Single-nucleus RNA sequencing reveals Cntnap2+ astrocytes triggering a hypertensive effect in the rostral ventrolateral medulla by disrupting glutamate uptake

Shuai Zhang; Lei Tong; Zhangyan Ren; Tengteng Dai; Linping Wang; Dongfei Zhu; Haisheng Liu; Liucun Zhu; Dong Huang; Dongshu Du

doi:10.1016/j.jare.2025.06.002

Single-nucleus RNA sequencing reveals Cntnap2⁺ astrocytes triggering a hypertensive effect in the rostral ventrolateral medulla by disrupting glutamate uptake

J Adv Res. 2025 Jun 2:S2090-1232(25)00388-1. doi: 10.1016/j.jare.2025.06.002. Online ahead of print.

Authors

Shuai Zhang¹, Lei Tong², Zhangyan Ren², Tengteng Dai², Linping Wang², Dongfei Zhu³, Haisheng Liu⁴, Liucun Zhu⁵, Dong Huang⁶, Dongshu Du⁷

Affiliations

¹ International Cooperation Laboratory of Molecular Medicine, Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China; College of Agriculture and Bioengineering, Heze University, Heze, Shandong 274015, China.
² School of Life Sciences, Shanghai University, Shanghai 200444, China.
³ Shanghai Jiao Tong University Affiliated Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200233, China.
⁴ College of Agriculture and Bioengineering, Heze University, Heze, Shandong 274015, China.
⁵ School of Life Sciences, Shanghai University, Shanghai 200444, China. Electronic address: zhuliucun@shu.edu.cn.
⁶ Shanghai Jiao Tong University Affiliated Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200233, China. Electronic address: huangdong1004@126.com.
⁷ School of Life Sciences, Shanghai University, Shanghai 200444, China; College of Agriculture and Bioengineering, Heze University, Heze, Shandong 274015, China; Shaoxing Institute of Shanghai University, Shaoxing, Zhejiang 312000, China. Electronic address: dsdu@shu.edu.cn.

PMID: 40466760
DOI: 10.1016/j.jare.2025.06.002

Abstract

Introduction: The rostral ventrolateral medulla (RVLM) is a vital vasomotor nucleus involved in hypertension control. However, the molecular intricacies within RVLM cells, particularly in non-neuronal cell populations such as astrocytes, under hypertensive conditions remain to be systematically investigated.

Objectives: We aimed to elucidate cellular heterogeneity in the RVLM at single-cell resolution, with a focus on astrocytes, to further characterize its role in hypertension.

Methods: Transcriptional changes in cells in the RVLM after hypertension were examined by using single-nucleus RNA sequencing (snRNA-seq). Further experiments, like radiotelemetry and immunofluorescence assays, were conducted to support the findings of snRNA-seq.

Results: In total, 36 982 high-quality nuclei were obtained, with identification of nine cell types. Three transcriptomically distinct subtypes of astrocytes were defined within the astrocyte cell population: Cntnap2⁺, Pld5⁺, and Ppp1r16b⁺. The expression of Cntnap2 in RVLM astrocytes was notably increased to approximately 2.0-fold (P < 0.001) upon hypertension. Astrocyte-specific Cntnap2 overexpression in RVLM of WKY rats significantly enhanced neuronal excitability (∼21.5-fold, P < 0.001), sympathetic outflow (∼4.2-fold, P < 0.001), and blood pressure (BP, ∼1.5-fold, P < 0.001). Astrocyte-specific Cntnap2 knockdown in RVLM of SHRs resulted in 71 % reduction in neuronal excitability (P < 0.001), 51.6 % decrease in sympathetic outflow (P < 0.01), and 12.6 % drop in BP (P < 0.01). Elevated levels of Cntnap2 in primary astrocytes markedly increased primary neuron excitability to approximately 8.2-fold (P < 0.001). Mechanistically, Cntnap2 upregulation decreased Eaat2 expression by 26.5 % (P < 0.001), impairing astrocytic glutamate uptake. Importantly, reintroducing Eaat2 into astrocytes blocked 90.5 % (P < 0.001) of Cntnap2-induced neuronal excitability.

Conclusion: Our findings suggest that Cntnap2 may impede glutamate homeostasis at least partially through regulating Eaat2 in astrocytes, which could consequently enhance RVLM neuronal excitability, increase sympathetic tone, and contribute to elevated BP.

Keywords: Astrocytic glutamate uptake; Cntnap2; Eaat2; Hypertension; RVLM; snRNA-seq.