Introduction
The retrotrapezoid nucleus (RTN) is a brainstem respiratory control region where rodent astrocyte Kir4.1 channels are inhibited by increased CO2/H+ and contribute to chemosensing and CO₂/H⁺-dependent respiratory drive. All astrocytes express high levels of the inwardly rectifying potassium channel Kir4.1 and the water channel AQP4 and their disruption affects [K+]o homeostasis, glutamate uptake, water and waste clearance, processes likely altered in Alzheimer’s disease (AD). Reduced Kir4.1 expression has been reported in several neurodegenerative mouse models, but few studies have assessed brainstem alterations. Furthermore, whether astrocyte Kir4.1 is expressed in human RTN tissue, or whether its expression changes with age, differs by sex, or is altered in AD models remains poorly understood. To address this, we compared astrocytic Kir4.1 expression in the RTN of young and aged male and female rodents, including AD models, and examined Kir4.1 expression in putative human RTN tissue.
Methods
Kir4.1 expression was assessed by fluorescent immunohistochemistry in RTN and hippocampal tissue from young adult (2 months) or aged (≥9 months) TgF344-AD rats and APP23 mice, as well as in post-mortem human brainstem. Astrocytes were identified using GFAP or Aldh1L1 labelling and Kir4.1 mRNA levels in TgF344 animals were also assessed by qPCR. Kir4.1 protein expression in astrocytic somata and processes was quantified from confocal images acquired using identical settings, and U87 astrocytoma cells were assessed for Kir4.1 and AQP4 expression by immunocytochemistry. Statistical analyses used unpaired t-tests or multiple comparisons as appropriate with a minimum of 2 animals/subjects per group. Animal procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986 and human post-mortem tissue was obtained from the Manchester Brain Bank under approval from the Manchester Brain Bank Management Committee (REC Reference 24/NE/0201).
Results
Kir4.1 protein levels were higher in RTN astrocytes than in hippocampal astrocytes, with consistently greater expression in females than males across age groups. Kir4.1 expression also increased with ageing in both RTN and hippocampal astrocytes. However, Kir4.1 protein levels were reduced in aged TgF344-AD rats compared with age-matched controls, including at astrocytic endfoot domains. In 12-month-old male APP23 mice, Kir4.1 expression was detected at higher levels in GFAP-positive astrocytes in the RTN compared to hippocampus and appeared to differ less between genotypes. Kir4.1 mRNA levels were also unchanged between AD models and controls but U87 astrocyte like cells expressed both Kir4.1 and AQP4. Finally, Kir4.1 protein was detected in astrocytes within the putative RTN of aged human ponto-medullary post-mortem tissue.
Conclusions
These findings demonstrate that astrocytic Kir4.1 expression in the RTN increases with normal aging but appears altered in aged AD models, and we show for the first time that human RTN astrocytes also express Kir4.1. Disruptions in astrocyte Kir4.1 protein expression may contribute to neurodegenerative disease mechanisms and potentially to sleep-disordered breathing in cognitive decline.