Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer's models methods
Aim. Evidence-backed execution summary for Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer's models methods from Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer's models.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Online Methods
reagent used in the protocol.
- Use
- Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated PKI 14-22, and poly-L-lysine were from Sigma-Aldrich (St. Louis, MO). Culture media, qPCR kits, Alexa-labeled Phalloidin, fluorescent secon...
LTP measurements
reagent used in the protocol.
- Use
- Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, placed on infusion chambers on aCSF (124 mM NaCl, 4.4 mM KCl, 1 mM Na 2 HPO 4, 25 mM NaHCO 3, 2 mM MgCl 2, 2mM CaCl 2, 10 mM glucose) and all...
Mature hippocampal cultures
reagent used in the protocol.
- Use
- Primary rat hippocampal neuronal cultures were maintained in Neurobasal medium supplemented with B27 (Life Technologies, CA), glutamine, and antibiotics according to established procedures,,, and were used after 18-21 days in vitro. Cultures were exposed to 500 nM Aβ oligomers or an equivalent volume of veh...
RNA extraction and quantitative RT-PCR
reagent used in the protocol.
- Use
- Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions. RNA from human brain tissue was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Purity and integrity of RNA preparations were checked by the 260/280 nm absor...
Extended Data
reagent used in the protocol.
- Use
- (a) Antibody blocking assay. Previous incubation of anti-FNDC5 (Abcam; ab131390) with increasing molar ratios of recombinant irisin (Adipogen; AG-40B-0136) reduces the signal of recombinant irisin detected by immunoblotting. The experiment was repeated 4 times with similar results. (b) Left panel: Immunoblot of mous...
Online Methods
Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated PKI 14-22, and poly-L-lysine were from Sigma-Aldrich (St. Louis, MO). Culture media, qPCR kits, Alexa-labeled Phalloidin, fluorescent secon...
- Use
- Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated PKI 14-22, and poly-L-lysine were from Sigma-Aldrich (St. Louis, MO). Culture media, qPCR kits, Alexa-labeled Phalloidin, fluorescent secon...
Brain infusions in mice
For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwell, MA) and were gently restrained only during the injection procedure, as described,. A 2.5 mm-long needle was unilaterally inserted 1 mm...
- Use
- For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwell, MA) and were gently restrained only during the injection procedure, as described,. A 2.5 mm-long needle was unilaterally inserted 1 mm...
RNA extraction and quantitative RT-PCR
Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions. RNA from human brain tissue was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Purity and integrity of RNA preparations were checked by the 260/280 nm absor...
- Use
- Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions. RNA from human brain tissue was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Purity and integrity of RNA preparations were checked by the 260/280 nm absor...
Study design and approval
Sample size for each experiment was estimated by performing pilot studies and by previous experience with different experimental approaches. No algorithm or software was used to randomize animal subjects. Animal subjects were assigned to experimental groups by the researcher. Experiments involving irisin detection i...
- Use
- Sample size for each experiment was estimated by performing pilot studies and by previous experience with different experimental approaches. No algorithm or software was used to randomize animal subjects. Animal subjects were assigned to experimental groups by the researcher. Experiments involving irisin detection i...
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Animals
Male C57BL/6 or Swiss mice were obtained from the animal facility at Federal University of Rio de Janeiro and were 2.5-3 month-old at the beginning of experiments. Male and female APPswe/PS1 ΔE9 mice on a C57BL/6 background were originally obtained from The Jackson Laboratories ( http://research.jax.org/repository/alzheimers.html ) and bred at our animal facility. Male and female double transgenic mice (APP/PS1 M146L) were obtained by crossing Tg2576 mice harboring mutant human APP (K670M:N671L) with mutant PS1 (M146L) mice. Wild-type littermates were used as controls. All animals had their genotypes confirmed prior to use. Animals were housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle, with controlled room temperature and humidity. Experiments using C57BL/6 mice are presented in the main text, while biochemical and behavioral ex...
Brain infusions in mice
For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwell, MA) and were gently restrained only during the injection procedure, as described,. A 2.5 mm-long needle was unilaterally inserted 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eye,,,. 10 pmol AβOs (or vehicle) was injected in a final volume of 3 µL and the needle was kept in place for an additional 30 seconds to prevent backflow. Mice that showed signs of misplaced injections or any sign of hemorrhage were excluded from further analysis. Recombinant irisin (75 pmol per site) was bilaterally delivered into the hippocampal CA1 region (stereotaxical coordinates relative to Bregma: 2.0 mm AP, ± 1.5 mm ML,...
LTP measurements
Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, placed on infusion chambers on aCSF (124 mM NaCl, 4.4 mM KCl, 1 mM Na 2 HPO 4, 25 mM NaHCO 3, 2 mM MgCl 2, 2mM CaCl 2, 10 mM glucose) and allowed to recover for 90-120 min. For experiments with recombinant irisin and/or AβOs in slices from WT mice, AβOs (200 nM) and/or recombinant irisin (25 nM) were diluted in aCSF and perfused for 20 min before stimulus. For experiments with exercised mice that received anti-FNDC5 (or not), animals were exercised for three sessions and received an i.p. injection of anti-FNDC5 antibody (or non-specific IgG) after each session. An additional anti-FNDC5 injection was performed before slice preparation and perfusion with AβOs or vehicle. For experiments with adenovi...
Mature hippocampal cultures
Primary rat hippocampal neuronal cultures were maintained in Neurobasal medium supplemented with B27 (Life Technologies, CA), glutamine, and antibiotics according to established procedures,,, and were used after 18-21 days in vitro. Cultures were exposed to 500 nM Aβ oligomers or an equivalent volume of vehicle (2% DMSO in PBS) for the time intervals indicated in each experiment. When present, lentiviral vectors targeting FNDC5 (10 6 /mL) were allowed to express for five days before cells were processed for immunocytochemistry. When present, recombinant irisin (25 nM) or forskolin (10 µM) was added to cultures 15 min before AβOs, and myristoylated PKI 14-22 (1 µM) was added 40 minutes before irisin.
Measurement outputs
What raw and processed outputs should exist?
Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
For Western blotting analyses, brain samples from non-cognitively impaired, early or late AD subjects (defined by combined pathological and cognitive assessment) were obtained f...
- 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
For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwel...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, place...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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
Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated...; For Western blotting analyses, brain samples from non-cognitively impaired, early or late AD subjects (defined by combined pathological and cognitive assessment) were obtained f...; For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwel...; Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, place....
from paperStatistical comparison
Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions. RNA from human brain tissue was e...
from paperReporting output
Report representative outputs alongside summary comparisons for Aβ 1-42 was from American Peptide (Sunnyvale, CA) or Anaspec (Fremont, CA). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), DMSO, anti-ATF4 antibody (#WH0000468M1), myristoylated..., For Western blotting analyses, brain samples from non-cognitively impaired, early or late AD subjects (defined by combined pathological and cognitive assessment) were obtained f..., For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwel..., Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, place....
inferred from protocolStructured statistical methods
Total RNA was extracted from cultures or animal tissues using SV Total RNA Isolation System (Promega, CA), following manufacturer instructions. RNA from human brain tissue was e...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
Male C57BL/6 or Swiss mice were obtained from the animal facility at Federal University of Rio de Janeiro and were 2.5-3 month-old at the beginning of experiments. Male and female APPswe/PS1 ΔE9 mice on a C57BL/6 background were originally obtained from The Jackson Laboratories ( http://research.jax.org/repository/alzheimers.html ) and bred at our animal facility. Male and female double transgenic mice (APP/PS1 M146L) were obtained by crossing Tg2576 mice harboring mutant human APP (K670M:N671L) with mutant PS1 (M146L) mice. Wild-type littermates were used as controls. All animals had their genotypes confirmed prior to use. Animals were housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle, with controlled room temperature and humidity. Experiments using C57BL/6 mice are presented in the main text, while biochemical and behavioral experiments in Swiss mice performed to validate and confirm our findings are presented as Extended Data Figures. All procedures followed the "Principles of Laboratory Animal Care" from the US National Institutes of Health.
For intracerebroventricular (i.c.v.) infusion of AβOs, mice were anesthetized with 2.5% isoflurane (Cristália; São Paulo, Brazil) using a vaporizer system (Norwell, MA) and were gently restrained only during the injection procedure, as described,. A 2.5 mm-long needle was unilaterally inserted 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eye,,,. 10 pmol AβOs (or vehicle) was injected in a final volume of 3 µL and the needle was kept in place for an additional 30 seconds to prevent backflow. Mice that showed signs of misplaced injections or any sign of hemorrhage were excluded from further analysis. Recombinant irisin (75 pmol per site) was bilaterally delivered into the hippocampal CA1 region (stereotaxical coordinates relative to Bregma: 2.0 mm AP, ± 1.5 mm ML, 1.5 mm DV), immediately before AβO injections. Adenoviral particles (1 × 10 8 ) harboring GFP (AdGFP) or FNDC5 (AdFNDC5) constructs were stereotaxically injected into the right lateral ventricle of WT C57BL/6 or APP/PS1 mice (coordinates relative to Bregma: 0.2 mm AP; 1.0 ML; 2.4 mm DV). Inj...
Mice were euthanized by cervical dislocation followed by decapitation, and their hippocampi were immediately removed. Transverse hippocampal slices (400 µm) were cut, placed on infusion chambers on aCSF (124 mM NaCl, 4.4 mM KCl, 1 mM Na 2 HPO 4, 25 mM NaHCO 3, 2 mM MgCl 2, 2mM CaCl 2, 10 mM glucose) and allowed to recover for 90-120 min. For experiments with recombinant irisin and/or AβOs in slices from WT mice, AβOs (200 nM) and/or recombinant irisin (25 nM) were diluted in aCSF and perfused for 20 min before stimulus. For experiments with exercised mice that received anti-FNDC5 (or not), animals were exercised for three sessions and received an i.p. injection of anti-FNDC5 antibody (or non-specific IgG) after each session. An additional anti-FNDC5 injection was performed before slice preparation and perfusion with AβOs or vehicle. For experiments with adenovirus-mediated FNDC5 expression, WT or APP/PS1 ΔE9 mice were i.c.v.-injected with AdGFP (control) or AdFNDC5 14 days prior to euthanasia and slice preparation. Field EPSPs were recorded from hippocampal CA1 region according to established procedures,.
Primary rat hippocampal neuronal cultures were maintained in Neurobasal medium supplemented with B27 (Life Technologies, CA), glutamine, and antibiotics according to established procedures,,, and were used after 18-21 days in vitro. Cultures were exposed to 500 nM Aβ oligomers or an equivalent volume of vehicle (2% DMSO in PBS) for the time intervals indicated in each experiment. When present, lentiviral vectors targeting FNDC5 (10 6 /mL) were allowed to express for five days before cells were processed for immunocytochemistry. When present, recombinant irisin (25 nM) or forskolin (10 µM) was added to cultures 15 min before AβOs, and myristoylated PKI 14-22 (1 µM) was added 40 minutes before irisin.
Machine-readable layer
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