In their recent Review (MacAulay, N. Molecular mechanisms of brain water transport. Nat. Rev. Neurosci. 22, 326–344 (2021))1, MacAulay highlights many open questions about how brain water transport is controlled. They posit that cotransport of water may bridge the gap in our understanding of cellular and barrier brain water transport. As the existence of the glymphatic system and its dependence upon the glial water channel aquaporin 4 (AQP4) have been controversial, MacAulay places them outside the scope of their Review. We agree that a lack of mechanistic insight into them represents a significant gap in current knowledge of the brain in health and disease. However, it is necessary to contextualize the role of AQP4 in glymphatic function (which we think deserves more attention) and address the need for tighter definitions when describing the fluids involved. As MacAulay’s review has such a broad title, our aim is to provide its reader with an appreciation of these important and, in some cases, emerging concepts in brain fluid dynamics.

As a detailed molecular mechanistic understanding of brain water transport is currently lacking, now is the time to carefully define the processes involved. The field has a tendency to discuss ‘water’ and ‘fluid’ in a manner that incorrectly suggests their interchangeability. As we describe in Fig. 1, in the glymphatic system, the clearance of brain waste occurs through paracellular flow. Classic tracer studies measure this paracellular flow, while the use of H217O captures both paracellular flow and diffusive transcellular exchange of water2. Importantly, both are AQP4 dependent — one directly and one indirectly.

Fig. 1: Aquaporin 4 has direct and indirect roles in controlling fluid flow in the brain.
figure 1

Astrocyte endfeet wrap around blood vessels at the blood–brain barrier, separated by a cerebrospinal fluid (CSF)-filled perivascular space. Para-arterial fluid influx, a trans-parenchymal pathway (where CSF exchanges with interstitial fluid to clear extracellular solutes) and para-venous efflux into the deep cervical lymph nodes define the glymphatic pathway. Glymphatic function clears brain metabolic waste. Solutes and water are transported paracellularly between astrocyte endfeet, while water can also be exchanged across the endfoot membrane via aquaporin 4 (AQP4). Physiological and pathophysiological factors regulate the relocalization of AQP4 between intracellular vesicular pools and the plasma membrane of astrocyte endfeet, dynamically regulating membrane water permeability.

Full size image

A comprehensive view of brain water transport must include the role of the glymphatic system (which refers to perivascular and paracellular fluid transport (Fig. 1)), especially in light of recent studies suggesting that perivascular cerebrospinal fluid (CSF) is a major source of oedema fluid that accumulates acutely following stroke3. The AQP4 dependence of perivascular flow is established: work in five independent laboratories4 has refuted the single study5 suggesting that this is an AQP4-independent process. The link between AQP4 and glymphatic function is compelling, not only from studies in Aqp4–/– mice, but also Snta1–/– and mdx mice. Together, these studies confirm a requirement for polarized AQP4 localization for rapid tracer transport from the CSF into the brain parenchyma. We agree with MacAulay’s view that a biophysical explanation for how AQP4 at astrocyte endfeet indirectly facilitates glymphatic flux is incompletely understood. However, we have shown that subcellular relocalization of AQP4, from intracellular vesicles to the plasma membrane, has a crucial role in the regulation of AQP4 function6 (Fig. 1). We6 and others7,8 have also shown that AQP4 subcellular relocalization is a dynamic process independent of changes in AQP4 expression. Following pathological dysregulation, AQP4 relocalization to astrocyte endfeet facilitates oedema formation, which can be pharmacologically inhibited6. A comprehensive review of brain water transport should therefore consider the dynamic relocalization of AQP4 channels as it provides a framework to address fundamental questions about water homeostasis in health and disease.

One approach to answering some of the outstanding questions in the field is to use specific AQP4 inhibitors. Notably, it is here that much of the literature lacks clarity. TGN-020 has been suggested to be an AQP4 inhibitor on the basis of Xenopus laevis oocyte swelling assays, as have diverse, structurally unrelated molecules such as acetazolamide, ethoxzolamide, topiramate, lamotrigine, zonisamide, acetylsulfanilamide, phenytoin, bumetanide, furosemide, tetraethylammonium and IMD-0354. The inhibitory action of the majority of these molecules has been challenged9 and many have AQP4-independent effects on brain water transport, confounding the interpretation of in vivo studies. To our knowledge, the off-target effects of TGN-020 remain completely unexplored. This highlights the need for well-validated inhibitors whose efficacies are reproducible between experimental assay systems and laboratories, and the need for greater understanding of the indirect effects of AQP4 knockout in the brain.

To conclude, we welcome MacAulay’s Review1; in combination with current knowledge of the glymphatic system, a new understanding for the role of dynamic AQP4 regulation and the search for specific inhibitors, understanding of the mechanisms of brain water transport can only improve.

There is a reply to this letter by MacAulay, N. Nat. Rev. Neurosci. https://doi.org/10.1038/s41583-021-00515-y (2021).