Hypothesis

Female mammals exhibit a longevity advantage because a subset of X‑linked OGT alleles escapes inactivation, generating mosaic populations of cells with differing OGT dosage. This mosaicism produces heterogeneous O‑GlcNAcylation levels that buffer against the uniform hexosamine biosynthetic pathway (HBP) flux dysregulation driving age‑related proteotoxic stress. In contrast, males (hemizygous) and females with skewed X‑inactivation lack this buffering, leading to earlier proteostasis collapse and reduced lifespan.

Mechanistic Basis

OGT, encoded on the X chromosome, modulates proteasome and autophagy activity and regulates aggregation‑prone proteins such as tau and polyglutamine expansions (3). HBP flux elevation— independent of hyperglycemia— increases O‑GlcNAcylation, disrupting proteostasis and promoting aggregation in tissues vulnerable to aging (2). If a proportion of OGT alleles escapes inactivation (~15‑30% of X‑linked genes in humans), females would possess two intermingled cell cohorts: one with near‑wild‑type OGT activity (lower O‑GlcNAcylation, enhanced clearance) and another with reduced OGT activity (higher O‑GlcNAcylation, potentially signaling stress responses). This creates a bet‑hedging scenario where the low‑OGT cells can clear aggregates while the high‑OGT cells activate adaptive stress pathways, collectively attenuating damage accumulation.

Testable Predictions

1. In genetically heterogeneous female mice, cells expressing the active maternal versus paternal X‑chromosome will show inversely correlated OGT protein levels and O‑GlcNAcylation signatures.
2. Tissue‑specific loss of X‑inactivation escape (e.g., via Xist deletion) will reduce OGT mosaicism and accelerate age‑related proteotoxicity and shorten lifespan in females to male‑like levels.
3. Increasing OGT dosage uniformly in males (transgenic overexpression) will not recapitulate the protective effect seen in heterozygous females, because uniform elevation lacks the protective low‑OGT subpopulation.
4. Pharmacological modulation of HBP flux (e.g., with GFAT inhibitors) will rescue proteostasis defects more effectively in female mice with intact OGT mosaicism than in those with forced uniform OGT expression.

Experimental Approach

• Generate a reporter mouse line where OGT expression is coupled to a fluorescent tag on each X allele (e.g., maternal X‑GFP, paternal X‑mCherry). Use flow cytometry and immunofluorescence to quantify OGT mosaicism in liver, brain, and muscle across ages.
• Cross this line with an inducible Xist‑knockout allele to abolish escape in adult tissues; monitor OGT distribution, O‑GlcNAcylation (RL2 blot), ubiquitin‑proteasome activity, and insoluble protein aggregates over time.
• Perform longitudinal survival analysis on four cohorts: (1) wild‑type females, (2) Xist‑KO females, (3) wild‑type males, (4) males with OGT heterozygous knockdown (to mimic hemizygous deficiency).
• Apply a low‑dose GFAT inhibitor (AZD‑6482) to a subset of each cohort and assess proteostatic readouts and lifespan extension.

Potential Confounds and Controls

Control for hormonal influences by gonadectomizing subsets and supplementing with physiologic hormone levels; verify that observed lifespan differences persist independent of gonadal status. Ensure that Xist deletion does not cause global transcriptional dysregulation unrelated to OGT by conducting RNA‑seq on sorted high‑ and low‑OGT populations.

If OGT mosaicism is causal, disrupting it should erase the female survival advantage, while preserving it should confer resilience even in males with matched OGT total dosage. This directly tests whether the X chromosome’s role in longevity stems from escape‑driven gene dosage rather than its sex‑determination function.