Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy methods
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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
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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
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- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
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- Relative expression values or fold-change comparisons across groups
Microscopy-based tissue or cell-state imaging
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- Field or section images captured from matched samples
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- Selected representative panels with quantified intensity, counts, or area measurements
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- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
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Acquisition
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inferred from protocolPreprocessing / cleaning
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inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Protein expression by western blot; Microscopy-based tissue or cell-state imaging.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Protein expression by western blot, Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
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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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