Double depletion rescue experiment: tau and MAP6 co-depletion

PRIMARY OUTCOME

morphological phenotypes compared to single depletions

EXPECTED OUTCOMES

- 1. Primary: Complete morphological rescue in tau+MAP6 co-depleted neurons, with dendritic complexity returning to >85% of control values (Cohen's d < 0.3 vs control, p > 0.05) - 2. Secondary: Tau depletion alone causes >40% reduction in dendritic complexity compared to controls (Cohen's d > 0.8, p < 0.001) - 3. Secondary: MAP6 depletion alone causes >35% increase in dendritic complexity compared to controls (Cohen's d > 0.8, p < 0.001) - 4. Mechanistic: Microtubule stability ratio (acetylated/tyrosinated tubulin) in co-depleted neurons returns to within 15% of control values - 5. Rescue quantification: Rescue index >70% for all morphological parameters in tau+MAP6 co-depletion group - 6. Quality control: Knockdown efficiency >70% for both tau and MAP6 maintained throughout experimental period - 7. Negative control: Non-targeting siRNA shows no significant difference from untreated controls for any measured parameter (p > 0.1)

SUCCESS CRITERIA

- • Statistical rescue: Co-depletion group not significantly different from controls (p > 0.05) while both single depletions remain significant (p < 0.001) - • Effect size threshold: Cohen's d < 0.4 between co-depletion and control groups for primary morphological parameters - • Rescue completeness: ≥70% rescue index for at least 4 out of 6 morphological parameters measured - • Data quality: >80% of transfected neurons showing appropriate morphology for analysis, RNA integrity number (RIN) >7.0 - • Reproducibility: Rescue effect demonstrated in ≥3 independent culture preparations with consistent direction - • Knockdown validation: Maintained >70% reduction in both target proteins confirmed by both qPCR and Western blot - • Statistical power: Final sample size achieves >90% power to detect Cohen's d = 0.6 difference between groups

PROTOCOL

**Phase 1: Primary Neuronal Culture Preparation and Transfection** — Days 1-7 Isolate primary cortical neurons from E18 Sprague-Dawley rat embryos using standard papain digestion protocol. Plate neurons at 150,000 cells/well in 24-well plates on poly-L-lysine (0.1 mg/ml, Sigma P2636) coated coverslips in Neurobasal medium (Thermo 21103049) with B27 supplement (Thermo 17504044) and 2mM glutamine. Allow neurons to mature for 5 days in vitro (DIV5) before transfection. Design and validate siRNA sequences: Tau siRNA (5'-GCAAGGAGAUUAAGCAGAA-3', Dharmacon), MAP6 siRNA (5'-GGACAUGCUGAACUACUAU-3', Dharmacon), and non-targeting control siRNA (Dharmacon D-001810-10). Perform transfections using Lipofectamine RNAiMAX (Thermo 13778075) at 50nM final concentration for each siRNA. Establish four experimental groups (n=6 wells each): control siRNA, tau siRNA alone, MAP6 siRNA alone, and tau+MAP6 co-depletion. **Phase 2: Knockdown Validation and Quality Control** — Days 8-10 At 48 hours post-transfection (DIV7), harvest one well from each group for knockdown validation. Extract total RNA using TRIzol reagent (Thermo 15596026) and perform RT-qPCR using TaqMan probes for rat Mapt (Rn00691205_m1), Map6 (Rn01464975_m1), and Gapdh (Rn01775763_g1). Calculate knockdown efficiency using ΔΔCt method with GAPDH normalization. Additionally, perform Western blot validation using anti-tau antibody (Dako A0024, 1:1000) and anti-MAP6 antibody (Novus NBP1-85939, 1:500) with β-actin loading control (Sigma A2228, 1:5000). Only proceed with morphological analysis if knockdown efficiency >70% for both targets. **Phase 3: High-Resolution Morphological Imaging** — Days 10-11 At 72 hours post-transfection (DIV8), fix neurons with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Permeabilize with 0.1% Triton X-100 and block with 10% normal goat serum. Perform immunofluorescence staining using mouse anti-MAP2 (Sigma M4403, 1:800) for dendritic morphology and rabbit anti-βIII-tubulin (Abcam ab18207, 1:1000) for overall neuronal structure. Use Alexa Fluor secondary antibodies (Thermo A11001 and A11008, 1:500). Image neurons using confocal microscopy (63x oil objective, 0.3μm z-steps) with identical acquisition settings across all groups. Acquire minimum 25 neurons per condition from 3 independent culture preparations. **Phase 4: Quantitative Morphological Analysis and Microtubule Assessment** — Days 11-14 Perform comprehensive morphological analysis using ImageJ and Sholl analysis plugin. Measure primary dendrite number, total dendritic length, dendritic complexity (Sholl intersections), and soma area for each neuron. Additionally, assess microtubule stability using immunofluorescence for acetylated α-tubulin (Sigma T7451, 1:800) as a marker of stable microtubules and tyrosinated α-tubulin (Abcam ab6160, 1:500) for dynamic microtubules. Calculate acetylated/tyrosinated tubulin ratio as an index of microtubule stability. Use live-cell imaging with SiR-tubulin (Cytoskeleton CY-SC002, 100nM) to assess microtubule dynamics in a subset of cultures (n=15 neurons per condition) with 2-minute intervals for 30 minutes. **Phase 5: Statistical Analysis and Data Integration** — Days 14-16 Perform power analysis to confirm adequate sample size (β=0.8, α=0.05, expected effect size d=0.8). Use one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons between groups. Apply Bonferroni correction for multiple morphological parameters (adjusted α=0.01). Perform principal component analysis (PCA) to identify patterns in morphological rescue. Calculate rescue index as: [(double depletion value - worst single depletion value) / (control value - worst single depletion value)] × 100%. Statistical analysis using R software with appropriate packages for morphological data analysis.

LINKED HYPOTHESES

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