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)

NOTCH3突变可引发常染色体显性遗传性脑动脉病伴皮质下梗死和白质脑病（cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy, CADASIL），这是最常见的单基因性小血管疾病。迄今为止，针对CADASIL的特异性治疗手段仍十分匮乏。本研究证实，CADASIL小鼠模型（NOTCH3R170C）的胶质淋巴系统（glymphatic system）流入与流出功能均受损，这会阻碍代谢废物清除并促进脑衰老。相应地，CADASIL患者的脑萎缩与血管周围间隙扩大相关，提示胶质淋巴系统功能障碍参与了CADASIL患者的脑衰老进展。CADASIL中的胶质淋巴功能异常归因于星形胶质细胞终足（astrocytic endfeet）上水通道蛋白4（aquaporin 4, AQP4）的表达下调，而AQP4是胶质淋巴活性的核心介导因子。机制上，AQP4的表达受RUNX1-CMYB信号通路调控，而NOTCH3突变会抑制该通路的激活。通过腺相关病毒（adeno-associated virus, AAV）介导的疗法增强星形胶质细胞中的AQP4表达，可恢复CADASIL小鼠的胶质淋巴功能，进而延缓脑衰老。因此，本研究提出通过增强AQP4表达以改善胶质淋巴功能，是一种极具前景的CADASIL治疗策略。

衰老相关迁移体（Senescence-associated Migrasomes, SAM）通过巨噬细胞凋亡抑制因子（Apoptosis Inhibitor of Macrophage, AIM）促进脑衰老。衰老是包括阿尔茨海默病（Alzheimer's disease, AD）在内的多种神经系统疾病的主要危险因素。在衰老过程中，衰老细胞会分泌促衰老因子，传递衰老信号并强化衰老进程。迁移体（Migrasomes）是近年发现的、由细胞迁移过程中形成的细胞器，可从母细胞脱离并介导细胞间通讯。本研究鉴定出边界相关巨噬细胞（border-associated macrophages, BAMs）是脑衰老过程中获得促衰老特性的主要细胞类型，这可能与其长期暴露于β淀粉样蛋白（amyloid beta, Aβ）有关。在促衰老的BAMs中，迁移体的生成量升高，可向邻近细胞传递促衰老信号。机制上，巨噬细胞凋亡抑制因子（AIM）被包裹进入迁移体；当受体细胞的CD16被激活后，AIM可保护受体细胞免于凋亡，进而诱导细胞衰老。对18月龄小鼠施用包裹Tspan4小干扰RNA（Tspan4-siRNA）的脂质体，可抑制促衰老BAMs的迁移体生成，延缓脑衰老并改善认知功能缺损。本研究结果表明，迁移体是促衰老信号的有效载体，可作为衰老干预治疗的潜在靶点。

实质边界巨噬细胞（Parenchymal border macrophages, PBMs）定位于中枢神经系统（central nervous system, CNS）与外周组织的界面，已知其可调控物质向脑内的转运效率。本研究证实，脑卒中后，β淀粉样蛋白（Aβ）会在同侧及对侧半球的脑血管周围蓄积。当PBMs被清除后，Aβ的胶质淋巴引流功能显著受损，并伴随认知功能恶化，这凸显了PBMs在脑卒中后Aβ清除中的关键作用。其潜在机制可能与中脑星形胶质细胞源性神经营养因子（mesencephalic astrocyte-derived neurotrophic factor, MANF）有关，已知MANF会阻碍Aβ的胶质淋巴清除。当向脑脊液中补充PBMs来源的MANF时，可抑制星形胶质细胞应激并维持胶质淋巴引流功能。在脑卒中慢性期，PBMs中MANF的表达下调，进而加剧胶质淋巴功能障碍，导致Aβ持续蓄积与认知功能下降。综上，补充MANF不仅可减轻PBM清除带来的不良影响，还可改善胶质淋巴系统功能，发挥治疗作用。因此，本研究提出该策略或可用于预防脑卒中后认知功能损害。

整体实验设计：分离16周龄NOTCH3野生型（NOTCH3-WT）或NOTCH3-R170C突变小鼠的脑组织，经消化、去除髓鞘后制备单细胞悬液，用于单细胞RNA测序（scRNAseq）。共设置4个样本；原始数据为多重测序数据。
样本1：NOTCH3-WT4m，生物学重复1，scRNAseq
样本2：NOTCH3-WT4m，生物学重复2，scRNAseq
样本3：NOTCH3-R170C，生物学重复1，scRNAseq
样本4：NOTCH3-R170C，生物学重复2，scRNAseq

*** 2025年7月30日，新增来自GSM8108141多重原始数据的处理数据，更新了记录：对6月龄雄性野生型（WT）小鼠脑组织进行单细胞RNA测序（scRNA-seq），包含2个生物学重复：WT6m, brain, male, rep1；WT6m, brain, male, rep2

*** 2025年9月24日，新增以下来自GSM8108141多重原始数据的处理数据，更新了记录：分离野生型、NOTCH3-R170C或短暂性大脑中动脉闭塞（transient middle cerebral artery occlusion, MCAO）小鼠的脑组织，经消化、去除髓鞘后制备单细胞悬液，用于单细胞RNA测序。共设置7个样本；原始数据为多重测序数据。
样本1：Wildtype 4m，生物学重复1，脑组织，雄性
样本2：Wildtype 4m，生物学重复2，脑组织，雄性
样本3：NOTCH3-R170C 4m，生物学重复1，脑组织，雄性
样本4：NOTCH3-R170C 4m，生物学重复2，脑组织，雄性
样本5：Wildtype 6m，生物学重复1，脑组织，雄性
样本6：Wildtype 6m，生物学重复1，脑组织，雄性
样本7：MCAO3d，脑组织，雄性

处理数据文件：barcodes.tsv.gz、features.tsv.gz、matrix.mtx.gz（详见readme_09242025.txt）