Evaluation of gliovascular functions of AQP4 readthrough isoforms

Shayna M Mueller; Kelli McFarland White; Stuart B Fass; Siyu Chen; Zhan Shi; Xia Ge; John A Engelbach; Seana H Gaines; Annie R Bice; Michael J Vasek; Joel R Garbow; Joseph P Culver; Zila Martinez-Lozada; Martine Cohen-Salmon; Joseph D Dougherty; Darshan Sapkota

doi:10.3389/fncel.2023.1272391

Evaluation of gliovascular functions of AQP4 readthrough isoforms

Front Cell Neurosci. 2023 Nov 23:17:1272391. doi: 10.3389/fncel.2023.1272391. eCollection 2023.

Authors

Shayna M Mueller^#^{1

2}, Kelli McFarland White^#^{1

2}, Stuart B Fass^{1

2}, Siyu Chen^{1

2

3}, Zhan Shi⁴, Xia Ge^{3

5}, John A Engelbach^{3

5}, Seana H Gaines³, Annie R Bice³, Michael J Vasek^{1

2}, Joel R Garbow^{3

5}, Joseph P Culver^{3

6

7

8

9}, Zila Martinez-Lozada¹⁰, Martine Cohen-Salmon¹¹, Joseph D Dougherty^{1

2

5}, Darshan Sapkota^{4

12}

Affiliations

¹ Department of Genetics, Washington University School of Medicine, Saint Louis, MO, United States.
² Department of Psychiatry, Washington University School of Medicine, Saint Louis, MO, United States.
³ Department of Radiology, Washington University School of Medicine, Saint Louis, MO, United States.
⁴ Department of Biological Sciences, University of Texas at Dallas, Richardson, TX, United States.
⁵ Intellectual and Developmental Disabilities Research Center, Washington University School of Medicine, Saint Louis, MO, United States.
⁶ Department of Physics, Washington University in St. Louis, Saint Louis, MO, United States.
⁷ Department of Biomedical Engineering, Washington University in St. Louis, Saint Louis, MO, United States.
⁸ Department of Neuroscience, Washington University School of Medicine, Saint Louis, MO, United States.
⁹ Imaging Science PhD Program, Washington University in St. Louis, Saint Louis, MO, United States.
¹⁰ Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States.
¹¹ Center for Interdisciplinary Research in Biology (CIRB), Collège de France, The National Centre for Scientific Research (CNRS), National Institute of Health and Medical Research (INSERM), Université PSL, Paris, France.
¹² Department of Neuroscience, University of Texas at Dallas, Richardson, TX, United States.

^# Contributed equally.

Abstract

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllX^Hom and AllX^Het mice to WT and to previously characterized AQP4 NoX^Het and NoX^Hom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoX^Hom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Keywords: AQP4; AQP4x; astrocyte; blood-brain barrier; glymphatic; readthrough.

Abstract

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