Hypothesis

In aged non‑immune tissues, chronic interferon signaling redirects STAT1/2 from the cytosol to the nucleus, where they become sequestered by enhanced importin‑α5–mediated nuclear import and reduced export. This nuclear retention sustains basal ISG transcription but depletes the phosphorylatable cytosolic STAT pool, so acute IFN fails to generate a robust pSTAT peak. The state is reversible by blocking nuclear import or boosting cytosolic phosphatase activity.

Mechanistic Basis

Chronic low‑grade IFN from transposable element derepression [1] maintains low‑level JAK‑STAT activity. Persistent signaling upregulates importin‑α5 (KPNA5) and downregulates exportin‑1 (XPO1), shifting the equilibrium toward nuclear STAT accumulation [2]. Nuclear STATs are preferentially dephosphorylated by TC‑PTP, creating a pool of unphosphorylated, DNA‑bound STATs that drive basal ISG expression without requiring fresh phosphorylation [3]. Consequently, upon an acute IFN challenge, the limited cytosolic STAT reservoir cannot be rapidly phosphorylated, producing blunted pSTAT kinetics while basal ISGs remain high [4].

Testable Predictions

1. Aged liver, muscle, and hippocampus will show a higher nuclear‑to‑cytosolic ratio of total STAT1/2 compared with young counterparts, irrespective of IFN stimulation.
2. Acute IFN‑β stimulation will elicit a lower peak and delayed kinetics of pSTAT1/Y701 in the cytosol of aged tissues, while nuclear pSTAT1 levels will already be elevated at baseline.
3. Pharmacological inhibition of importin‑α5 (e.g., with ivermectin) or genetic knockdown of KPNA5 in aged mice will restore the cytosolic STAT pool and rescue acute pSTAT responses without diminishing basal ISG expression.
4. Overexpression of the nuclear phosphatase TC‑PTP in young tissues will phenocopy the IFN‑aging signature (high basal ISG, blunted acute pSTAT), whereas its knockdown in aged tissues will ameliorate the defect.

Experimental Approach

• Isolate primary hepatocytes, myocytes, and hippocampal neurons from young (3 mo) and aged (24 mo) mice.
• Perform subcellular fractionation followed by Western blot for total STAT1/2 and pSTAT1/Y701 at 0, 5, 15, 30, 60 min after IFN‑β (100 U ml⁻¹) treatment.
• Quantify nuclear/cytosolic ratios using densitometry; confirm purity with lamin B1 and GAPDH controls.
• Use siRNA or AAV‑mediated shRNA to knock down KPNA5 in aged tissues; assess rescue of pSTAT kinetics and ISG induction (e.g., ISG15, MX1) by qPCR.
• Treat young cells with TC‑PTP overexpression vectors; measure basal ISG and acute pSTAT response.
• Include controls: JAK inhibitor (ruxolitinib) to verify pathway dependence, and USP18 overexpression to distinguish feedback effects.

If nuclear STAT sequestration is central, blocking import will normalize acute pSTAT while leaving basal ISG unchanged; conversely, enhancing nuclear export or reducing TC‑PTP will produce the same effect. Failure to observe these changes would falsify the hypothesis and point toward alternative mechanisms such as irreversible receptor degradation or metabolic limitation.

This framework directly addresses the field’s gap in quantitative STAT phosphorylation kinetics and offers a reversible, mechanistically distinct explanation for the IFN-aging phenotype beyond simple SOCS/USP18‑mediated tachyphylaxis.