Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Protein expression by western blot
- 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 raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Protein expression by western blot.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Protein expression by western blot.
inferred from protocolSource and audit
What supports the facts on this page?
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
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods",
"description": "Protocol for Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"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."
},
{
"@type": "HowToStep",
"position": 2,
"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..."
},
{
"@type": "HowToStep",
"position": 3,
"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 ̵..."
},
{
"@type": "HowToStep",
"position": 4,
"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..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease",
"datePublished": "2016",
"author": [
{
"@type": "Person",
"name": "Lars Tatenhorst"
},
{
"@type": "Person",
"name": "Katrin Eckermann"
},
{
"@type": "Person",
"name": "Vivian Dambeck"
},
{
"@type": "Person",
"name": "Luis Fonseca-Ornelas"
},
{
"@type": "Person",
"name": "Hagen Walle"
},
{
"@type": "Person",
"name": "Tomás Lopes da Fonseca"
},
{
"@type": "Person",
"name": "Jan C. Koch"
},
{
"@type": "Person",
"name": "Stefan Becker"
},
{
"@type": "Person",
"name": "Lars Tönges"
},
{
"@type": "Person",
"name": "Mathias Bähr"
},
{
"@type": "Person",
"name": "Tiago F. Outeiro"
},
{
"@type": "Person",
"name": "Markus Zweckstetter"
},
{
"@type": "Person",
"name": "Paul Lingor"
}
],
"identifier": "10.1186/s40478-016-0310-y"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease methods",
"item": "https://replicatescience.com/experiments/fasudil-attenuates-aggregation-of-945-synuclein-in-models-of-parkinson-s-disease-methods-lars-tatenhorst-pmc4840958/fasudil-attenuates-aggregation-of-synuclein-in-models-of-parkinson-s-disease-mlph8opg"
}
]
}
]