NOTCH3 Mutation Causes Glymphatic Impairment and Promotes Brain Senescence in CADASIL
NOTCH3 mutation results in development of cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL), the most common monogenic small vessel disease. Up to date, specific treatment against CADASIL is scarce. Here, we report that both glymphatic influx and efflux are impaired in CADASIL mouse models (Notch3R170C), which impedes waste clearance and promotes brain senescence. In accordance, brain atrophy in CADASIL patients is associated with perivascular space enlargement, indicating that glymphatic impairment contributes to advanced brain senescence in CADASIL. The glymphatic malfunction in CADASIL is attributed to diminished AQP4 expression in astrocytic endfeet, which is the core mediator of glymphatic activity. Mechanistically, AQP4 expression is regulated by RUNX1-CMYB signaling whose activation is suppressed as a result of NOTCH3 mutation. Reinforcing AQP4 expression in astrocytes by AAV-based therapy resumes the glymphatic functions in CADASIL mice, which further prevents brain senescence. Therefore, we propose that to improve glymphatic function by reinforcing AQP4 expression is a promising therapeutic strategy in CADASIL. Senescence-associated Migrasomes (SAM) Promote Brain Ageing through Apoptosis Inhibitor of Macrophage (AIM) Aging is a major risk factor for various neurological disorders, including Alzheimer's disease (AD). During aging, senescent cells produce pro-aging factors that disseminate aging signals and reinforce the senescence process. Migrasomes, newly discovered organelles formed during cellular migration, detach from parent cells and mediate intercellular communication. In this study, we identify border-associated macrophages (BAMs) as the primary cells that acquire pro-senescent properties during brain aging, likely due to prolonged exposure to amyloid beta (A?). In pro-senescent BAMs, migrasome production is elevated, conveying pro-aging signals to neighboring cells. Mechanistically, the apoptosis inhibitor of macrophage (AIM) is packaged into migrasomes. Upon activation of CD16 in recipient cells, AIM protects them from apoptosis, thereby inducing senescence. Treatment with Tspan4-siRNA-encapsulated liposomes in 18-month-old mice suppresses migrasome production in pro-senescent BAMs, slows brain aging, and ameliorates cognitive deficits. Our findings suggest that migrasomes are potent carriers of pro-aging signals and represent a promising target for senomorphic therapy. Parenchymal border macrophages (PBMs) reside at the interface between the central nervous system and the periphery and are known to mediate the accessibility of the substances to the brain. Here, we report that amyloid-beta (A?) accumulates along brain blood vessels after stroke in both the ipsilateral and contralateral hemispheres. When PBMs were depleted, glymphatic drainage of A? was markedly reduced and this was accompanied by deterioration of cognitive function, highlighting a critical role for PBMs in post-stroke A? disposal. A possible mechanism relates to mesencephalic astrocyte-derived neurotrophic factor (MANF), which is known to impede glymphatic clearance of A?. MANF derived from PBMs suppressed astrocytic stress and maintained glymphatic drainage when supplemented into the cerebral spinal fluid. In the chronic phase of stroke, MANF production in PBMs was down-regulated and consequently, glymphatic impairments were exacerbated, which lead to on-going A? accumulation and cognitive decline. In summary, supplementation of MANF not only mitigates the adverse impacts of PBM depletion, but also exerts therapeutic effects that improve glymphatic system function. We thus propose that this represents a promising strategy to prevent post-stroke cognitive impairment. Overall design: Brain of NOTCH3-WT or NOTCH3-R170C mice (16w of age) were isolated and digested, myelin was removed, and single cell suspension were prepared for single cell sequencing. 4 samples; Raw data is multiplexed. sample 1: NOTCH3-WT4m, replicate 1, scRNAseq sample 2: NOTCH3-WT4m, replicate 2, scRNAseq sample 3: NOTCH3-R170C, replicate 1, scRNAseq sample 4: NOTCH3-R170C, replicate 2, scRNAseq *** The records have been updated with the additional "WT6m, brain, male" processed data (generated from the GSM8108141 multiplexed raw data) on Jul 30, 2025: scRNA-seq profiling of wildtype (WT) 6-month-old male mice with two biological replicates: WT6m, brain, male, rep1 WT6m, brain, male, rep2 *** The records have been updated with the following data (generated from the GSM8108141 multiplexed raw data) on Sep 24, 2025: Brain of Wildtype, NOTCH3-R170C or transient middle cerebral artery occlusion mice were isolated and digested, myelin was removed, and single cell suspension were prepared for single cell sequencing. 7 samples; Raw data is multiplexed. sample 1: Wildtype 4m, replicate 1, brain, male sample 2: Wildtype 4m, replicate 2, brain, male,q sample 3: NOTCH3-R170C 4m, replicate 1, brain, male sample 4: NOTCH3-R170C 4m, replicate 2, brain, male sample 5: Wildtype 6m, replicate 1, brain, male sample 6: Wildtype 6m, replicate 1, brain, male sample 7: MCAO3d, brain, male processed data files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz (detailed in the readme_09242025.txt)