Design Experiments From Reported Results

 Long Analysis Plan Copied

Evidence grounding the design

Niche-restricted TNF receptor deletion is a proven strategy: oligodendroglial TNFR2 conditional knockout accelerated immune infiltration in EAE (onset 14.2 vs 16.7 days; cumulative disease index 37.2 vs 26.2) and raised BBB permeability (Evans blue, p=0.0238, n=10/group), showing cell-specific TNFR2 loss is detectable against a systemic inflammatory challenge Oligodendrocyte TNFR2 in EAE. Receptor biology differs sharply: TNFR1 drives inflammatory/differentiation programs while TNFR2 supports regulatory programs in T cells TNFR1 vs TNFR2 in Treg differentiation, and cell-specific TNFR2 deletion is feasible and effective (CD11c-Cre reduced TNFR2+ CD11c+ cells from ~8.07% to ~0.87%, blunting serum IL-12, TNF-α, IL-23, IL-17A) DC-specific TNFR2 deletion reduces inflammation. Prior hippocampal cell-specific TNFR2 work (NexCreERT2:TNFR2 fl/flox) shows conditional ablation can abolish a TNFR2-dependent phenotype, but sex-dependent effects and RNA-seq done only in males are caveats Neuron-specific TNFR2 conditional KO.

Experimental design

Animals (4 genotypes, both sexes, power n≥10/group for histology): (1) TNFR1 fl/fl × TNFR2 fl/fl (TNFRSF1A/1B floxed) with GFAP-CreERT2 (astrocyte niche); (2) same flox background with PDGFRβ-CreERT2 or NG2-CreERT2 (perivascular niche); (3) floxed Cre-negative littermates (controls); (4) tamoxifen-only Cre-negative controls. Validate recombination with RNAscope for TNFRSF1A/1B on GLAST+/ACSA-2+ astrocytes and PDGFRβ+ pericytes; expect partial CNP-Cre-style efficiency (~90% mature cells, 30-40% progenitors) as a known limitation Conditional deletion efficiency limits.

Challenge: poly I:C 10 mg/kg i.p. daily × 3 days (or poly-ICLC as a clinically stabilized analog used in human trials such as NCT01299662 in healthy volunteers) starting 48 h after tamoxifen washout. Groups (2×3 factorial): genotype {WT-flox control, astrocyte-cKO, perivascular-cKO} × treatment {poly I:C, vehicle}; plus a systemic anti-TNF/TNFR1-blocking antibody arm as positive control, and a Tag7-Hsp70–like TNFR1-only ligand arm is optional given TNFR1-selective agonism is structurally achievable (R1-6 mutant, PDB 2ZJC, >1800× TNFR1 selectivity) TNFR1-selective TNF mutant structure.

Readouts (D1, D3, D7 post-challenge): primary — BrdU/EdU+ Ki-67+ cells and DCX+/PSA-NCAM+ neuroblasts in the dentate gyrus (stereological counts); secondary — Iba1/MHCII microglia, GFAP and vascular TNF mRNA, peripheral cytokines (ELISA), hippocampal bulk + single-cell RNA-seq (DESeq2/Scanpy). Decision rule: if poly I:C suppresses DCX+ cells in Cre-negative controls but not in either cKO line, local TNF signaling is necessary; suppression retained in cKOs implicates systemic (e.g., circulating TNF acting on surviving receptor populations, or IL-6/IFN-β via TLR3 directly). Counterpoint to plan for: TLR3 activation induces type-I IFN and inflammatory cytokines independent of TNF; the positive-control anti-TNF arm is what attributes any rescued neurogenesis specifically to TNF rather than IFN pathways.

Bioinformatics/coding pipeline: Snakemake workflow → FastQC/Cutadapt → STAR+HTSeq or Salmon → DESeq2 (design: ~ genotype + treatment + genotype:treatment + sex, pairwise interaction contrasts astro-cKO×polyI:C and perivascular-cKO×polyI:C) → gseapy (GO neurogenesis, TNF/NF-κB, interferon-α/β Hallmark sets) → Scanpy on 10x data to segment astrocyte/perivascular/V-SVZ and SGZ clusters and test whether poly I:C-induced TNF-response scores (NF-κB target gene module) are lost specifically in recombined cells — the single-cell-level causal check. Report effect sizes with sex-stratified estimates given known sex-divergent TNFR2 signaling Sex-dependent TNFR2 signaling.

Known blindspots: Cre off-targets (GFAP-Cre hits neural stem cells; NG2-Cre hits pericytes broadly), incomplete recombination, and the fact that knock-in hTNFR2 models reveal receptor-specific effects that mouse-only systems may miss TNFR2 targeting translational caveats. What would falsify the hypothesis: cKO lines showing fully preserved poly I:C suppression of DCX+ neurogenesis identical to controls.