Cx43 in Cdh5+ cells maintains blood-brain barrier integrity by NAD+-dependent stabilization of mitophagy during aging and inflammation

Vascular dysfunction of the blood-brain barrier (BBB) contributes to brain aging, cognitive impairment and neurodegenerative diseases. It is unclear how the BBB function deteriorates during natural aging. Our single-nucleus transcriptomics analysis identified decreased expression of Cx43 in Cdh5+ cells of cerebral vasculature in natural aging mice. Heterozygous global knockout of Cx43 or Cdh5+ cell-specific depletion of Cx43 in mice exacerbated BBB dysfunction in aging or chronic inflammation. Pharmacological and metabolomic studies revealed that the Cx43-dependent effect was not due to the gap junction canonical function of Cx43 on cell membrane, but associated with the effects of Cx43 influencing intercellular NAD+ levels. Cx43 deficiency reduced NAD+ levels in a cell-autonomous manner, impaired Sirt3-associated mitophagy, caused mitochondrial dysfunction, and compromised Cdh5+ cell proliferation. Long-term dietary supplementation with nicotinamide mononucleotide (NMN) in Cx43-deficient mice to increase NAD+ levels, stabilized mitochondrial function in Cdh5+ cells, and rescued BBB leakage in aging and chronic inflammation. These findings established a central role of the Cx43-dependent metabolic pathway in regulating Cdh5+ cells mitophagy and mitochondrial function during vascular aging, and identified a dietary intervention as a therapeutic strategy to protect against aging-induced disruption of BBB integrity and cognition functions. To investigate BBB-related transcriptomic changes across different cell types during natural aging, we studied the cortex from young (2-3-month-old) and old (18-22-month-old) mice (n = 3 pooled per group) using snRNA-seq with the 10x Genomics Chromium platform (Hu et al., 2017).血脑屏障（blood-brain barrier, BBB）功能异常参与脑衰老、认知障碍及神经退行性疾病的病理进程。目前尚不清楚自然衰老过程中血脑屏障功能的衰退机制。我们通过单细胞核转录组学分析，在自然衰老小鼠的脑血管Cdh5阳性（Cdh5+）细胞中发现连接蛋白43（connexin 43, Cx43）的表达水平显著下调。在小鼠中实施Cx43杂合全局敲除，或特异性敲除Cdh5+细胞中的Cx43，均会加剧衰老或慢性炎症状态下的血脑屏障功能异常。药理学与代谢组学研究表明，Cx43的调控效应并非源于其在细胞膜上的经典间隙连接功能，而是与Cx43影响细胞间烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD+）水平的作用密切相关。Cx43缺失会以细胞自主的方式降低细胞内NAD+水平，损害Sirt3相关的线粒体自噬过程，引发线粒体功能异常，并削弱Cdh5+细胞的增殖能力。对Cx43缺失小鼠长期膳食补充烟酰胺单核苷酸（nicotinamide mononucleotide, NMN）以提升体内NAD+水平，可稳定Cdh5+细胞的线粒体功能，并修复衰老与慢性炎症状态下的血脑屏障渗漏。本研究确立了Cx43依赖的代谢通路在血管衰老过程中调控Cdh5+细胞线粒体自噬与线粒体功能的核心作用，并明确了一种膳食干预策略，可用于防治衰老引发的血脑屏障完整性破坏与认知功能损伤。为探究自然衰老过程中不同细胞类型的血脑屏障相关转录组变化，我们利用10x Genomics Chromium平台的单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）技术，对年轻（2~3月龄）与老年（18~22月龄）小鼠的大脑皮层组织进行了测序分析（每组合并3只小鼠的样本，n=3），实验方法参考Hu等人2017年的研究。