Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods
Aim. Evidence-backed execution summary for Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods from Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- Recombinant α-Syn was expressed and purified as described previously [ ]. Samples containing α-Syn were diluted to a concentration of 100 µM in a 50 mM HEPES (Sigma, Taufkirchen, Germany) buffer with 100 mM NaCl (Sigma) and 0.03 % NaN 3 (Sigma) at pH 7.4 in a 500 µl...
Electron microscopy of α-Syn fibrils
reagent used in the protocol.
- Use
- A solution containing protein was applied to glow-discharged carbon coated grids and stained with 1 % uranyl acetate (Sigma). Images were taken in a Philips CM120 electron microscope (Philips, Amsterdam, The Netherlands) at a defocus of 2.3 µm using a TemCam 224A slow scan CCD camera (TVIPS, Gauting,...
NMR spectroscopy of α-Syn
reagent used in the protocol.
- Use
- Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600...
Treatment, transfection, and immunocytochemistry of H4 cells
reagent used in the protocol.
- Use
- H4 human neuroglioma cells (HTB-148, ATCC, Manassas, VA) were plated on poly-D-lysine (Sigma)-coated glass cover slips at a density of 35,000/cm 2 and pre-incubated with 5 and 20 µM Fasudil or Y-27632 in DMEM (Gibco, Karlsruhe, Germany) supplemented with 10 % fetal bovine serum (PAA Laboratories, Pasc...
Western blot analysis of H4 cells
reagent used in the protocol.
- Use
- H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', R...
SEC-HPLC and dotblot analysis
reagent used in the protocol.
- Use
- H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged for 10 min at 10,000 g. 2-3 mg of total protein in a maximum volume of 500 &...
Immunohistochemistry
reagent used in the protocol.
- Use
- Mice were sacrificed by exposure to carbon dioxide and directly afterwards perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA; Applichem) in PBS (pH 7.4). Brains were dissected, post-fixed in PFA overnight at 4 °C, and finally embedded into paraffin. Brains were coronally sec...
Fasudil attenuates α-Syn aggregation by direct C-terminal interaction
reagent used in the protocol.
- Use
- α-Syn is a natively unfolded protein that upon exposure to aggregation-prone conditions forms amyloid fibrils that are reactive with Thioflavin T (ThT) and are visible by electron microscopy (EM). To probe the influence of Fasudil on the aggregation kinetics of α-Syn, we monitored the change in ThT fluores...
Electron microscopy of α-Syn fibrils
A solution containing protein was applied to glow-discharged carbon coated grids and stained with 1 % uranyl acetate (Sigma). Images were taken in a Philips CM120 electron microscope (Philips, Amsterdam, The Netherlands) at a defocus of 2.3 µm using a TemCam 224A slow scan CCD camera (TVIPS, Gauting,...
- Use
- A solution containing protein was applied to glow-discharged carbon coated grids and stained with 1 % uranyl acetate (Sigma). Images were taken in a Philips CM120 electron microscope (Philips, Amsterdam, The Netherlands) at a defocus of 2.3 µm using a TemCam 224A slow scan CCD camera (TVIPS, Gauting,...
NMR spectroscopy of α-Syn
Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600...
- Use
- Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600...
Treatment, transfection, and immunocytochemistry of H4 cells
H4 human neuroglioma cells (HTB-148, ATCC, Manassas, VA) were plated on poly-D-lysine (Sigma)-coated glass cover slips at a density of 35,000/cm 2 and pre-incubated with 5 and 20 µM Fasudil or Y-27632 in DMEM (Gibco, Karlsruhe, Germany) supplemented with 10 % fetal bovine serum (PAA Laboratories, Pasc...
- Use
- H4 human neuroglioma cells (HTB-148, ATCC, Manassas, VA) were plated on poly-D-lysine (Sigma)-coated glass cover slips at a density of 35,000/cm 2 and pre-incubated with 5 and 20 µM Fasudil or Y-27632 in DMEM (Gibco, Karlsruhe, Germany) supplemented with 10 % fetal bovine serum (PAA Laboratories, Pasc...
Western blot analysis of H4 cells
H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', R...
- Use
- H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', R...
SEC-HPLC and dotblot analysis
H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged for 10 min at 10,000 g. 2-3 mg of total protein in a maximum volume of 500 &...
- Use
- H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged for 10 min at 10,000 g. 2-3 mg of total protein in a maximum volume of 500 &...
Animal experiments
Animals were treated according to the regulations of the local animal research council and legislation of the State of Lower Saxony, Germany (33.19-42502-04-12/0884). Heterozygous breeding couples of the transgenic mouse strain B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J (short: α-Syn A53T ) were bought from Jax Labs (J004...
- Use
- Animals were treated according to the regulations of the local animal research council and legislation of the State of Lower Saxony, Germany (33.19-42502-04-12/0884). Heterozygous breeding couples of the transgenic mouse strain B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J (short: α-Syn A53T ) were bought from Jax Labs (J004...
Animal experiments
Blood of randomly selected mice was analyzed to monitor for alterations of standard clinical chemistry and hematology parameters due to Fasudil treatment. Approximately 200 µl blood were collected for hematological analysis from the retrobulbar venous plexus and transferred to ethylene diamine tetra-acetic...
- Use
- Blood of randomly selected mice was analyzed to monitor for alterations of standard clinical chemistry and hematology parameters due to Fasudil treatment. Approximately 200 µl blood were collected for hematological analysis from the retrobulbar venous plexus and transferred to ethylene diamine tetra-acetic...
Rotarod
To measure motor balance and coordination, mice were tested on an accelerated rotarod (rotarod for mice 47600, Ugo Basile, Comerio, Italy) ranging from 5 to 40 rpm within 5 min as described before [, ]. All mice were pre-trained on the rotarod in order to reach a stable performance. Rotarod test was perfo...
- Use
- To measure motor balance and coordination, mice were tested on an accelerated rotarod (rotarod for mice 47600, Ugo Basile, Comerio, Italy) ranging from 5 to 40 rpm within 5 min as described before [, ]. All mice were pre-trained on the rotarod in order to reach a stable performance. Rotarod test was perfo...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analyses were conducted using Kyplot software (Version 2.0, KyensLab Incorporated, Tokyo, Japan). Comparisons of two groups were done by unpaired Student's T -Test, multiple group comparisons by one-way ANOVA with Dunnett post-hoc test. The statistical test and number of in vitro experiments or ani...
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NMR spectroscopy of α-Syn
Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600 MHz spectrometer. The temperature was set to 20 °C. Data processing was performed using the software packages Topspin (Bruker, Billerica, MA) and CCPN Analysis [ ]. 15 N- 1 H heteronuclear single quantum coherence (HSQC) amide cross-peaks affected during compound addition were identified by comparison of their chemical shift values with those of the same cross-peaks in the data set of samples lacking the compound. Perturbations in the chemical shift values for 1 H and 15 N were calculated as [(Δδ 1 H) 2 + (Δδ 15 N/10) 2 ] 1/2.
Western blot analysis of H4 cells
H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', Roche, Basel, Switzerland). Briefly, protein content of the samples was determined by BCA assay (ThermoScientific, Rockford, IL, USA), and equal amounts of protein (20 µg) were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane (Applichem). After blocking with 5 % nonfat milk (Applichem) in Tris-buffered saline/Tween 20 (TBS-T, Applichem) for 1 h membranes were incubated with respective primary antibodies for 18 h at 4 °C in TBS-T and 5 % milk. Finally, membran...
Rotarod
To measure motor balance and coordination, mice were tested on an accelerated rotarod (rotarod for mice 47600, Ugo Basile, Comerio, Italy) ranging from 5 to 40 rpm within 5 min as described before [, ]. All mice were pre-trained on the rotarod in order to reach a stable performance. Rotarod test was performed after 100 d, 200 d, and from day 220 on twice a week, each time in three sessions with an intertrial interval of 30 min to reduce stress and fatigue. Rotarod performance in all three runs was recorded and the average time on the rotarod was evaluated. The continuous rotarod testing twice a week from day 220 on was used to detect the onset of clinical pathology described for this mouse model [ ] as early as possible for each animal. When rotarod performance of a single mouse dropped below 50 % of its long term average, the respective animal was termed as ̵...
Immunohistochemistry
Mice were sacrificed by exposure to carbon dioxide and directly afterwards perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA; Applichem) in PBS (pH 7.4). Brains were dissected, post-fixed in PFA overnight at 4 °C, and finally embedded into paraffin. Brains were coronally sectioned into 5 µm slices, mounted on slides and deparaffinized by xylene (Sigma) treatment for 2x 10 min, followed by a descending ethanol (EtOH) row (2x 3 min 100 % EtOH (Applichem), 96 %, 90 %, 70 %, 50 % EtOH for 3 min each). For immunohistochemistry, sections were incubated for 30 min in citrate buffer (10 mM citric acid (Sigma), 0.05 % Tween 20 (Applichem), pH 6.0 at 80 °C. After cooling down, sections were either treated with 10 µg/ml proteinase K (PK, Applichem) in PBS at 55...
Catwalk gait analysis reveals motor improvement after Fasudil treatment
To detect more subtle differences in gait of α-Syn A53T mice after treatment with Fasudil, mice were tested with the Catwalk gait analysis system, since this method is more sensitive than the rotarod [ ]. α-Syn A53T mice were tested after 'disease onset' as detected by rotarod failure (Fig. ). Footprints of the mice were automatically detected, and various gait parameters were analyzed (Fig. b, c). Before disease onset at day 200, no significant differences between the groups were apparent (data not shown). Acute Fasudil treatment also did not have any significant effects in the multiparametric gait analysis (Additional file: Figure S4). After mice displayed clinical symptoms, long-term Fasudil treatment significantly improved gait performance of α-Syn A53T mice as compared to untreated controls. In detail, run average speed was improved dose-de...
Measurement outputs
What raw and processed outputs should exist?
Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 m...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Tr...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Animals were treated according to the regulations of the local animal research council and legislation of the State of Lower Saxony, Germany (33.19-42502-04-12/0884). Heterozygo...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Statistical analyses were conducted using Kyplot software (Version 2.0, KyensLab Incorporated, Tokyo, Japan).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 m...; H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Tr...; H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail...; Animals were treated according to the regulations of the local animal research council and legislation of the State of Lower Saxony, Germany (33.19-42502-04-12/0884). Heterozygo....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Statistical analyses were conducted using Kyplot software (Version 2.0, KyensLab Incorporated, Tokyo, Japan). Comparisons of two groups were done by unpaired Student's T -...; To investigate the influence of long-term Fasudil treatment on α-Syn pathology in vivo we used a mouse model expressing human α-Syn A53T under the prion protein (Prnp)...; α-Syn is a natively unfolded protein that upon exposure to aggregation-prone conditions forms amyloid fibrils that are reactive with Thioflavin T (ThT) and are visible by e...; To address the influence of Y133 and Y136 on α-Syn aggregation in the H4 cell culture model in vitro, both residues were substituted with alanine in human synT by site-dir...
from paperReporting output
Report representative outputs alongside summary comparisons for Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 m..., H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Tr..., H4 neuroglioma cells were collected 24 h after transfection in a phosphate buffer (1X PBS with 0.5 % TritonX-100) freshly supplemented with protease inhibitor cocktail..., Animals were treated according to the regulations of the local animal research council and legislation of the State of Lower Saxony, Germany (33.19-42502-04-12/0884). Heterozygo....
inferred from protocolStructured statistical methods
Statistical analyses were conducted using Kyplot software (Version 2.0, KyensLab Incorporated, Tokyo, Japan). Comparisons of two groups were done by unpaired Student's T -...; To investigate the influence of long-term Fasudil treatment on α-Syn pathology in vivo we used a mouse model expressing human α-Syn A53T under the prion protein (Prnp)...; α-Syn is a natively unfolded protein that upon exposure to aggregation-prone conditions forms amyloid fibrils that are reactive with Thioflavin T (ThT) and are visible by e...; To address the influence of Y133 and Y136 on α-Syn aggregation in the H4 cell culture model in vitro, both residues were substituted with alanine in human synT by site-dir...
source structuredSource and audit
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Evidence quotes (5)
Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600 MHz spectrometer. The temperature was set to 20 °C. Data processing was performed using the software packages Topspin (Bruker, Billerica, MA) and CCPN Analysis [ ]. 15 N- 1 H heteronuclear single quantum coherence (HSQC) amide cross-peaks affected during compound addition were identified by comparison of their chemical shift values with those of the same cross-peaks in the data set of samples lacking the compound. Perturbations in the chemical shift values for 1 H and 15 N were calculated as [(Δδ 1 H) 2 + (Δδ 15 N/10) 2 ] 1/2.
H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', Roche, Basel, Switzerland). Briefly, protein content of the samples was determined by BCA assay (ThermoScientific, Rockford, IL, USA), and equal amounts of protein (20 µg) were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane (Applichem). After blocking with 5 % nonfat milk (Applichem) in Tris-buffered saline/Tween 20 (TBS-T, Applichem) for 1 h membranes were incubated with respective primary antibodies for 18 h at 4 °C in TBS-T and 5 % milk. Finally, membranes were incubated with corresponding horseradish peroxidase-coupled secondary antibodies (1:4000 for 1 h at room temperature) and chemiluminescence signal was visualized and quantified using ECL (Immobilon Western, Millipore, Billerica, MA, USA) and ChemiDoc XRS+ with Image Lab Software (BIO-RA...
To measure motor balance and coordination, mice were tested on an accelerated rotarod (rotarod for mice 47600, Ugo Basile, Comerio, Italy) ranging from 5 to 40 rpm within 5 min as described before [, ]. All mice were pre-trained on the rotarod in order to reach a stable performance. Rotarod test was performed after 100 d, 200 d, and from day 220 on twice a week, each time in three sessions with an intertrial interval of 30 min to reduce stress and fatigue. Rotarod performance in all three runs was recorded and the average time on the rotarod was evaluated. The continuous rotarod testing twice a week from day 220 on was used to detect the onset of clinical pathology described for this mouse model [ ] as early as possible for each animal. When rotarod performance of a single mouse dropped below 50 % of its long term average, the respective animal was termed as 'disease onset' and ultimately tested in Catwalk gait analysis and novel object recognition test.
Mice were sacrificed by exposure to carbon dioxide and directly afterwards perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA; Applichem) in PBS (pH 7.4). Brains were dissected, post-fixed in PFA overnight at 4 °C, and finally embedded into paraffin. Brains were coronally sectioned into 5 µm slices, mounted on slides and deparaffinized by xylene (Sigma) treatment for 2x 10 min, followed by a descending ethanol (EtOH) row (2x 3 min 100 % EtOH (Applichem), 96 %, 90 %, 70 %, 50 % EtOH for 3 min each). For immunohistochemistry, sections were incubated for 30 min in citrate buffer (10 mM citric acid (Sigma), 0.05 % Tween 20 (Applichem), pH 6.0 at 80 °C. After cooling down, sections were either treated with 10 µg/ml proteinase K (PK, Applichem) in PBS at 55 °C for 12 min to quantify α-Syn pathology, or just washed with PBS, followed by incubation in 0.1 g sudan black (Applichem) per 100 ml 70 % EtOH. After rinsing with water and PBS washing, sections were incubated for 20 min in 25 mM glycine (Applichem) in PBS....
To detect more subtle differences in gait of α-Syn A53T mice after treatment with Fasudil, mice were tested with the Catwalk gait analysis system, since this method is more sensitive than the rotarod [ ]. α-Syn A53T mice were tested after 'disease onset' as detected by rotarod failure (Fig. ). Footprints of the mice were automatically detected, and various gait parameters were analyzed (Fig. b, c). Before disease onset at day 200, no significant differences between the groups were apparent (data not shown). Acute Fasudil treatment also did not have any significant effects in the multiparametric gait analysis (Additional file: Figure S4). After mice displayed clinical symptoms, long-term Fasudil treatment significantly improved gait performance of α-Syn A53T mice as compared to untreated controls. In detail, run average speed was improved dose-dependently as compared to α-Syn A53T control, with high Fasudil dosage reaching significance (wt ctrl: 28.6 ± 2.11 cm/s; A53T ctrl: 13.6 ± 1.20 cm/s, p = 7.05x10 -6, T -test; A53T Fas10: 19.7 ± 1.85 cm/s; A53T Fas30:...
Machine-readable layer
[
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"@type": "HowTo",
"name": "Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods",
"description": "Evidence-backed execution summary for Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods from Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.",
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{
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"name": "NMR spectroscopy of α-Syn",
"text": "Recombinant 15 N-labeled α-Syn was expressed and purified as described previously [ ]. NMR samples contained 0.1 mM 15 N-labeled wild-type (wt) α-Syn in 50 mM HEPES buffer, 100 mM NaCl, pH 7.4 and 90 % H 2 O/10 % D 2 O. NMR experiments were recorded on a Bruker Avance 600 MHz spectrometer. The temperature was set to 20 °C. Data processing was performed using the software packages Topspin (Bruker, Billerica, MA) and CCPN Analysis [ ]. 15 N- 1 H heteronuclear single quantum coherence (HSQC) amide cross-peaks affected during compound addition were identified by comparison of their chemical shift values with those of the same cross-peaks in the data set of samples lacking the compound. Perturbations in the chemical shift values for 1 H and 15 N were calculated as [(Δδ 1 H) 2 + (Δδ 15 N/10) 2 ] 1/2."
},
{
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"name": "Western blot analysis of H4 cells",
"text": "H4 cells were lysed 24 h after transfection in RIPA buffer consisting of 20 mM sodium phosphate (pH7.4; Roth, Karlsruhe, Germany), 150 mM NaCl (Roth), 1 % Triton X-100 (Roth), 0.5 % sodium deoxycholate (Sigma), 0.5 % SDS (Roth), and protease inhibitors ('Complete tablets', Roche, Basel, Switzerland). Briefly, protein content of the samples was determined by BCA assay (ThermoScientific, Rockford, IL, USA), and equal amounts of protein (20 µg) were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane (Applichem). After blocking with 5 % nonfat milk (Applichem) in Tris-buffered saline/Tween 20 (TBS-T, Applichem) for 1 h membranes were incubated with respective primary antibodies for 18 h at 4 °C in TBS-T and 5 % milk. Finally, membran..."
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{
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"name": "Rotarod",
"text": "To measure motor balance and coordination, mice were tested on an accelerated rotarod (rotarod for mice 47600, Ugo Basile, Comerio, Italy) ranging from 5 to 40 rpm within 5 min as described before [, ]. All mice were pre-trained on the rotarod in order to reach a stable performance. Rotarod test was performed after 100 d, 200 d, and from day 220 on twice a week, each time in three sessions with an intertrial interval of 30 min to reduce stress and fatigue. Rotarod performance in all three runs was recorded and the average time on the rotarod was evaluated. The continuous rotarod testing twice a week from day 220 on was used to detect the onset of clinical pathology described for this mouse model [ ] as early as possible for each animal. When rotarod performance of a single mouse dropped below 50 % of its long term average, the respective animal was termed as ̵..."
},
{
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"name": "Immunohistochemistry",
"text": "Mice were sacrificed by exposure to carbon dioxide and directly afterwards perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA; Applichem) in PBS (pH 7.4). Brains were dissected, post-fixed in PFA overnight at 4 °C, and finally embedded into paraffin. Brains were coronally sectioned into 5 µm slices, mounted on slides and deparaffinized by xylene (Sigma) treatment for 2x 10 min, followed by a descending ethanol (EtOH) row (2x 3 min 100 % EtOH (Applichem), 96 %, 90 %, 70 %, 50 % EtOH for 3 min each). For immunohistochemistry, sections were incubated for 30 min in citrate buffer (10 mM citric acid (Sigma), 0.05 % Tween 20 (Applichem), pH 6.0 at 80 °C. After cooling down, sections were either treated with 10 µg/ml proteinase K (PK, Applichem) in PBS at 55..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Catwalk gait analysis reveals motor improvement after Fasudil treatment",
"text": "To detect more subtle differences in gait of α-Syn A53T mice after treatment with Fasudil, mice were tested with the Catwalk gait analysis system, since this method is more sensitive than the rotarod [ ]. α-Syn A53T mice were tested after 'disease onset' as detected by rotarod failure (Fig. ). Footprints of the mice were automatically detected, and various gait parameters were analyzed (Fig. b, c). Before disease onset at day 200, no significant differences between the groups were apparent (data not shown). Acute Fasudil treatment also did not have any significant effects in the multiparametric gait analysis (Additional file: Figure S4). After mice displayed clinical symptoms, long-term Fasudil treatment significantly improved gait performance of α-Syn A53T mice as compared to untreated controls. In detail, run average speed was improved dose-de..."
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"name": "Western blot analysis of H4 cells"
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{
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"name": "SEC-HPLC and dotblot analysis"
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"supply": [
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{
"@type": "HowToSupply",
"name": "Electron microscopy of α-Syn fibrils"
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{
"@type": "HowToSupply",
"name": "NMR spectroscopy of α-Syn"
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{
"@type": "HowToSupply",
"name": "Treatment, transfection, and immunocytochemistry of H4 cells"
},
{
"@type": "HowToSupply",
"name": "Western blot analysis of H4 cells"
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{
"@type": "HowToSupply",
"name": "SEC-HPLC and dotblot analysis"
},
{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
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{
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"name": "Fasudil attenuates α-Syn aggregation by direct C-terminal interaction"
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"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease",
"datePublished": "2016",
"author": [
{
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"name": "Lars Tatenhorst"
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