intrinsic ability to inhibit release. The inability of PD-associated A30P αsyn to inhibit transmitter release in neurons raises questions about the relevance of this activity to the pathogenesis of PD, but the reduced effect of the A30P mutant may in fact account for the late onset of disease and incomplete penetrance observed in families with the A30P mutation relative to those with A53T (Kruger et al., 2001
How does αsyn influence synaptic transmission? Since hypertonic sucrose elicits release independent of calcium, the ability of synuclein to inhibit release stimulated by hypertonic sucrose excludes an effect of synuclein on calcium entry or the calcium-dependent triggering of release. In addition, αsyn does not alter the kinetics of either synaptic vesicle exo- or endocytosis. Rather, αsyn reduces the size of the synaptic vesicle recycling pool assessed either with FM 4-64 or by stimulation in the presence of the H+ pump inhibitor bafilomycin. Since the readily releasable pool shows a proportionate reduction in size, we infer that this reduction simply reflects the change in recycling pool. Further, the analysis of VGLUT1-pHluorin in the presence of NH4Cl, the quantitative analysis of synaptic vesicle proteins, and the electron microscopy show that αsyn does not reduce the total number of synaptic vesicles. Thus, synuclein affects specifically the size of the synaptic vesicle recycling pool.
Transgenic over-expression of αsyn also increases the paired-pulse ratio. Although changes in PPR are generally considered to reflect changes in calcium accumulation, the manipulation of release probability independent of calcium has also been shown to influence PPR (Rosahl et al., 1993
; Augustin et al., 2001
). In addition, the changes in PPR produced by synuclein expression are much smaller than those produced by manipulating calcium, presumably because synuclein reduces the entire pool of recycling vesicles as well as the readily releasable pool, limiting the availability of vesicles for the second pulse.
The analysis of transgenic mice over-expressing αsyn shows a reduced number of synaptic vesicles adjacent to the active zone, consistent with a defect in vesicle mobilization rather than fusion. The transgenic mice indeed show a general reduction in synaptic vesicle clustering, suggesting a physical basis for the defect in mobilization. How does αsyn control synaptic vesicle clustering and the size of the recycling pool? The biochemical analysis suggests several possibilities.
The transgenic mice over-expressing αsyn show a 20–45% reduction in the amount of multiple synapsins, and previous work has implicated the synapsins in synaptic vesicle mobilization (Hilfiker et al., 1999
). The knockout of synapsin I reduces recycling pool size by 30–40% (Ryan et al., 1996
).However, the ultrastructural analyses of synapses from mice lacking either synapsin I or synapsin II show a reduction in the total number of synaptic vesicles, distinct from the primary defect in recycling pool size observed with αsyn over-expression (Rosahl et al., 1995
). In addition, the over-expression of αsyn appears to cause a more severe defect in the recycling pool than the complete loss of synapsins, and the transgenic mice show only a partial reduction in synapsins. The effect of αsyn over-expression is thus unlikely to reflect simply the loss of synapsins. Rather, αsyn and the synapsins both seem to influence related processes involved in vesicle mobilization.
Over-expression of αsyn also reduces the amount of complexin 2 in the transgenic mice. A change in the level of complexins, which are implicated in a step at or close to fusion with the plasma membrane (Sudhof and Rothman, 2009
), might thus be consistent with a proposed role for synuclein as chaperone for SNARE proteins (Chandra et al., 2005
). However, a change in SNARE protein function seems unlikely to account for the observed defect in synaptic vesicle mobilization, or the reduced clustering of synaptic vesicles near the active zone. On the other hand, α-/β-synuclein double KO mice show a 30% increase in complexins (particularly complexin 2) (Chandra et al., 2004
), suggesting that the reduction in complexin 2 observed in the transgenic mice may reflect a gain in the normal function of αsyn.
The imaging of VGLUT1-pHluorin provides indirect evidence that synuclein impairs the reclustering of synaptic vesicles after endocytosis. To visualize this directly, we used a cytoplasmic fusion of VGLUT1 to GFP rather than the lumenal fusion to ecliptic pHluorin. Although the steady-state dispersion of synaptic vesicles observed by electron microscopy might have resulted from a subtle defect in reclustering that becomes evident only over time, we observe a clear defect in reclustering after only a single period of stimulation. The imaging of GFP-VGLUT1 thus provides information complementary to the pHluorin fusion, and direct evidence for a defect in synaptic vesicle reclustering that presumably accounts for the synaptic vesicle dispersion observed by electron microscopy, and the reduction in recycling pool size observed by imaging.