CORRESPONDENCE also Snta1–/– and mdx mice. Together, these studies confirm a requirement for polarized Aquaporin 4 and glymphatic flow AQP4 localization for rapid tracer transport from the CSF into the brain parenchyma. We have central roles in brain fluid agree with MacAulay’s view that a biophys- ical explanation for how AQP4 at astrocyte homeostasis endfeet indirectly facilitates glymphatic flux is incompletely understood. However, we have shown that subcellular relocalization of AQP4, Mootaz M. Salman , Philip Kitchen, Jeffrey J. Iliff and Roslyn M. Bill from intracellular vesicles to the plasma mem- brane, has a crucial role in the regulation of In their recent Review (MacAulay, N. currently lacking, now is the time to carefully AQP4 function6 (Fig. 1). We 6 and others7,8 Molecular mechanisms of brain water define the processes involved. The field has a have also shown that AQP4 subcellular relo- transport. Nat. Rev. Neurosci. 22, 326–344 tendency to discuss ‘water’ and ‘fluid’ in calization is a dynamic process independent (2021))1, MacAulay highlights many a manner that incorrectly suggests their of changes in AQP4 expression. Following open questions about how brain water interchangeability. As we describe in FIG. 1, pathological dysregulation, AQP4 relocali- transport is controlled. They posit that in the glymphatic system, the clearance of zation to astrocyte endfeet facilitates oedema cotransport of water may bridge the gap in brain waste occurs through paracellular formation, which can be pharmacologically our understanding of cellular and barrier flow. Classic tracer studies measure this inhibited6. A comprehensive review of brain 17 brain water transport. As the existence of the paracellular flow, while the use of H2 O water transport should therefore consider the glymphatic system and its dependence upon captures both paracellular flow and diffusive dynamic relocalization of AQP4 channels as it the glial water channel aquaporin 4 (AQP4) transcellular exchange of water2. Importantly, provides a framework to address fundamental have been controversial, MacAulay places both are AQP4 dependent — one directly and questions about water homeostasis in health them outside the scope of their Review. We one indirectly. and disease. agree that a lack of mechanistic insight into A comprehensive view of brain water trans- One approach to answering some of the them represents a significant gap in current port must include the role of the glympha tic outstanding questions in the field is to use spe- knowledge of the brain in health and disease. system (which refers to perivascular and par- cific AQP4 inhibitors. Notably, it is here that However, it is necessary to contextualize the acellular fluid transport (Fig. 1)), especially in much of the literature lacks clarity. TGN-020 role of AQP4 in glymphatic function (which light of recent studies suggesting that peri- has been suggested to be an AQP4 inhibitor we think deserves more attention) and vascular cerebrospinal fluid (CSF) is a major on the basis of Xenopus laevis oocyte swelling address the need for tighter definitions when source of oedema fluid that accumulates assays, as have diverse, structurally unrelated describing the fluids involved. As MacAulay’s acutely following stroke3. The AQP4 depen- molecules such as acetazolamide, ethoxzola- review has such a broad title, our aim is to dence of perivascular flow is established: work mide, topiramate, lamotrigine, zonisamide, provide its reader with an appreciation of in five independent laboratories4 has refuted acetylsulfanilamide, phenytoin, bumetanide, these important and, in some cases, emerging the single study5 suggesting that this is an furosemide, tetraethylammonium and IMD- concepts in brain fluid dynamics. AQP4-independent process. The link between 0354. The inhibitory action of the major- As a detailed molecular mechanistic AQP4 and glymphatic function is compelling, ity of these molecules has been challenged9 understanding of brain water transport is not only from studies in Aqp4–/– mice, but and many have AQP4- independent effects on brain water transport, confounding the interpre tation of in vivo studies. To our Parenchyma knowledge, the off- target effects of TGN-020 Vesicle remain completely unexplored. This high- lights the need for well- validated inhibitors whose efficacies are reproducible between experimental assay systems and laborato- ries, and the need for greater understanding AQP4 of the indirect effects of AQP4 knockout in Perivascular the brain. space Astrocyte To conclude, we welcome MacAulay’s Review1; in combination with current Solutes Vasculature know ledge of the glymphatic system, a new Water understanding for the role of dynamic AQP4 Blood regulation and the search for specific inhibi- tors, understanding of the mechanisms of | Fig. 1 Aquaporin 4 has direct and indirect roles in controlling fluid flow in the brain. Astrocyte brain water transport can only improve. endfeet wrap around blood vessels at the blood–brain barrier, separated by a cerebrospinal fluid There is a reply to this letter by MacAulay, (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 N. Nat. Rev. Neurosci. https://doi.org/10.1038/ cervical lymph nodes define the glymphatic pathway. Glymphatic function clears brain metabolic s41583-021-00515-y (2021). waste. Solutes and water are transported paracellularly between astrocyte endfeet, while water can Mootaz M. Salman 1,2 ✉ , Philip Kitchen3 ✉ , also be exchanged across the endfoot membrane via aquaporin 4 (AQP4). Physiological and patho- Jeffrey J. Iliff4,5,6 ✉ and Roslyn M. Bill 3 ✉ physiological factors regulate the relocalization of AQP4 between intracellular vesicular pools and 1Department of Physiology, Anatomy and Genetics, the plasma membrane of astrocyte endfeet, dynamically regulating membrane water permeability. University of Oxford, Oxford, UK. 650 | OCTOBER 2021 | VOLUME 22 www.nature.com/nrn 0123456789();: CORRESPONDENCE 2Oxford Parkinson’s Disease Centre, 4. Mestre, H. et al. Aquaporin-4-dependent glymphatic the fluorescent particles will not “follow the University of Oxford, Oxford, UK. solute transport in the rodent brain. eLife 7, e40070 (2018). resulting osmotic gra dient into the interstit- 3School of Biosciences, College of Health and Life 5. Smith, A. J., Yao, X., Dix, J. A., Jin, B.-J. & ium through intercellular clefts”6 as water Sciences, Aston University, Verkman, A. S. Test of the ‘glymphatic’ Birmingham, UK. hypothesis demonstrates diffusive and follows particles by osmosis, not the opposite. 4Department of Psychiatry and Behavioral Sciences, aquaporin-4-independent solute transport Last, osmosis occurs across a semipermeable University of Washington School of Medicine, in rodent brain parenchyma. eLife 6, e27679 (2017). membrane and not through intercellular clefts. Seattle, WA, USA. 6. Kitchen, P. et al. Targeting aquaporin-4 subcellular 5 To my knowledge, AQP4- dependent Department of Neurology, localization to treat central nervous system edema. University of Washington School of Medicine, Cell 181, 784–799 (2020). water transport through AQP4 has not been Seattle, WA, USA. 7. Ciappelloni, S. et al. Aquaporin-4 surface trafficking documented in the glymphatic hypothesis, 6VISN 20 Mental Illness Research, Education and regulates astrocytic process motility and synaptic activity in health and autoimmune disease. Cell Rep. which, however, does not prevent AQP4 Clinical Center, VA Puget Sound Health Care System, 27, 3860–3872 (2019). from serving a structural role in the system. Seattle, WA, USA. 8. Lisjak, M., Potokar, M., Rituper, B., Jorgačevski, J. –/– ✉e- mail: [email protected]; & Zorec, R. AQP4e- based orthogonal arrays regulate Notably, Aqp4 mice display severely [email protected]; [email protected]; rapid cell volume changes in astrocytes. J. Neurosci. reduced expression of protein anchoring 37, 10748–10756 (2017). [email protected] 7 9. Yang, B., Zhang, H. & Verkman, A. S. Lack complexes in the astrocytic endfeet , which https://doi.org/10.1038/s41583-021-00514- z of aquaporin-4 water transport inhibition by may affect endfoot polarization of other antiepileptics and arylsulfonamides. Bioorg. Med. 1. MacAulay, N. Molecular mechanisms of brain Chem. 16, 7489–7493 (2008). astrocytic membrane proteins and thereby water transport. Nat. Rev. Neurosci. 22, 326–344 indirectly influence astrocyte function in (2021). Competing interests 2. Alshuhri, M. S., Gallagher, L., Work, L. M. & The authors declare no competing interests. a manner that could affect the paracellular Holmes, W. M. Direct imaging of glymphatic transport flow of fluorescent particles. A specific 17 using H2 O MRI. JCI Insight 6, e141159 (2021). Peer review information 3. Mestre, H. et al. Cerebrospinal fluid influx drives Nature Reviews Neuroscience thanks R. Keep and the other, and efficient inhibitor of AQP4 would acute ischemic tissue swelling. Science 367, anonymous, reviewer(s) for their contribution to the peer provide the tool to reveal a requirement for eaax7171 (2020). review of this work. AQP4- dependent transcellular water flux to support paracellular flow of fluorescent molecules. The authors rightly point out the futile search for such inhibitors. However, Reply to ‘Aquaporin 4 and glymphatic TGN-020 is among the most promising of its kind: it displays near- absent binding flow have central