Hypothesis

Senescent mesenchymal cells in aged male mouse livers upregulate Wif1 and Smoc1, enzymes that remodel extracellular matrix and modulate Wnt signaling [1]. We propose that this matrix remodeling changes the binding affinity of heparan sulfate proteoglycans for chemokines such as CXCL10, thereby altering systemic chemokine gradients that guide immune cell trafficking. In aged brains, CXCL10‑dependent CD8⁺ T cell accumulation around demyelinated axons is observed [2], but the source of the chemokine gradient is unclear. We hypothesize that soluble factors released from liver senescent niches travel through the circulation, reprogramming the extracellular matrix of cerebral vasculature and white matter, which stabilizes CXCL10 at perivascular sites and sustains T‑cell recruitment.

Testable predictions

1. Reducing Wif1/Smoc1 activity in liver senescent mesenchymal cells will normalize hepatic matrix composition and decrease circulating matrix‑modulating activity.
2. This normalization will flatten CXCL10 gradients in aged brain white matter, leading to fewer infiltrating CD8⁺ T cells.
3. Conversely, overexpression of Wif1/Smoc1 in young liver mesenchymal cells will induce an aged‑like CXCL10 gradient and increase brain T‑cell infiltration.

Experimental approach

Cell isolation – Senescent mesenchymal cells will be harvested from livers of 24‑month‑old male mice using FiNi‑seq‑based enrichment [6].

Genetic manipulation – Two groups will be generated: (a) control (wild‑type) and (b) CRISPR‑mediated knockdown of Wif1 and Smoc1. Validation will be by qPCR and western blot.

Conditioned media assay – Organotypic brain slices from aged mice will be exposed to conditioned media from each group for 48 h. CXCL10 distribution will be mapped by MERFISH (single‑cell resolution, ~100 nm) [4] and quantified alongside immunofluorescence for CD8⁺ T cells.

In vivo validation – AAV‑delivered shRNA targeting Wif1/Smoc1 under a PDGFRa‑Cre driver will restrict knockdown to hepatic mesenchymal cells. After 4 weeks, brains will be processed for MERFISH‑based CXCL10 mapping and flow cytometry for CD8⁺ T cells.

Controls – (i) AAV‑scramble shRNA, (ii) young (3‑month‑old) mice receiving aged liver conditioned media to test sufficiency, (iii) heparinase treatment to probe heparan sulfate dependence.

Readouts – Primary: CXCL10 intensity perivascularly (mean fluorescence intensity) and CD8⁺ T cell density (cells/mm²). Secondary: hepatic hydroxyproline content (fibrosis) and plasma levels of Wnt antagonists.

Falsifiability

If knockdown of Wif1/Smoc1 in liver senescent mesenchymal cells fails to alter brain CXCL10 levels or CD8⁺ T cell infiltration compared with controls, the hypothesis is refuted. A significant reduction (≥30 %) in perivascular CXCL10 signal and T‑cell numbers would support the proposed liver‑brain axis.

Broader impact

Demonstrating a remote organ‑to‑brain axis mediated by senescent‑cell‑driven matrix remodeling would reveal a novel therapeutic avenue: targeting hepatic senescent mesenchymal cells could mitigate neuroinflammatory processes in aging without direct CNS intervention.