Hypothesis

Aged white matter fibers experience increased mechanical stiffness due to myelin loss and extracellular matrix remodeling. This altered biomechanical environment activates integrin‑β1 signaling in microglia, driving a transition to a white‑matter‑associated microglia (WAM) and disease‑associated microglia (DAM2) state that amplifies inflammatory and senescence programs. Because estrogen modulates integrin‑β1 expression and downstream FAK/Src signaling, female mice exhibit a stronger microglial response, accounting for the observed sex bias.

Testable Predictions

1. Inhibition of integrin‑β1 in microglia will blunt the WAM/DAM2 transcriptional gradient in aged white matter.

   • Experiment: Cross Cx3cr1‑CreER mice with Itgb1^fl/fl mice, administer tamoxifen at 100 weeks of age, and perform spatial transcriptomics (Visium) on corpus callosum at 126 weeks. Expect a significant reduction (>30 %) of LGALS3, AIF1, TREM2, and senescence panel genes compared with Itgb1^+/+ controls.

2. Pharmacological suppression of FAK/Src signaling will recapitulate the genetic inhibition phenotype.

   • Experiment: Treat aged wild‑type mice with the FAK inhibitor PF‑573228 (10 mg/kg i.p., three times weekly for 4 weeks). Assess microglial activation via immunofluorescence (IBA1/LGALS3) and myelin integrity (luxol fast blue). Predict decreased microglial nodular density and preserved myelin thickness relative to vehicle.

3. Estrogen deficiency will exaggerate the integrin‑β1‑dependent microglial response in males, while estrogen replacement will attenuate it in females.

   • Experiment: Orchidectomize male mice at 80 weeks and supplement with vehicle or estradiol; ovariectomize female mice at 80 weeks with similar treatments. After 8 weeks, measure WAM/DAM2 signatures. Expect orchidectomy‑induced increase (mimicking female phenotype) and ovariectomy‑induced decrease (mimicking male baseline).

4. Rescuing matrix stiffness normalizes microglial activation independent of integrin‑β1.

   • Experiment: Inject hyaluronidase‑low molecular weight into the corpus callosum of aged mice to reduce tissue stiffness. Assess whether microglial gradient is attenuated even when integrin‑β1 remains intact, indicating that stiffness is the upstream cue.

Mechanistic Rationale

The Visium data show tight spatial coupling of microglial activation markers (LGALS3, AIF1) with senescence signatures and myelin loss in fiber tracts. Mechanical strain is known to activate integrin‑β1, triggering FAK/Src‑dependent NF‑κB and STAT3 pathways that promote pro‑inflammatory phenotypes and senescence-associated secretory phenotype (SASP). Estrogen receptors can bind integrin‑β1 subunits and modulate their affinity for ligands, providing a plausible molecular basis for sex‑specific differences. By positioning integrin‑β1 as a mechanosensor that translates altered white‑matter mechanics into a transcriptional program, we link the observed transcriptomic gradients to a physical driver rather than solely to soluble cytokine cascades.

Falsifiability

If integrin‑β1 loss or FAK/Src inhibition fails to reduce WAM/DAM2 gene expression or myelin preservation in aged mice, the hypothesis would be refuted. Likewise, if manipulating estrogen levels does not shift the microglial response as predicted, the sex‑specific mechanism would be unsupported. Positive outcomes, however, would support a novel biomechanical axis of brain aging that can be targeted therapeutically.