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

Journal: Oncotarget

Article Title: Impaired GABA B -mediated presynaptic inhibition increases excitatory strength and alters short-term plasticity in synapsin knockout mice

doi: 10.18632/oncotarget.21405

Figure Lengend Snippet: (A) , (B) Effects of sustained and high frequency stimulation of Schaffer collaterals (20 s at 20 Hz) in CA1 pyramidal neurons from WT (left) and TKO (right) mice in the absence (black traces/symbols) or presence (red traces/symbols) of CGP55845. A sustained facilitation was present in WT mice, which became transient and associated with late depression in the presence of CGP. On the contrary, the profound depression following a short-lived facilitation observed in TKO mice was unaffected by CGP. Representative traces of eEPSCs in response to the 1 st , 2 nd and last action potential in the train are shown, normalized to the amplitude of the first response (WT, 97.9 pA; WT+CGP, 249.6 pA; TKO, 430.2 pA; TKO+CGP, 408.0 pA) (A). In the main plots, the amplitude of eEPSCs, normalized to the mean baseline value is plotted as a function of time (B). (C) Mean (± sem) percentage of depression of eEPSC amplitude as a function of the genotype in the absence (left; black bars) or presence of CGP55845 (right; red bars). Quantitative analysis of depression was carried out by measuring the first eEPSC 0.5 s after the peak of facilitation (I p ) and 10 s after the start of the train stimulation (I 10 s ), according to the formula: (I p -I 10s )/I p * 100 (see panel B). The effects of GABA B R block by CGP55845 in the two genotypes were analyzed using the unpaired Mann-Whitney's U -test; * p<0.05; ** p<0.01. (D) , (E) The time course of recovery, studied for 600 s after the end of the train by lowering the stimulation frequency from 20 to 0.1 Hz, is shown for WT (left) and TKO (right) slices in the absence (black) or presence (red) of CGP55845. Representative traces of eEPSCs at various times of recovery are shown after normalization to the baseline (bsl) amplitudes (WT, 87.5 pA; WT+CGP, 239.4 pA; TKO, 427.1 pA; TKO+CGP, 400.3 pA) (D). In the main plots, the amplitude of eEPSCs, normalized to the mean baseline value, is plotted as a function of time (E). * p<0.05, #p<0.05, for untreated and CGP-treated samples, respectively; one-tailed paired Student's t -test vs mean steady-state values calculated in the last 5 min of recording (shaded area). WT, n=11 and n=10; TKO, n=6 and n=10; for vehicle and CGP, respectively.

Article Snippet: TKO mice were re-derived on a C57BL/6J background (Charles River, Calco, Italy), obtaining single and multiple Syn KO strains up to the triple Syn knockout (TKO) and matched wild type (WT) mice.

Techniques: Blocking Assay, MANN-WHITNEY, One-tailed Test

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic showing pH-sensitive sensor sypHy and principle of pHluorin experiments to quantitatively evaluate the SV cycle (see main text and methods for more details). ( B ) Elimination of all synapsins block α-syn functionality at synapses. Left : Schematic showing design of pHluorin experiments. WT or synapsin TKO cultured hippocampal neurons were co-transduced at 5 days in vitro (DIV) with h-α-syn:mCherry (or mCherry as control) and sypHy, and imaged at 14–15 DIV. Right : Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 s – for clarity, symbols only mark every other mean ± SEM ΔF/F 0 value in all sypHy traces). Note that while h-α-syn over-expression (orange) attenuated sypHy fluorescence in WT neurons, there was no effect in neurons from mice lacking all synapsins (TKO). All sypHy data quantified in ( C ). ( C ) Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). A total of 12–19 coverslips were analyzed for each condition, from at least three separate cultures (***p=9e-7, ns p=0.45, student’s t-test). ( D ) Domain structure of the five main synapsin isoforms. ( E ) Experimental design to identify the synapsin isoform that reinstated α-syn functionality, Synapsin TKO neurons were co-transduced at 5 DIV with each synapsin isoform, h-α-syn, and sypHy; and imaged at 14–15 DIV. ( F ) SypHy fluorescence traces (mean ± SEM). Note that h-α-syn(orange) attenuates SV recycling only if the neurons are also co-expressing the ‘a’ isoforms – synapsins Ia, IIa, and IIIa (300 action potentials at 20 Hz, delivered at t=0 sec). Data quantified in G. ( G ) Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). 13–26 coverslips from at least three separate cultures were analyzed for each condition (from left to right: ***p=0.0009, ns p=0.62, ***p=0.00005, ns p=0.99, **p=0.004, student’s t test). Figure 1—source data 1. Tabular data and statistical analyses for graphs presented in panels B, C, F, G.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Blocking Assay, Cell Culture, In Vitro, Fluorescence, Over Expression, Expressing

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Data from sypHy experiments where reacidification was blocked by bafilomycin (Baf), allowing isolated evaluation of exocytosis only (also see results). Note that h-α-syn over-expression attenuated synaptic exocytosis in WT neurons (left), while there was no effect in synapsin TKO neurons (middle). Reintroduction of tagBFP:synapsin Ia reinstated the h-α-syn-mediated synaptic attenuation (right). All pHluorin data quantified in ( B ). 9–22 coverslips from at least three independent cultures (***p=1.9e-5 Mann-Whitney test, p=0.22 Student’s t-test, **p=0.008 Student’s t-test). ( C ) A representative trace showing how the fluorescence decay was quantified to evaluate endocytosis in the sypHy experiments (also see Results). ( D–E ) Fluorescence decay analyses of h-α-syn over-expression in WT and synapsin TKO neurons ( D ), as well as in synapsin TKO neurons where each synapsin isoform was reintroduced ( E ). Note that there were no significant differences in any of these groups. All data in this figure are represented as mean +/- SEM. Ten to 26 coverslips from at least three independent cultures (D: p=0.36; E: p=0.85 both Kruskal Wallis ANOVA). Figure 1—figure supplement 1—source data 1. Tabular data and statistical analyses for graphs presented in panels A, B, D, and E.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Isolation, Over Expression, MANN-WHITNEY, Fluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Workflow for co-immunoprecipitation experiments in neuro2a cells. ( B ) Western blots from co-immunoprecipitation experiments show that the synapsin isoforms Ia, IIa, and IIIa associate more robustly with h-α-syn (top panel), when compared to synapsins Ib and IIb (a non-specific band is marked with an asterisk). ( C ) Quantification of blots in ( B ) n=5, all data presented as mean ± SEM (a vs. b isoform, **p=0.003, ***p=0.0003, Student’s t-test). ( D ) Schematic showing synapsin isoforms and their variable domains. Note that the E-domain is common between synapsins Ia, IIa, and Iia. ( E ) Workflow for pulldown of GST-tagged h-α-syn WT/deletions/scrambled mutations after incubation with mouse brain lysates. Equivalent amounts of immobilized GST α-syn variants were used. ( F ) Schematic showing α-syn regions that were scrambled (amino acids between 96–140 and 96–110). ( G ) Top : Samples from GST-pulldown were analyzed by NuPAGE and immunoblotted with an antibody against synapsin I (top panel). Bottom : Ponceau staining shows equivalent loading of fusion proteins. Note that full-length h-α-syn bound synapsin I from mouse brains (lane 2), while deletion of the h-α-syn C-terminus (amino acids 96–140, lane 3) eliminated this interaction. Lanes 4–7 show that the sequence within amino acids 96–110 of h-α-syn is critical for binding to synapsin I. All western blots are quantified below (n=3). Data presented as mean ± SEM (**p=0.003, **p=0.002, ns p=0.99, ns p=0.98, **p=0.004, **p=0.004, comparing to full-length h-α-syn, one-way ANOVA with Tukey’s posthoc test). Figure 2—source data 1. Tabular data and statistical analyses for graphs shown in panels C and G. Figure 2—source data 2. Full western blots for segments shown in panel B. Figure 2—source data 3. Full western blots for segments shown in panel G.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Immunoprecipitation, Western Blot, Incubation, Staining, Sequencing, Binding Assay

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic showing synapsin Ia scrambled E-domain sequence (synapsin Ia scr-E ). Numbers depict amino acid positions, letters in the inset depict amino-acids. Note that the WT amino acids are randomized in the scrambled mutant. ( B ) Design of sypHy experiments co-expressing synapsin Ia scr-E and h-α-syn in cultured neurons from synapsin TKO mice. ( C ) Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 sec). Note that while h-α-syn attenuated sypHy fluorescence in synapsin TKO neurons expressing synapsin Ia, h-α-syn had no effect in neurons expressing synapsin Ia scr-E . Insets: Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). Ten to 16 coverslips from at least three separate cultures were analyzed for each condition (***p=0.0007, ns p=0.67, one-way ANOVA with Tukey’s posthoc analysis). ( D ) Top : Schematic for co-immunoprecipitation experiments, to test the interaction of h-α-syn with WT synapsin Ia or synapsin Ia scr-E . Neuro2a cells were co-transfected with myc-tagged α-syn and respective YFP-tagged synapsin Ia, and the YFP was immunoprecipitated. Bottom : Note that h-α-syn co-immunoprecipitated with synapsin Ia, but not synapsin Ia scr-E ; quantification of the gels below (n=4, all data are means ± SEM ***p<0.001, Student’s t test – a non-specific band is marked with an asterisk). ( E ) Schematic of experiments to test if the synapsin E-domain is sufficient to enable α-syn functionality in synapsin TKO neurons. Synapsin-E (a 46 amino acid sequence) was fused to the C-terminus of sypHy, so that upon expression in neurons, the E-domain would be present on the cytosolic surface of Svs. ( F ) SypHy fluorescence traces (mean ± SEM). Note that while h-α-syn (orange) was unable to attenuate SV recycling in synapsin TKO neurons (as expected), diminished synaptic responses were seen when the E-domain was present. Insets: Quantification of peak ΔF/F 0 sypHy values (bars: mean ± SEM). Twelve 19 coverslips from at least three separate cultures were analyzed for each condition (ns p=0.89, ***p=2.8e-7, one-way ANOVA with Tukey’s posthoc analysis). Figure 3—source data 1. Tabular data and statistical analyses for graphs shown in panels C, D and F. Figure 3—source data 2. Full western blots for segments shown in panel D.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Sequencing, Mutagenesis, Expressing, Cell Culture, Fluorescence, Immunoprecipitation, Transfection, Western Blot

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic of experiments to evaluate quantitative localization of tagBFP:synapsin Ia and tagBFP:synapsin Ia Scr-E in synapsin TKO neurons (with and without h-α-syn over-expression). Neurons were immunostained for synapsin I (for reliable visualization of the transduced synapsin constructs), as well as for the SV-marker vGlut1 (to confidently identify synapses). ( B ) Representative images showing equivalent immunofluorescence of synapsin Ia and synapsin Ia Scr-E at synapses. Over-expression of h-α-syn did not affect their synaptic fluorescence. ( C ) Quantified synaptic fluorescence data, represented as mean +/- SEM, 15–16 coverslips from at least three independent cultures were analyzed for each condition. ns p=0.52, ns p=0.14, one-way ANOVA. Figure 3—figure supplement 1—source data 1. Tabular data and statistical analyses for graph shown in panel C. Figure 3—figure supplement 1—source data 2. Raw images. Full images of anti-synapsin I and anti-vGlut1 immunofluorescence channels containing the details shown in panel B (marked with white dashed square).

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Over Expression, Construct, Marker, Immunofluorescence, Fluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Schematic of experiments to evaluate synaptic targeting of synapsin E-domain constructs in synapsin TKO neurons. Note that EGFP is tagged to the E-domain in these experiments. ( B ) The EGFP:E-domain construct was diffusely distributed in neurons and not enriched to synapses (marked by immunostaining of VGlut1). A representative image showing that the E-domain construct is not targeted to synapses (green: EGFP:E-domain, magenta: vGlut1). ( C ) Over-expression of synapsin E-domain in the context of excessive α-syn did not have any effect on SV recycling (as determined by sypHy experiments), presumably because the E-domain fails to enrich at synapses. Data shown as mean mean ± SEM, 11–20 coverslips from at least 3 independent cultures were analyzed for each condition (p=0.16, Kruskal-Wallis ANOVA). ( D ) Representative images illustrating synaptic localization of the E-domain tagged to sypHy (green: sypHy:E-domain, magenta: vGlut1). ( E ) Expression of sypHy:E-domain in synapsin TKO neurons enhances the synaptic enrichment of h-α-syn. Synaptic enrichment (see Methods section) of h-α-syn was measured in synapsin TKO neurons expressing either sypHy or sypHy-E-domain. We observed significantly higher enrichment of h-α-syn in the latter. Data shown as mean ± SEM, 23 to 25 coverslips from three independent cultures were analyzed for each condition (**p=0.009, Mann-Whitney U-test). Figure 3—figure supplement 2—source data 1. Tabular data and statistical analyses for graphs shown in panels C and E. Figure 3—figure supplement 2—source data 2. Raw images. Full images of EGFP:E-domain and anti-vGlut1 immunofluorescence channels containing the details shown in panel B, and the full images of sypHy:E-domain and anti-vGlut1 immunofluorescence channels containing the details shown in panel D (marked with white dashed square).

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Construct, Immunostaining, Over Expression, Expressing, MANN-WHITNEY, Immunofluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet: ( A ) Representative images from WT or synapsin TKO neurons immunostained with an SV marker (vGlut1); zoomed insets marked by yellow boundaries. Note that the compact clustering of SVs is lost in synapsin-null neurons. ( B ) FWHM as a quantitative means to determine spreading of fluorophores at synapses (also see Results). Note that an increase in FWHM corresponds to a decrease in intensity and increased spreading of fluorescence within a bouton. ( C ) Quantification of synaptic fluorescence in WT and synapsin TKO neurons. Overall intensities are decreased in TKO synapses (left), and FWHM is increased (right), compared to WT synapses; consistent with a spreading of SVs in the synapsin null setting. ( D ) Experimental plan to determine effects of h-α-syn over-expression on the overall distribution of SVs in WT and synapsin TKO neurons. ( E ) FWHM of vGlut1 staining at synapses is augmented by h-α-syn over-expression in WT neurons, but not in neurons from synapsin TKO mice. Reintroduction of synapsins Ia/IIa (but not Ib/IIb) in the setting of h-α-syn over-expression rescues the changes in vGlut1-FWHM ( F ). All data in this figure are represented as mean +/-SEM. Nine to 28 coverslips from at least three independent cultures were analyzed for C, E, and F (C, left: ***p=0.0006, Mann-Whitney U-test; right: see E; E: ***p=4e-8, ns p=0.92, one-way ANOVA with Tukey’s posthoc analysis; F, left: ***p=2.7e-4, ns p=1.0, Kruskal-Wallis ANOVA with Dunn’s posthoc test; F, right: **p=0.001, ns p=0.52, one-way ANOVA with Tukey’s posthoc analysis). Figure 4—source data 1. Raw images. Full images of anti-vGlut1 immunofluorescence channels in WT and synapsin TKO neurons, containing the details shown in panel A (marked with white dashed square). Figure 4—source data 2. Tabular data and statistical analyses for graphs shown in panels C, E, and F.

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Marker, Fluorescence, Over Expression, Staining, MANN-WHITNEY, Immunofluorescence

Journal: eLife

Article Title: Synapsin E-domain is essential for α-synuclein function

doi: 10.7554/eLife.89687

Figure Lengend Snippet:

Article Snippet: Synapsin triple knock-out (TKO) mice (RRID: MMRRC_041434-JAX ) were backcrossed onto the C57BL/6 background as described previously ( ; ; ), and C57BL/6JRccHsd mice (RRID: IMSR_ENV:HSD-043 ) served as WT controls.

Techniques: Recombinant, Software, Transduction, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Simultaneous Disruption of Mouse ASIC1a, ASIC2 and ASIC3 Genes Enhances Cutaneous Mechanosensitivity

doi: 10.1371/journal.pone.0035225

Figure Lengend Snippet: A , Sample recording traces showing responses of A-mechanonociceptors (AM) from WT and ASIC TKO mice to mechanical stimuli. The upper, middle and lower panels show the digitized oscilloscope tracing, the spike density histograms (bin width = 1 s), and the force stimuli applied, respectively. CV = conduction velocity. B , Stimulus-response function of AM from ASIC TKO ( n = 40, ○) vs . WT ( n = 35, •), showing enhanced mechanosensitivity in ASIC TKO mice ( p = 0.006 between genotypes by two-way ANOVA with repeated measures on one factor. ** p < 0.01; † p < 0.001 vs . WT by unpaired t-test). C , D , When AM fibers were subclassified based on CV, not Aβ-AM ( C ) but Aδ-AM ( D ) showed a significant increase in the stimulus-response function in ASIC TKO mice ( p = 0.028 between genotypes by two-way ANOVA with repeated measures on one factor. * p < 0.05; ** p < 0.01 vs . WT by unpaired t-test). E-H , Stimulus-response function of C-fibers ( E ), D-hair receptors (DHs) ( F ), rapidly adapting (RAs) ( G ), and slowly adapting low-threshold mechanoreceptors (SAs) ( H ). Data are presented as mean ± SEM.

Article Snippet: Combined behavioral and electrophysiological experiments were performed on 53 ASIC TKO mice (27 females and 26 males) and 59 age-matched WT C57BL/6J control mice (30 females and 29 males, Jackson Laboratory, Bar Harbor, ME).

Techniques:

Journal: PLoS ONE

Article Title: Simultaneous Disruption of Mouse ASIC1a, ASIC2 and ASIC3 Genes Enhances Cutaneous Mechanosensitivity

doi: 10.1371/journal.pone.0035225

Figure Lengend Snippet: A-H , Mechanical stimulus response function of A-mechanonociceptors (AM), D-hair receptors (DHs), rapidly adapting (RAs) and slowly adapting low-threshold mechanoreceptors (SAs) before and during exposure to pH 5.0 lactic acid plus ATP or control K-H solution. * p < 0.05; ** p < 0.01 vs . baseline by paired t-test. Data are presented as means ± SEM. WT = wild-type. TKO = triple-knockout. LA = lactic acid; K-H = Krebs-Henseleit.

Article Snippet: Combined behavioral and electrophysiological experiments were performed on 53 ASIC TKO mice (27 females and 26 males) and 59 age-matched WT C57BL/6J control mice (30 females and 29 males, Jackson Laboratory, Bar Harbor, ME).

Techniques: Control, Triple Knockout

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome expression in the brains of young and old mice. ( A ) A western blot analysis of immunoproteasome distribution in the spleen, thymus, liver and brain of 2-month-old female WT mice. A representative western blot is shown (left side). The bar graphs display a relative abundance of low - molecular-weight protein 2 (LMP2) and low-molecular-weight protein 7 (LMP7) normalized to the proteasome subunit α3 ( n = 3–4). The statistical analysis was performed by using one-way ANOVA. Data are presented as mean ± SD, * P = 0.01–0.05, *** P < 0.001. Uncropped blots are shown in . ( B ) A western blot of polyubiquitination in the hippocampi of 2-month-old and 1-year-old female WT mice. A representative western blot is shown (left side), and the bar graphs display a relative polyubiquitin signal normalized to β-actin ( n = 3). The statistical analysis was performed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. ( C ) A western blot of 2- and 8-month-old female WT and TKO mice displays an increase in the polyubiquitination of TKO hippocampi compared with WT hippocampi. A representative western blot is shown. The bar graphs display polyubiquitination levels normalized to β-actin ( n = 3–4). Statistical significance for ( B ) and ( C ) was analysed by using an unpaired t -test. Data are presented as mean ± SD, * P = 0.01–0.05. Uncropped blots are shown in . ( D) A fluorescence staining of LMP7 (green) and NeuN (magenta, a neuronal marker) in the brains of young and old WT mice. A staining of the brains of old TKO mice is shown as a negative control. ( E ) A fluorescence staining of phospho-tau (AT8 = green, white arrows) in the CA3 region of the brains of WT versus TKO mice. The neuronal nuclear protein (NeuN, magenta) serves as a neuronal marker, and DAPI (blue) stains the nuclei. ( F ) A diaminobenzidine staining of phospho-tau (AT8) in the hippocampi of old WT and TKO mice (CA1).

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Expressing, Western Blot, Molecular Weight, Fluorescence, Staining, Marker, Negative Control

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: Immunoproteasome deficiency increases excitability and the risk of developing epilepsy. ( A ) TKO mice develop epileptic seizures at an age of 5–10 months. Female mice ( n = 141) were scored over 20 months. The graph displays the occurrence of seizures in per cent. Statistical significance was analysed by performing a Mantel–Cox test **** P < 0.0001. ( B–G ) Seizure susceptibility of female TKO mice was tested via an i.p. injection of KA (10 mg/kg). The mice were monitored for 60 min after injection. ( B ) A representative EEG of female WT, LMP7 KO and TKO mice. ( C ) The Racine score was estimated in intervals of 5 min. Statistical significance was analysed by using one-way ANOVA (Turkey’s multiple comparisons test, n = 7 mice/group): WT versus LMP7 KO n.s., WT versus TKO **** P < 0.0001, LMP7 KO versus TKO **** P < 0.0001. ( D ) The number of seizures per 5 min was calculated by counting the occurrence of seizures over 40 min ( n = 7 mice/group). Statistical significance was analysed by using one-way ANOVA: WT versus LMP7 n.s., LMP7 versus TKO n.s., WT versus TKO * P = 0.0122. ( E ) The graph displays the survival of animals ( n = 7 mice/group). Statistical significance was analysed by using the Mantel–Cox test: WT versus TKO **** P < 0.0001, WT versus LMP7 KO n.s., LMP7 KO versus TKO ** P = 0.0031. ( F ) The behavioural score of young female WT and TKO animals after an injection of KA ( n = 4 mice/group). Statistical significance was analysed by performing a paired t -test. *** P = 0.0001. ( G ) The Survival of young and old female TKO mice after an injection of KA ( n = 4–7 mice per group). Statistical significance was analysed by using the Mantel–Cox test ** P = 0.003.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Injection

Journal: Brain Communications

Article Title: Immunoproteasome deficiency results in age-dependent development of epilepsy

doi: 10.1093/braincomms/fcae017

Figure Lengend Snippet: TKO animals display several neurological disorders apart from recurrent seizures. ( A ) Female TKO animals show increased anxiety in the open field study compared with age-matched WT mice (>1-year-old mice; n = 5 mice/group). The statistical analysis was performed by using an unpaired t -test. ( B ) A gait analysis of >1-year-old female TKO and WT mice ( n = 5 mice/group). The stride length, stride width and toe spread were measured. The SD value of each mouse was calculated. ( C ) Calbidin staining in female WT and TKO mice shows a significant loss of Purkinje cells in TKO animals ( n = 4 mice/group). The number of Purkinje cells per 10 mm was calculated. The statistical analysis was performed by using the unpaired t -test. * P = 0.01–0.05; ** P = 0.001–0.01.

Article Snippet: The TKO mice were kindly provided by Kenneth Rock (University of Massachusetts Medical School) and Regeneron Pharmaceuticals.

Techniques: Staining