␣ -Synuclein interacts transiently with synaptic components. A , A single, isolated hippocampal bouton expressing GFP– ␣ -synuclein was photobleached at high laser power, and recovery was monitored every 400 ms thereafter. GFP– ␣ -synuclein recovers rapidly after photobleaching. The two synapses outside the bleached box show a reduction in fluorescence during recovery of the bleached region. The color scale is shown to the right. Scale bar, 2 ␮ m. B , The intensity of fluorescence at the bleached synapse was quantified and expressed as a percentage of initial fluorescence, with fluorescence after the bleach defined as zero. GFP (green) and GFP–A30P (blue) recover with similar, rapid kinetics after photobleaching, consistent with their behavior as soluble proteins. In contrast, GFP– ␣ -synuclein (red) recovers more slowly, indicating a distinct but rapidly reversible interaction with synaptic components. GFP–synapsin (GFPsyp; purple) recovers more slowly than ␣ -synuclein, consistent with a higher affinity for synaptic vesicles. GFP–SV2 (black) does not recover after photobleaching, indicating that synaptic vesicles do not exchange between synapses, at least during this time scale. Data shown are averages Ϯ SEM of 30 – 40 boutons per construct. 

Contexts in source publication

Context 1

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...

Context 2

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...

Context 3

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...

Context 4

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...

Context 5

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...

Context 6

... steady-state, ␣ -synuclein exhibits synaptic enrichment similar in extent to that observed for polytopic membrane proteins of the synaptic vesicle such as VGLUT1 ( Fig. 1 A ) (Withers et al., 1997; Fortin et al., 2004; Specht et al., 2005). To understand how this protein, which does not contain a transmembrane domain or known lipid anchor, localizes to the nerve terminal, we studied the dynamics of ␣ -synuclein in live hippocampal neurons. Transfection of hippocampal neurons with human ␣ -synuclein fused at its N terminus to enhanced GFP (GFP– ␣ -synuclein) leads to a modest ϳ 50% overexpression compared with untransfected cells as determined by quantitative immunofluorescence using an an- tibody that recognizes both rat and human proteins: 1545 Ϯ 39 arbitrary fluorescence units (AFU) in transfected cells ( n ϭ 320 boutons from eight different neurons) versus 1050 Ϯ 21 AFU in untransfected cells ( n ϭ 409 boutons from the same eight fields). This level of overexpression does not perturb the enrichment of either human or endogenous rat ␣ -synuclein at the nerve terminal of transfected neurons (Fig. 1 B ). In addition, the overexpression does not appear to affect the development of transfected neurons or the localization of other presynaptic proteins includ- ing synaptophysin, SV2, synapsin, and VGLUT1 (data not shown). The synaptic localization of GFP– ␣ -synuclein also strongly suggests that the GFP fusion does not influence the behavior of ␣ -synuclein or its interactions at the nerve terminal. Other N-terminal tags such as glutathione S -transferase or the calmodulin-binding protein CBP do not interfere with the membrane binding of ␣ -synuclein in vitro and do not change the requirement for acidic phospholipid headgroups or the ␣ -helical transition of synuclein during membrane binding (Perrin et al., 2000; Kubo et al., 2005). Although we cannot completely exclude some difference from the untagged protein that has eluded detec- tion in vitro and in vivo , GFP– ␣ -synuclein thus appears to serve as an accurate, nondisruptive reporter for the endogenous protein. To assess the mobility of ␣ -synuclein in live cells, we used FRAP (Lippincott-Schwartz et al., 2001). After photobleaching GFP– ␣ -synuclein at a single synapse, we observed rapid fluorescence recovery in the bleached region (Fig. 2 A ), suggesting that wild-type human ␣ -synuclein is highly mobile in hippocampal neurons, an unexpected finding given its strong synaptic enrichment. No fluorescence recovery was observed after photobleaching fixed neurons, confirming that the conditions used here irre- versibly bleach GFP– ␣ -synuclein and that recovery results from the movement of unbleached molecules into the bleached area (data not shown). In addition, fluorescence recovery at the bleached synapse was typically accompanied by a decrease in fluorescence at neighboring boutons (24.3 Ϯ 0.8%; n ϭ 57 boutons), consistent with adjacent synapses acting as the source of mobilized GFP– ␣ -synuclein (Fig. 2 A ). Recovery after photobleaching was best fit with a single exponential, supporting the behavior of GFP– ␣ -synuclein as a homogeneous population. GFP– ␣ -synuclein thus exchanges rapidly between neighboring synapses. Rapid recovery after photobleaching makes it unlikely that ␣ -synuclein remains tightly associated with synaptic vesicles. Synaptic vesicles can exhibit mobility within a single terminal under certain conditions (Henkel et al., 1996), but there is no precedent for their movement between terminals at rest (Henkel et al., 1996; Kraszewski et al., 1996). However, spontaneous, sus- tained network activity can lead to the mixing of synaptic vesicles between terminals (Li and Murthy, 2001). To exclude activity- dependent vesicle mobility as a cause for the rapid recovery of ␣ -synuclein, we used the glutamate receptor antagonists CNQX and AP-V in the FRAP experiments described above. In addition, we measured the recovery after photobleaching of a GFP fusion to SV2, an integral membrane protein of synaptic vesicles (Lowe et al., 1988). The fluorescence of GFP–SV2 does not recover from photobleaching in the time course of these experiments (Fig. 2 B ), consistent with the relative immobility of synaptic vesicles. The rapid recovery of ␣ -synuclein after FRAP thus excludes the tight association of ␣ -synuclein with synaptic vesicles. A fusion of GFP to synapsin I, another peripheral membrane protein associated with synaptic vesicles, also recovers more slowly from photobleaching than ␣ -synuclein. The FRAP recovery kinetics thus indicates that ␣ -synuclein binds more weakly to synaptic vesicles than synapsin (Fig. 2 B ) (Phair and Misteli, 2001). Despite its rapid recovery after photobleaching, wild-type ␣ -synuclein recovers more slowly than GFP alone. The slower recovery of GFP– ␣ -synuclein does not reflect its larger mass, because a fusion of GFP to the PD-associated A30P ␣ -synuclein (GFP–A30P), which has es- sentially the same molecular weight as GFP– ␣ -synuclein, recovers as rapidly as GFP alone (Fig. 2 B ). The recovery kinetics of GFP–A30P is consistent with its lack of synaptic enrichment and its behavior as a soluble protein in neurons (Fortin et al., 2004). The synaptic localization of wild- type ␣ -synuclein thus reflects rapidly reversible interactions that enable exchange of the protein between adjacent synapses. Nonetheless, these transient interactions are sufficient for the steady-state enrichment of wild-type ␣ -synuclein at the nerve terminal, and their disruption by the A30P mutation eliminates synaptic localization. To determine whether a small fraction of ␣ -synuclein might be immobile, we have assessed the extent as well as the rate of recovery after photobleaching. GFP and GFP–A30P do not recover fully after photobleaching, presumably reflecting the limited local pool of protein from which recovery can occur (Elson and Qian, 1989). The extent of their recovery ( ϳ 80%) was therefore considered the maximal recovery for a soluble protein. The reduced re- Figure 4. ␣ -Synuclein covery of GFP– ␣ -synuclein ( ϳ 70%) relative diately fixed (stimulated), or to that of GFP and GFP–A30P suggests that a VGLUT1 staining and are small proportion ( ϳ 10%) of the protein ization. Unlike synapsin, synapsin has reclustered in may be immobile (Fig. 2 B ). To assess more timeframe.Scalebar,2 ␮ m. accurately the existence of an immobile fraction, we repeatedly photobleached the same Scale bar, 2 ␮ m. C , No synapse. In the case of a true immobile frac- in the absence of calcium, tion, a second photobleach will be followed dispersion of ␣ -synuclein by more complete recovery than the first sentative cell. D , Average photobleach, because the fluorescence is normalized to that observed at the start n ϭ 138 synapses from three of each bleach, thereby eliminating the con- tribution of the immobile fraction to recovery from the second bleach (Lippincott-Schwartz et al., 2003). In the case of GFP–synapsin, recovery is substantially greater after the second photobleach (Fig. 3), suggesting the presence of a significant immobile fraction. In contrast, the fluorescence of GFP– ␣ -synuclein recovers to the same extent after the first and second photobleach (Fig. 3), excluding a significant immobile fraction and indicating that the entire pool of protein is mobile and can exchange between adjacent synapses. Neural activity modulates the localization of multiple synaptic proteins. For example, synapsin disperses from the synapse early in the course of stimulation, suggesting dissociation from synaptic vesicles before their fusion with the plasma membrane (Chi et al., 2001, 2003). Similarly, depolarization of hippocampal neurons with high K ϩ influences the distribution of ␣ -synuclein observed in fixed cells. Before stimulation, endogenous ␣ -synuclein concentrates at synaptic boutons where it colocalizes with synapsin and VGLUT1 (Fig. 4 A , rest). Depolarization with high K ϩ results in the loss of ␣ -synuclein from synaptic boutons (Fig. 4 A , stimulated, arrowheads). Unlike synapsin, however, ␣ -synuclein does not increase substantially in the axon after stimulation (Fig. 4 A , stimulated, arrows), and synaptic ␣ -synuclein does not recover within 10 min after cessation of the stimulus (Fig. 4 A , recovery, arrowheads). To characterize the activity-dependent dispersion of ␣ -synuclein, we imaged the GFP fusion in living hippocampal neurons. Similar to the endogenous protein, the amount of GFP– ␣ -synuclein at the synapse decreases by ϳ 20% after a 600 action potentials stimulus (Fig. 4 B–D ). GFP– ␣ -synuclein does not appear to accumulate in the axon, and very little returns to the boutons after the stimulus has stopped (Fig. 4 B , C ). The loss of synaptic GFP– ␣ -synuclein produced by activity depends on ex- ternal calcium (Fig. 4 C , D ), indicating a requirement for calcium entry. GFP and GFP–A30P show no change in fluorescence after stimulation, consistent with their behavior as soluble proteins and supporting the significance of the dispersion observed for GFP– ␣ -synuclein (Fig. 5 A ). The PD-associated A53T mutant, which localizes normally to the nerve terminal (Fortin et al., 2004), exhibits the same activity-dependent dispersion as wild- type ␣ -synuclein (Fig. 5 B ). The kinetics of dispersion differs for the two peripheral membrane proteins synapsin and ␣ -synuclein (Fig. 5 A ). The activity- dependent dispersion of ␣ -synuclein appears somewhat slower than that of synapsin (Fig. 5 A ) (Chi et al., 2001). ␣ -Synuclein may thus disperse at a later step during the evoked release of neurotransmitter. In addition, the synaptic fluorescence of GFP–synapsin rapidly recovers to prestimulation levels, whereas GFP– ␣ -synuclein ...