synuclein triple knockout (tko) mice  (Jackson Laboratory)

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: (a) Chromaffin cells from wt mice were transduced with lentiviruses encoding BDNF-pHluorin and either human α-synuclein (SYN) or empty vector (wt). Over-expression of α-synuclein reduces the number of exocytotic events evoked over 50 s by depolarization with 45 mM K + . Cells from the synuclein TKO show no difference from wt cells. *, p = 0.01 by one-way ANOVA (F(2, 54) = 4.991). n = 19 cells for each group from 3 independent cultures (b) Synuclein affects the rise time of exocytotic events. For each exocytotic event, the time to reach 90% maximum fluorescence was determined. Inset shows the average rise time of a single representative cell from each group (wt, n = 46 events; SYN, n = 34 events; TKO, n = 30 events). The histogram represents the frequency of events with rise time in the 50 ms bin indicated (p < 0.0001 by Kolmogorov-Smirnov test). wt, n = 473 events; SYN, n = 256 events; TKO, n = 518 events (c) Exocytotic events belong to four distinct classes (left). In full decay, the fluorescence immediately decays to baseline. In plateau-decay, the fluorescence decay begins after a variable latency. In decay-closure, the fluorescence decays with no latency but the decay arrests before return to baseline. Plateau-decay-closure involves both a latency before decay and incomplete decay. The diagram (upper right) illustrates our interpretation of the traces. The proportion of event types differed among all three groups (p < 0.0001 by Chi-square for pair-wise as well as the comparison of all three groups). (d) Synuclein influences the rate of BDNF release. For all full decay events, the time constant of fluorescence decay (τdecay) was determined by fitting to a single exponential. The histogram represents the distribution of events with different τdecay (p < 0.0001 for WT versus SYN and TKO vs SYN; p < 0.001 for WT vs TKO by Kolmogorov-Smirnov test). wt, n = 266 events; SYN, n = 167 events; TKO, n = 237 events (e) For all events with non-zero latency to decay, the time from reaching 90% maximal fluorescence to the onset of decay was determined (wt, n = 134 events; SYN, n = 66 events; TKO, n = 218 events). ****, p < 0.0001 by Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test; H = 55.22 (d) and 39.45 (e)

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Transduction, Plasmid Preparation, Over Expression, Fluorescence, Comparison

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: (a) Wild type chromaffin cells were transduced with lentiviruses encoding VMAT2-pHluorin and either human α-synuclein or empty vector and depolarized 3–5 days later with 45 mM K + in the presence of H + pump inhibitor bafilomycin to inhibit vesicle reacidification. The kymographs of two exocytotic events illustrate the observed variation in fluorescence time course and spread. after depolarization with 45 mM K + . Bar indicates 0.5 s. (b) α-Synuclein over-expression reduces the number of VMAT2-pHluorin exocytotic events (p = 0.0154 by unpaired, two-tailed t test; t(32) = 2.560). con, n = 16 cells; SYN, n = 18 cells from 3 independent cultures (c) Synuclein over-expression also reduces the latency to fluorescence decay (p = 0.0347 by Mann-Whitney; U = 42.00). con, n = 153 events; SYN, n = 128 events (d) Representative traces showing a VMAT2-pHluorin event quenched (left) and not quenched (right) by pH 5.5. (e) After depolarization for 30 s in 45 mM K + , the chromaffin cells were challenged at pH 5.5. Over-expression of α-synuclein reduced the proportion of events protected from quenching at low pH (p = 0.0005 by unpaired t-test; t(30) = 3.863). con, n = 222 events from 15 cells; SYN, n = 133 events from 17 cells (f) The time constant of fluorescence decay shortens with α-synuclein over-expression (p = 0.0012 by Mann-Whitney; U = 44.00). con, n = 348 events; SYN, n = 267 events. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Transduction, Plasmid Preparation, Fluorescence, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: (a) Rodent hippocampal neurons were transfected with BDNF-pHluorin and imaged 14–20 days later, stimulating at 50 Hz for 5 s followed immediately by quenching of the cell surface fluorescence at pH 5.5. The arrow indicates an event quenchable at low pH, and the arrowheads events resistant to quenching. Scale bar, 5 μm. (b) Sample BDNF-pHluorin traces show sensitivity to quenching by pH 5.5 applied between the dashed lines (left) and resistance to quenching (right). (c) Average event frequency per coverslip (mean ± SEM) for BDNF-pHluorin expressing rat hippocampal neurons co-transfected with α-synuclein (SYN) or empty vector (con) and stimulated as in (a) (above) (p = 0.13 by unpaired, two-tailed t test; t(16) = 1.582). n = 9 cells from 2 independent cultures (d) Classifying events as either already decayed at the time of acid exposure or if not, unquenched or quenched by low pH, α-synuclein over-expression reduces the proportion of unquenched events (p < 0.0001 by Chi-square test) (left panel). n = 281 events (con), 158 events (α-syn). Synuclein over-expression also increases the proportion of quenchable events per coverslip independent of those already decayed (right panel). *, p < 0.05 by unpaired t-test (t(15) = 2.145) (e) Mouse neurons transfected with BDNF-pHluorin were stimulated at 50 Hz for 5 s and superfused with rapidly oscillating (1.33 Hz) Tyrode’s solutions at pH 7.8 and 6.4. Top trace shows an exocytotic event with oscillation that persists until fluorescence decay, indicating that the fusion pore remains open until the peptide is released. Middle trace shows an event that does not decay completely but shows oscillation throughout, indicating that the fusion pore does not close. Bottom trace shows an event where the oscillation stops (arrow) before full peptide release, indicating pore closure. (f) The proportion of event types differs in wt and synuclein TKO neurons (p = 0.01 by Chi-square). n = 100 events from 7 (wt) and 9 (TKO) coverslips (g) Among events with pore closure, the cumulative frequency distribution shows no significant difference between wt and synuclein TKO neurons in time to pore closure (p = 0.63 by Kolmogorov-Smirnov). Inset shows mean ± SEM (n.s., not significant). n = 44 events for wt and 63 events for TKO

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Transfection, Fluorescence, Expressing, Plasmid Preparation, Two Tailed Test, Over Expression

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: (a) Sample fluorescence traces of NPY-pHluorin in cultured rodent neurons stimulated at 50 Hz for 5 s. Individual traces were fit to a plateau with single exponential decay. Events that failed to exhibit fluorescence decay (right) were scored as no decay. (b,d) The latency to decay was combined with non-parametric “no decay” data and the cumulative frequency distribution plotted. (b) Overexpression of α-synuclein (SYN) in rat neurons significantly decreased latency to decay compared to controls transfected with empty vector (con) (p < 0.0001 by Kolmogorov-Smirnov). control, n = 652 events / 5 coverslips; α-syn, n = 586 events / 8 coverslips / 2 cultures (d) Latency to decay of NPY-pHluorin events increased in neurons from synuclein TKO mice relative to wt controls (p < 0.0001). wt, n = 928 events / 10 coverslips; TKO, n = 769 events / 11 coverslips / 3 cultures (c,e) Cumulative frequency histograms for the time constants of fluorescence decay (τ decay ) by NPY-pHluorin, including events with no decay. (c) Overexpression of α-synuclein in rat neurons reduced latency to decay and τ decay (p < 0.001 by Kolmogorov-Smirnov). (e) Loss of synuclein in neurons from TKO mice increases NPY-pHluorin τ decay relative to neurons from wild type animals (p < 0.0001). Insets in (c) and (e) indicate mean ± SEM for the latency to decay and τ decay for decaying events in rat (c) and mouse (e) neurons. ****, p < 0.0001 by Mann-Whitney; U = 130046 (latency) and 149764 (tau) in (c); U = 221111 (latency) and 227995 (tau) in (e)

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Fluorescence, Cell Culture, Over Expression, Transfection, Plasmid Preparation, Control, MANN-WHITNEY

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: (a) Chromaffin cells from wt or synuclein TKO mice were transduced with lentivirus encoding either human α-synuclein (SYN) or empty vector, cultured for 72 h and immunostained for α-synuclein (H3C, green) as well as the dense core vesicle protein secretogranin II (SgII, red) The images were obtained using structured illumination and shown here as reconstructions of a 120 nm-thick slice located within 0.5 μm of the cell-coverglass interface. Size bar, 2.5 μm. (b) The extent of SgII colocalization with synuclein was quantified using Pearson’s correlation coefficient (R) and Manders overlap coefficient (M1). The extent of wt synuclein colocalization with the mitochondrial protein TOM20 is shown in green. n = 7 cells for wt, 5 cells for SYN, 6 cells for TKO and 3 cells for TOM20 (c) Similar colocalization measures for a slice located 0.5–1.0 μm deeper inside the cell shows that the localization of synuclein to secretory vesicles is not limited to the docked pool; n = 3 cells. Values in b and c indicate mean ± SEM

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Transduction, Plasmid Preparation, Cell Culture

Journal: Nature neuroscience

Article Title: α-Synuclein Promotes Dilation of the Exocytotic Fusion Pore

doi: 10.1038/nn.4529

Figure Lengend Snippet: Chromaffin cells from wt mice were transduced with lentiviruses encoding BDNF-pHluorin and either mutant human α-synuclein (A30P, A53T), human β-synuclein (β-syn), human γ-synuclein (γ-syn) or empty vector (con). (a) Relative to control, over-expression of mutant α-, β- or γ-synuclein all caused a reduction in the number of exocytotic events evoked over 50 s by depolarization with 45 mM K + (**, p = 0.0046 by one-way ANOVA with Tukey’s post hoc test; F(4, 60) = 4.027). con, n = 17 cells; A30P, n = 14 cells; A53T, n = 10 cells; β-syn, n = 11 cells; γ-syn, n = 13 cells from 3 independent cultures (b) β- and γ-Synuclein both accelerate the kinetics of individual BDNF-pHluorin release events. However, the two PD-associated mutants do not affect release kinetics. The cumulative frequency distribution includes the decay constants for all BDNF-pHluorin events that decayed to baseline. Expression of either β- or γ- but not mutant α-synuclein, shifted the decay constants to shorter values relative to control (p < 0.0001 by Kolgomorov-Smirnov test for con vs. β-syn and con vs. γ-syn). The inset shows a 10–90 percentile box and whisker plot of the decay constants (mean represented as “+”). ****, p < 0.0001 by Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test (H = 76.76), con vs. β-syn and con vs. γ-syn; n = 610 events for control, 296 for A30P, 172 for A53T, 210 for β-synuclein and 265 for γ-synuclein (c,d) Chromaffin cells from wild type mice infected with either empty vector or lentivirus encoding A30P or A53T human α-synuclein were immunostained for α-synuclein (H3C, green) as well as SgII (red) and visualized by TIRF microscopy (c) . Size bar, 2.5 μm. (d) The extent of SgII colocalization with synuclein was assessed using the Manders coefficient. n.s., not significant (p = 0.6147 by one-way ANOVA; F(4, 21) = 0.6781); n = 3 cells for TKO, 7 for control, 5 for A30P, 6 for A53T, 4 for TKO-A30P and 4 for TKO-A53T (e) The expression of wild type and mutant human α-synuclein was assessed by immunofluorescence with the syn-1 antibody (left) and human α-, β- and γ-synuclein with a pan-synuclein antibody (right). n = 6 cells for control, 8 for A30P, 7 for A53T using the syn-1 antibody, and 7 for control, 7 for β-synuclein and 7 for γ-synuclein using the pan-synuclein antibody

Article Snippet: Synuclein triple knockout (TKO) mice were produced by crossing α/β-synuclein double KO mice (Jackson Laboratory, stock # 006390) to a γ-synuclein KO line generously provided by L. Lustig.

Techniques: Transduction, Mutagenesis, Plasmid Preparation, Control, Over Expression, Expressing, Whisker Assay, Infection, Microscopy, Immunofluorescence