IF a temporally sequenced "Prime-then-Purge" protocol — specifically, ambroxol (oral, ~100 mg/kg/day for 4 weeks, a BBB-penetrant approved GCase chaperone), co-administered with trehalose (2% drinking water, mTOR-independent autophagy inducer) as the PRIME phase, followed by a PURGE phase of fisetin (25 mg/kg i.p., 3 days on / 2 weeks off × 2 cycles; a CNS-penetrant senolytic with superior quercetin-analog bioavailability) — is administered to aged (22–24 month) male and female C57BL/6J mice harboring established α-synuclein pathology (induced by stereotactic striatal α-synuclein preformed fibril [PFF] injection at 18 months), representing a post-RMR1 residual pathology model (i.e., animals that have already received D+Q senolytic treatment and show partial senescent cell reduction but persistent lysosomal dysfunction),

THEN compared to senolytic-alone (fisetin without priming), treated animals will show:

• ≥50% reduction in phospho-Ser129–α-synuclein (pSyn) immunoreactivity within GFAP+ and Iba1+ cells in the substantia nigra and striatum (quantified by confocal immunofluorescence and ELISA),
• ≥40% reduction in seeding-competent extracellular α-synuclein in brain interstitial fluid (measured by real-time quaking-induced conversion assay, RT-QuIC),
• Preservation of dopaminergic neuron density (TH+ cells in SN pars compacta, stereological count) at levels ≥25% higher than senolytic-alone controls,
• Reduced post-senolytic extracellular seeding signal (RT-QuIC of conditioned medium from dissociated glia collected 48 hours post-fisetin), confirming that the Prime phase depleted intracellular aggregate cargo before cell death,

BECAUSE the following causal chain is operative:

1. Senescent astrocytes and microglia accumulate phosphorylated, insoluble α-synuclein intracellularly because the senescence program suppresses lysosomal function — including GCase (GBA1) activity — creating an "engulf-but-stall" phenotype in which endocytosed fibrils are not degraded. (Microglia normally clear neuron-released α-synuclein through ATG7-dependent selective autophagy, termed synucleinphagy; when this pathway is compromised in senescent glia, fibrils accumulate)[https://doi.org/10.1038/s41467-020-15119-w].

2. Ambroxol, as a pharmacological chaperone for GCase, restores lysosomal GCase enzymatic activity in senescent glia [SPECULATIVE as to magnitude in senescent-specific glia], reducing glucosylceramide substrate accumulation, relieving lysosomal membrane stress, and permitting re-acidification sufficient to restore cathepsin and GCase-dependent α-synuclein proteolysis. This is supported by the broader mechanistic evidence that GBA1/GCase deficiency impairs autophagy-lysosomal flux specifically in the context of synucleinopathy (GBA1 is cited among PD-associated genes disrupting autophagy-related processes, alongside PINK1, PARKIN, and LRRK2)[https://doi.org/10.1038/s41467-020-15119-w].

3. **Trehalose, acting via mTOR-independent TFEB nucl...

SENS category: LysoSENS

Key references: • doi.org/10.1038/s41467-020-15119-w]. • doi.org/10.1111/bpa.12373]. • doi.org/10.1111/bpa.12373], • doi.org/10.1038/s41467-020-15119-w] • doi.org/10.1111/bpa.12373]