Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy methods
Aim. Evidence-backed execution summary for Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy methods from Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- We used phosphorylation-independent anti-tau antibodies BR133 and BR134 which recognize both murine and human tau isoforms. Phosphorylation-dependent anti-tau antibodies AT8 and AT100 (Innogenetics) were also used. AT8 recognizes tau protein phosphorylated at S202 and T205 ( ), whereas AT100 detects tau phosphorylat...
Tissue extraction
reagent used in the protocol.
- Use
- To extract sarkosyl-insoluble proteins from brain and spinal cord, frozen tissues were weighed and homogenized in cold extraction buffer [10 mM Tris-HCl, pH 7.4, 800 mM NaCl, 1 mM EGTA, 0.1 M phenylmethylsulphonyl fluoride, 10% sucrose and one tablet of complete protease inhibitor cocktail (R...
Immunoblot analysis
reagent used in the protocol.
- Use
- After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room temperature in 0.1 M phosphate buffer, pH 7.4, 0.2% Tween 20 and 5% milk, followed by an overnight incubation with the primary antibody...
Immunohistochemistry
reagent used in the protocol.
- Use
- Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryoprotection in PBS containing 30% sucrose for at least 24 h. Serial sagittal brain sections (30 µm) were cut...
Effects of trehalose on tau pathology and neuronal survival
reagent used in the protocol.
- Use
- Total soluble tau and soluble tau phosphorylated at the AT8 epitope were not affected by trehalose treatment ( Supplementary Fig. 2 ). In contrast, quantification of sarkosyl-insoluble tau in the brain indicated that trehalose decreased the amount of insoluble tau compared with untreated controls (-53%, P R...
Animals and treatment
Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per group for immunohistochemistry and stereology experiments). From weaning onwards, the animals were treated with 2% trehalose or 2% sucrose,...
- Use
- Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per group for immunohistochemistry and stereology experiments). From weaning onwards, the animals were treated with 2% trehalose or 2% sucrose,...
Quantification of proteins
Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble tau was normalized relative to the tissue weight. Data were expressed as means ± SEM. Statistical analysis was performed usi...
- Use
- Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble tau was normalized relative to the tissue weight. Data were expressed as means ± SEM. Statistical analysis was performed usi...
Immunohistochemistry
Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryoprotection in PBS containing 30% sucrose for at least 24 h. Serial sagittal brain sections (30 µm) were cut...
- Use
- Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryoprotection in PBS containing 30% sucrose for at least 24 h. Serial sagittal brain sections (30 µm) were cut...
Stereology
The Stereo Investigator 9 (MBF Bioscience) was used to quantify the number of NeuN-positive cells. Sections through the brain were sampled in a systematic random manner using 1:12 series for quantification of NeuN-positive and AT100-positive neurons. The thickness of each section was determined using the Stereo Inve...
- Use
- The Stereo Investigator 9 (MBF Bioscience) was used to quantify the number of NeuN-positive cells. Sections through the brain were sampled in a systematic random manner using 1:12 series for quantification of NeuN-positive and AT100-positive neurons. The thickness of each section was determined using the Stereo Inve...
Quantification of proteins
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble tau was normalized relative to the tissue weight. Data were expressed as means ± SEM. Statistical analysis was performed usi...
Stereology
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The Stereo Investigator 9 (MBF Bioscience) was used to quantify the number of NeuN-positive cells. Sections through the brain were sampled in a systematic random manner using 1:12 series for quantification of NeuN-positive and AT100-positive neurons. The thickness of each section was determined using the Stereo Inve...
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Animals and treatment
Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per group for immunohistochemistry and stereology experiments). From weaning onwards, the animals were treated with 2% trehalose or 2% sucrose, which were used as the control. A separate group received normal drinking water (no treatment). The water solutions were changed twice weekly. Animals were checked daily and the quantity of water drunk and the animals' weights were assessed twice a week. P301S tau transgenic mice develop motor deficits at ∼3-4 months of age, as shown using Rotarod ( ). We routinely evaluated these deficits by visual inspection, as they form in a stereotypical manner consisting of an unstable gait followed by general stiffness and hindlimb paralysis. Furthermore, at 3-...
Tissue extraction
To extract sarkosyl-insoluble proteins from brain and spinal cord, frozen tissues were weighed and homogenized in cold extraction buffer [10 mM Tris-HCl, pH 7.4, 800 mM NaCl, 1 mM EGTA, 0.1 M phenylmethylsulphonyl fluoride, 10% sucrose and one tablet of complete protease inhibitor cocktail (Roche)]. The homogenates were spun at 6000 g for 20 min and the supernatants incubated with 1% sarkosyl for 1 h at room temperature. After a 1 h centrifugation at 80 000 g, the supernatants were discarded and the pellets resuspended in 50 mM Tris-HCl, pH 7.4 (300 µl/g tissue). For investigation of soluble tau, brains and spinal cords were homogenized in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride and one tablet of complete protease inh...
Immunoblot analysis
After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room temperature in 0.1 M phosphate buffer, pH 7.4, 0.2% Tween 20 and 5% milk, followed by an overnight incubation with the primary antibody in blocking buffer. Membranes were then washed in 0.1 M phosphate buffer, pH 7.4, 0.2% Tween 20 and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody (Pierce) in blocking solution. After washing, the blots were developed using enhanced chemiluminescence (Amersham Biosciences).
Immunohistochemistry
Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryoprotection in PBS containing 30% sucrose for at least 24 h. Serial sagittal brain sections (30 µm) were cut on a Leica SM2400 microtome (Leica Microsystems) and stored at 4°C in PBS containing 0.1% sodium azide. For fluorescence labelling, sections were permeabilized for 5 min with cold PBS containing 0.5% Triton X-100 and washed three times with PBS, 0.1% Triton X-100 (PBST). After 2 h blocking at room temperature with PBST containing 3% bovine serum albumin, sections were incubated with the primary antibodies in blocking solution for 24 h at 4°C. This was followed by three washes and a 2 h incubation at room temperature with Alexa Fluor®...
Measurement outputs
What raw and processed outputs should exist?
Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per...
- 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
After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room te...
- 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
Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble t...
- 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
To investigate autophagy activation, we measured the conversion of LC3-I into LC3-II, a marker of autophagic vacuole formation (;; ). We observed the conversion of 17.5...
- 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
Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per...; After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room te...; Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble t...; To investigate autophagy activation, we measured the conversion of LC3-I into LC3-II, a marker of autophagic vacuole formation (;; ). We observed the conversion of 17.5....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble t...; The Stereo Investigator 9 (MBF Bioscience) was used to quantify the number of NeuN-positive cells. Sections through the brain were sampled in a systematic random manner using 1:...; Immunohistochemistry revealed the co-localization of p62/SQSTM1 and AT100-positive tau aggregates in P301S tau transgenic mice ( D-F), while no co-staining was observed in...
from paperReporting output
Report representative outputs alongside summary comparisons for Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per..., After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room te..., Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble t..., To investigate autophagy activation, we measured the conversion of LC3-I into LC3-II, a marker of autophagic vacuole formation (;; ). We observed the conversion of 17.5....
inferred from protocolStructured statistical methods
Band intensities were quantified (Chemidoc™ XRS and Quantity One® software, Bio-Rad) and the amounts of soluble proteins normalized with respect to GAPDH. Insoluble t...; The Stereo Investigator 9 (MBF Bioscience) was used to quantify the number of NeuN-positive cells. Sections through the brain were sampled in a systematic random manner using 1:...; Immunohistochemistry revealed the co-localization of p62/SQSTM1 and AT100-positive tau aggregates in P301S tau transgenic mice ( D-F), while no co-staining was observed in...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
Homozygous human P301S tau transgenic mice ( ) and age-matched wild-type C57BL/6 mice were used ( n = 6 per group for western blot analysis; n = 3 per group for immunohistochemistry and stereology experiments). From weaning onwards, the animals were treated with 2% trehalose or 2% sucrose, which were used as the control. A separate group received normal drinking water (no treatment). The water solutions were changed twice weekly. Animals were checked daily and the quantity of water drunk and the animals' weights were assessed twice a week. P301S tau transgenic mice develop motor deficits at ∼3-4 months of age, as shown using Rotarod ( ). We routinely evaluated these deficits by visual inspection, as they form in a stereotypical manner consisting of an unstable gait followed by general stiffness and hindlimb paralysis. Furthermore, at 3-4 months of age, the human P301S tau mice are unable to extend their hindlimbs when lifted by the tail ( ). In this study, we assessed the motor deficits by visual inspection and used the time when all the animals had developed hindleg stiffness and were unable to extend their hindlimbs when suspen...
To extract sarkosyl-insoluble proteins from brain and spinal cord, frozen tissues were weighed and homogenized in cold extraction buffer [10 mM Tris-HCl, pH 7.4, 800 mM NaCl, 1 mM EGTA, 0.1 M phenylmethylsulphonyl fluoride, 10% sucrose and one tablet of complete protease inhibitor cocktail (Roche)]. The homogenates were spun at 6000 g for 20 min and the supernatants incubated with 1% sarkosyl for 1 h at room temperature. After a 1 h centrifugation at 80 000 g, the supernatants were discarded and the pellets resuspended in 50 mM Tris-HCl, pH 7.4 (300 µl/g tissue). For investigation of soluble tau, brains and spinal cords were homogenized in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride and one tablet of complete protease inhibitor cocktail. Samples were centrifuged at 80 000 g for 15 min. The resulting supernatant constituted the soluble fraction. The samples were analysed by SDS-PAGE.
After SDS-PAGE, the gels were blotted for 1 h at room temperature onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h at room temperature in 0.1 M phosphate buffer, pH 7.4, 0.2% Tween 20 and 5% milk, followed by an overnight incubation with the primary antibody in blocking buffer. Membranes were then washed in 0.1 M phosphate buffer, pH 7.4, 0.2% Tween 20 and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody (Pierce) in blocking solution. After washing, the blots were developed using enhanced chemiluminescence (Amersham Biosciences).
Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryoprotection in PBS containing 30% sucrose for at least 24 h. Serial sagittal brain sections (30 µm) were cut on a Leica SM2400 microtome (Leica Microsystems) and stored at 4°C in PBS containing 0.1% sodium azide. For fluorescence labelling, sections were permeabilized for 5 min with cold PBS containing 0.5% Triton X-100 and washed three times with PBS, 0.1% Triton X-100 (PBST). After 2 h blocking at room temperature with PBST containing 3% bovine serum albumin, sections were incubated with the primary antibodies in blocking solution for 24 h at 4°C. This was followed by three washes and a 2 h incubation at room temperature with Alexa Fluor® 488 (Molecular Probes) or Cy5 (Abcam) secondary antibodies in blocking solution. After washing and DAPI (4,6-diamidino-2-phenylindole) staining, the sections were mounted using Vectashield mounting medium (Vector Laboratories). They were analysed using a Leica DMRB fluorescence microscope (Leica) or...
Machine-readable layer
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