Chi3l1 deletion in APP/PS1 mouse model of Alzheimer's disease

Validation Score: 0.950 Price: $0.50 Alzheimer's disease APP/PS1 x Chi3l1 knockout mice Status: proposed

What This Experiment Tests

Validation experiment designed to validate causal mechanisms targeting Chi3l1 in APP/PS1 x Chi3l1 knockout mice. Primary outcome: amyloid plaque burden and microglial phagocytic activity

Description

A comprehensive animal model study using APP/PS1 transgenic mice to investigate the role of Chi3l1 in Alzheimer's disease pathogenesis. Chi3l1 knockout mice were crossed with APP/PS1 mice to examine effects on amyloid plaque burden and microglial phagocytic function. The study measured amyloid plaque load and periplaque expression of CD68, a microglial lysosomal marker. Results demonstrated that Chi3l1 deletion decreased amyloid plaque burden and increased CD68 expression around plaques, suggesting that Chi3l1 normally suppresses glial phagocytic activity.

TARGET GENE

Chi3l1

MODEL SYSTEM

APP/PS1 x Chi3l1 knockout mice

ESTIMATED COST

TIMELINE

0 months

PATHWAY

amyloid clearance and neuroinflammation

SOURCE

extracted_from_pmid_33328329

PRIMARY OUTCOME

amyloid plaque burden and microglial phagocytic activity

Scoring Dimensions

📖 Wiki Pages

CHI3L1 Protein (YKL-40)protein CHI3L1 Genegene APP Processing and Amyloid-Beta Productionmechanism APP-Overexpressing Neuronscell APP→Amyloid-beta→Plaque→Alzheimer's Disease Causalpathway APP-BACE1-Fe65 Complexmechanism APP Genegene APP Swedish Mutation (APPswe)mutation APP — Amyloid Precursor Proteingene CD68 Genegene APP Arctic Mutation (APP Arctic)disease APP Dutch Mutation (APP Dutch)disease APP Flemish Mutation (APP Flemish)disease APP Amyloid Pathway in Alzheimer's Diseasemechanism APP Gene Dosage Reduction Therapy for Down Syndromidea

Protocol

Study Design

Model: APP/PS1 (APPswe/PSEN1dE9) x Chi3l1 knockout (C57BL/6J background)
Animal Numbers: n=12/genotype/group (power=0.80, α=0.05, expected effect size d=1.2)
Sex: Both sexes, balanced per group
Housing: Specific pathogen-free, 12h light/dark cycle

PHASE 1: Colony Establishment & Genotyping (Week 0–4)

Timepoints: 3–4 weeks of age (wean)

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Study Design

Model: APP/PS1 (APPswe/PSEN1dE9) x Chi3l1 knockout (C57BL/6J background)
Animal Numbers: n=12/genotype/group (power=0.80, α=0.05, expected effect size d=1.2)
Sex: Both sexes, balanced per group
Housing: Specific pathogen-free, 12h light/dark cycle

PHASE 1: Colony Establishment & Genotyping (Week 0–4)

Timepoints: 3–4 weeks of age (wean)

Methods:

Cross APP/PS1 hemizygous mice with Chi3l1^+/- breeders on C57BL/6J
Genomic DNA extraction from ear punch: 50mM NaOH, 95°C 30min, neutralization
Genotyping PCR:
APP/PS1: primers 5'-GACTTGATGTTCTGTGTTCTGC-3' / 5'-TGACCACTCGACCAGGTT-3' (300bp mutant band)
Chi3l1 knockout: primers 5'-CTTCCTCGTGCTTTACGGTAT-3' / 5'-CCATGATGAAAGCTGGAAAG-3' (400bp WT, 600bp KO)
Reagents: DreamTaq PCR Master Mix (Thermo), 2% agarose gel, ethidium bromide

Controls:

Positive control: known APP/PS1 and Chi3l1 KO DNA
Negative control: water template
Internal loading control: GAPDH primer pair

PHASE 2: Baseline Behavioral Assessment (Week 8–10)

Timepoints: 2 months of age, pre-intervention

Methods:

Open Field Test: 40cm × 40cm arena, 5min session, automated tracking (Ethovision XT). Measure center time, total distance, velocity.

Y-Maze Spontaneous Alternation: 3-arm maze, 5min session, alternation % as hippocampal-independent working memory indicator.

Nest Building Test: 1g compressed cotton nest, 48h scoring (1–5 scale) as proxy for hippocampus-dependent behavior.

Equipment: Noldus Ethovision XT 16, ANY-maze 7.0

PHASE 3: Experimental Intervention (Week 12–48)

Timepoints: 3, 6, 9 months of age (early, mid, late pathology)

Methods:

APP/PS1;Chi3l1^+/+ (positive group): Standard chow
APP/PS1;Chi3l1^-/- (experimental group): Standard chow
WT;Chi3l1^+/+ (negative control): Standard chow
WT;Chi3l1^-/- (knockout control): Standard chow

Reagents:

Diet: Envigo Teklad 2918 irradiated chow
No pharmacological intervention (genetic deletion only)

Endpoint allocation: 4 mice/sex/genotype at each timepoint (3, 6, 9 months)

PHASE 4: In Vivo Microglial Imaging (Week 11, 23, 35)

Timepoints: 1 week before sacrifice (2, 5, 8 months of age)

Methods:

TPH-1 Microglial Imaging (5 animals/timepoint):

5µg anti-Iba1-FITC (Wako #019-19741) + 50µg anti-P2RY12 (Abcam ab155624) IV
Window chamber implantation over somatosensory cortex
Intravital 2-photon microscopy (Zeiss LSM 7 MP, 910nm Ti:Sapphire)
Parameters: 1024×1024 pixels, 0.5Hz line scan, 60×1.0NA water immersion objective
Measures: microglial process velocity (µm/min), branchpoints/animal, dystrophy score (0–4)

CSF1R-PET (remaining animals, alternate week):

250µCi ^64Cu-DOTA-anti-CSF1R (generated in-house, specific activity 1.2 GBq/µmol)
PET/CT scan at 2h post-injection (Siemens Inveon)
Standardized uptake value (SUV) normalized to muscle

PHASE 5: Tissue Collection & Processing (Week 12, 24, 36)

Timepoints: 3, 6, 9 months of age

Methods:

Perfusion: 4% PFA in PBS, 50mL cardiac perfusion

Brain dissection: left hemisphere for biochemistry, right hemisphere for histology

Cryopreservation: 30% sucrose/PBS overnight, OCT-embedded, 40µm sagittal sections

Tissue allocation:

Biochemical: cortex, hippocampus (flash-frozen in liquid N2, stored -80°C)
Histology: 12 sections per animal (Bregma -0.5 to -3.5mm)

PHASE 6: Biochemical & Histological Analysis (Week 13–38)

6A: Amyloid Burden (ELISA)

Reagents:

Amyloid Beta 40/42 High Sensitivity ELISA Kit (Thermo #010-020)
Sample: hippocampal homogenate in 8M urea/0.4% DEA
Protocol: 100µg protein per well, duplicate, overnight coating with anti-Aβ antibody 6E10

Readout: EnSpire 2300 plate reader, 450nm

6B: Thioflavin-S Amyloid Plaque Staining

Reagents:

Thioflavin-S (Sigma T1892): 0.001% in PBS, 8min, destain 70%/50% EtOH 2min each

Quantification: 5 regions × 3 sections per animal

Cortex (layers 4–6)
Hippocampus (CA1, CA3, dentate gyrus)
Subiculum

Equipment: Zeiss Axio Imager Z2, 10 fields/region, automated particle analysis (ImageJ)

6C: Microglial Phagocytic Assay

Ex vivo assay:

100µg synaptically-enriched fraction from 6-month-old APP/PS1 brains
Incubate with 1×10^6 BV-2 cells (authenticated, mycoplasma-free) + 50µg FITC-labeled Aβ1-42 (rPeptide A-1003-1)
24h incubation at 37°C, 5% CO2

Reagents:

Aβ1-42 fibrils: 2µM in PBS, 37°C 72h pre-aggregation
Phagocytosis inhibitor: Cytochalasin D 10µM (Sigma C8273) as control

Readout:

Flow cytometry (BD FACSCanto II): FITC+ BV-2 cells (phagocytosis %)
Confocal microscopy: Aβ internalization colocalization with LAMP-1 (lysosome marker)

Markers by immunohistochemistry:

Iba1 (1:500, Wako #019-19741)
CD68 (1:200, Abcam ab31636)
YKL-40 (Chi3l1) (1:200, R&D Systems AF2596)
6E10 Aβ (1:1000, BioLegend #803001)

Secondary antibodies: Alexa Fluor 488/594 (1:500, Invitrogen)

Expected Outcomes

Amyloid plaque burden (Thioflavin-S) in APP/PS1;Chi3l1^-/- will be 35–45% lower than APP/PS1;Chi3l1^+/+ at 6 months (expected: 12.4±3.2 vs 20.1±4.1 plaques/mm², p<0.01, Cohen's d=1.4)

Aβ42 concentration (hippocampal ELISA) in APP/PS1;Chi3l1^-/- will be 28–38% reduced at 6 months (expected: 847±156 vs 1182±203 pg/mg protein, p<0.005, d=1.6)

Microglial process velocity (2-photon imaging) will be 55–70% faster in APP/PS1;Chi3l1^-/- vs APP/PS1;Chi3l1^+/+ at 6 months (expected: 2.3±0.4 vs 1.2±0.3 µm/min, p<0.001, d=2.1)

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Success Criteria

Primary: Thioflavine-S plaque burden reduction ≥30% in APP/PS1;Chi3l1^-/- vs APP/PS1;Chi3l1^+/+ at 6 months (two-tailed t-test, p<0.01, effect size d≥1.0)
Secondary: Ex vivo phagocytosis assay fold-change ≥2.0 in BV-2 cells (one-way ANOVA with Tukey post-hoc, F(3,44)=18.7, p<0.0001)
Microglial morphology: Process velocity increase ≥50% (Mann-Whitney U test, p<0.01) and branchpoint count increase ≥40%
Biochemical validation: Hippocampal Aβ42 reduction ≥25% (ELISA, p<0.01 after Bonferroni correction for 3 comparisons)

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Related Hypotheses (5)

TREM2-mediated microglial tau clearance enhancement0.800

Integrated Biomarker Panel for Therapeutic Window Identification0.784

LRP1-Dependent Tau Uptake Disruption0.747

Extracellular Vesicle Biogenesis Modulation0.635

Microglial TREM2 downregulation impairs damage-associated response in late-stage Alzheimer's disease0.628

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