Hypothesis

Aged fibroblast‑ and plasma‑derived exosomes cause a pathological over‑consolidation of synaptic networks by delivering hexceramides and TGFB1 that jointly increase postsynaptic membrane rigidity and stabilize AMPA‑receptor complexes, thereby suppressing depotentiation and favoring hyper‑stable prediction models.

Mechanistic rationale

1. Hexceramide enrichment – Senescent cell exosomes carry elevated hexceramides that insert into the plasma membrane, raising lipid order and decreasing lateral diffusion of synaptic proteins (see aging‑us.com article). This biophysical stiffening reduces the mobility of PSD‑95 and associated scaffolding molecules, favoring a locked‑in state.
2. TGFB1‑SMAD signaling – Exosomal TGFB1 activates neuronal SMAD2/3 pathways, leading to transcriptional up‑regulation of PSD‑95 and GluA1 subunits (supported by MSC‑exosome plasticity data, PMC12729007). Increased PSD‑95 density stabilizes surface AMPA receptors, weakening activity‑dependent AMPA‑receptor removal that underlies LTD.
3. Combined effect on plasticity – The rise in membrane order limits the conformational changes needed for AMPA‑receptor endocytosis, while elevated PSD‑95 anchors receptors in the synapse. Consequently, synapses exhibit enhanced LTP‑like strength and impaired LTD, manifesting as reduced reversal learning and heightened perceptual rigidity—behavioral signatures of over‑consolidation.

Testable predictions

• Prediction 1: Applying isolated exosomes from old mice (or old human plasma) to young hippocampal slices will decrease paired‑pulse facilitation (indicating lower release probability) and simultaneously increase the amplitude of evoked fEPSPs after a low‑frequency stimulation protocol that normally induces LTD.
• Prediction 2: Pharmacological inhibition of acid sphingomyelinase (ASM) with desipramine during exosome incubation will rescue LTD magnitude without altering baseline transmission.
• Prediction 3: Genetic knockdown of neuronal TGFBRI in vivo will block the exosome‑induced increase in PSD‑95 puncta and prevent the decline in reversal learning observed in aged‑exosome‑treated mice.

Experimental design

1. Exosome preparation: Isolate exosomes from plasma of 24‑month‑old mice and young controls using ultracentrifugation; quantify hexceramide content by LC‑MS and TGFB1 by ELISA.
2. In vitro slice physiology: Apply exosomes (10 µg ml⁻¹) to acute hippocampal slices from 2‑month‑old mice while recording fEPSPs in CA1. Measure paired‑pulse ratio, LTP (100 Hz train), and LTD (1 Hz 900 pulses) with and without ASM inhibitor.
3. In vivo behavior: Stereotaxically inject aged exosomes into the hippocampus of young mice; assess reversal learning in a water‑maze task after 2 weeks. Cohorts receive ASM inhibitor or neuronal TGFBRI shRNA via AAV.
4. Readouts: Western blot for PSD‑95, p‑SMAD2/3, and GluA1 surface biotinylation; immunostaining for PSD‑95 cluster size; electron microscopy to evaluate postsynaptic density thickness.

Potential outcomes and falsifiability

• If aged exosomes reduce LTD and increase PSD‑95 stability, and these effects are reversed by ASM blockade or TGFBRI knockdown, the hypothesis is supported.
• If exosome application fails to alter LTD or PSD‑95 levels, or if ASM/TGFBRI manipulations do not rescue plasticity, the hypothesis would be falsified, indicating that other cargo or mechanisms dominate age‑related synaptic rigidity.

This framework directly links exosome‑borne lipid and protein signals to a measurable synaptic phenotype, distinguishing over‑consolidation from outright neurodegeneration and offering a clear route for therapeutic intervention targeting membrane ceramide metabolism or TGFB1 signaling.