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: After obtaining the brain and Neuro2A lysates, we measured protein concentration (DC Protein Assay Kit II, Biorad), and samples were used in subsequent experiments.

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: After obtaining the brain and Neuro2A lysates, we measured protein concentration (DC Protein Assay Kit II, Biorad), and samples were used in subsequent experiments.

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:

Article Snippet: After obtaining the brain and Neuro2A lysates, we measured protein concentration (DC Protein Assay Kit II, Biorad), and samples were used in subsequent experiments.

Techniques: Recombinant, Software, Transduction, Fluorescence, Microscopy

Journal: microPublication Biology

Article Title: cnnm-5 knockdown improves proteostasis of mutant Huntingtin protein in C. elegans

doi: 10.17912/micropub.biology.001497

Figure Lengend Snippet: A) Day 1 EAK103 adults developed on L4440 (control) or cnnm-5 i at 25°C, zoomed in on the head region. Puncta indicate accumulation of mHTT tagged with YFP. Fluorescent images were taken using the GFP filter on the stereoscope at 63x. Scale bar indicates 500μm. B) MAH215 nematodes were imaged on Day 1 of adulthood after development at 25°C on L4440 (control) or cnnm-5 i. The green channel shows autophagosomes only (AP), and the red channel shows both autophagosomes and autolysosomes (AP + AL). Average autophagy levels in MAH215 L4440 or cnnm-5 i were quantified using ImageJ, p<0.05. Images in A & B are representative of three trials. C) Levels of ubiquitinated proteins in WT (lanes 1 & 2-biological replicates) and VC4126 (triple mutant strain that includes a nonsense mutation in cnnm-5 ) (lanes 3 & 3, technical replicates) on OP50 and EAK102 and EAK103 on control (L4440) ( EAK102 lanes 4-6, EAK103 lanes 10-12, biological triplicates) or cnnm-5 i ( EAK102 lanes 7-9, EAK103 lanes 13-15, biological triplicates). Beta-actin is shown as the loading control. The average ubiquitin signal was quantified and normalized to beta-actin. Two-tailed Student's t -test was performed to determine p value: N2 vs VC4126 p=0.65; EAK102 L4440 vs cnnm-5 i p=0.03 (*significant); EAK103 L4440 vs cnnm-5 i p=0.27. Fold change of normalized ubiquitin signal is based on comparison to the appropriate non- cnnm-5 -modified control (color matched). D) Survival curve of nematodes that survived each day of adulthood at 25°C. N2 and VC4126 were grown on OP50 , and EAK102 and EAK103 were developed and maintained on control (L4440) or cnnm-5 i. n=100 worms per condition. A chi-square test was performed to determine significance, *p<0.05. E) Percent of GMC101 nematodes that were paralyzed at adulthood Day 4 after 72 hours under heat stress (25°C) developed and grown on control (L4440) or cnnm-5 i. Each dot represents a separate trial (n>20 per trial). Bar within box represents the mean of all trials, and the whiskers represent standard error. One-tailed Student's t -test was performed to determine significance, *p=0.042.

Article Snippet: Worm bleach: 0.22M Sodium Hypochlorite and 0.786M Potassium Hydroxide in Millipore-filtered water Protein Quantification Assay Reagents (DC BCA): Bio Rad Catalog #5000112 Ubiquitin antibody: Proteintech Catalog #10201-2-AP Beta-actin antibody: Proteintech Catalog #81115-1-RR WesternSure Goat anti-rabbit HRP secondary: Licor Catalog #926-80011 SuperSignalTM West Femto Maximum Sensitivity Substrate: Fisher Catalog #34094 Sodium Azide : Fisher Catalog #S0489100G C. elegans strains: All strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Techniques: Control, Mutagenesis, Ubiquitin Proteomics, Two Tailed Test, Comparison, Modification, One-tailed Test