Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Mechanisms of aquaporin-4 vesicular trafficking in mammalian cells.

The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Astrocytes in functional recovery following central nervous system injuries.

Astrocytes are increasingly recognised as partaking in complex homeostatic mechanisms critical for regulating neuronal plasticity following central nervous system (CNS) insults. Ischaemic stroke and traumatic brain injury are associated with high rates of disability and mortality. Depending on the context and type of injury, reactive astrocytes respond with diverse morphological, proliferative and functional changes collectively known as astrogliosis, which results in both pathogenic and protective effects. There is a large body of research on the negative consequences of astrogliosis following brain injuries. There is also growing interest in how astrogliosis might in some contexts be protective and help to limit the spread of the injury. However, little is known about how astrocytes contribute to the chronic functional recovery phase following traumatic and ischaemic brain insults. In this review, we explore the protective functions of astrocytes in various aspects of secondary brain injury such as oedema, inflammation and blood-brain barrier dysfunction. We also discuss the current knowledge on astrocyte contribution to tissue regeneration, including angiogenesis, neurogenesis, synaptogenesis, dendrogenesis and axogenesis. Finally, we discuss diverse astrocyte-related factors that, if selectively targeted, could form the basis of astrocyte-targeted therapeutic strategies to better address currently untreatable CNS disorders.

Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis.

Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood-spinal cord and blood-brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.

Signaling Mechanisms and Pharmacological Modulators Governing Diverse Aquaporin Functions in Human Health and Disease.

The aquaporins (AQPs) are a family of small integral membrane proteins that facilitate the bidirectional transport of water across biological membranes in response to osmotic pressure gradients as well as enable the transmembrane diffusion of small neutral solutes (such as urea, glycerol, and hydrogen peroxide) and ions. AQPs are expressed throughout the human body. Here, we review their key roles in fluid homeostasis, glandular secretions, signal transduction and sensation, barrier function, immunity and inflammation, cell migration, and angiogenesis. Evidence from a wide variety of studies now supports a view of the functions of AQPs being much more complex than simply mediating the passive flow of water across biological membranes. The discovery and development of small-molecule AQP inhibitors for research use and therapeutic development will lead to new insights into the basic biology of and novel treatments for the wide range of AQP-associated disorders.

Biological insights from SMA-extracted proteins.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein-lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein-protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Advances in Applying Computer-Aided Drug Design for Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease are incurable and affect millions of people worldwide. The development of treatments for this unmet clinical need is a major global research challenge. Computer-aided drug design (CADD) methods minimize the huge number of ligands that could be screened in biological assays, reducing the cost, time, and effort required to develop new drugs. In this review, we provide an introduction to CADD and examine the progress in applying CADD and other molecular docking studies to NDs. We provide an updated overview of potential therapeutic targets for various NDs and discuss some of the advantages and disadvantages of these tools.

Expansion of HIV testing in Eswatini: stakeholder perspectives on reaching the first 90.

Achieving the United Nations' 90-90-90 goals has proven challenging in most settings and the ambitious 95-95-95 goals seem even more elusive. However, in Eswatini - a lower-middle-income country in sub-Saharan Africa with the highest HIV prevalence in the world - an estimated 92% of people living with HIV know their status. We conducted 26 in-depth interviews with stakeholders from policy, implementation, donor, local advocacy and academic sectors to elicit the facilitators and inhibitors to HIV testing uptake in Eswatini. Background data and related reports and policy documents (n = 57) were also reviewed. Essential facilitators included good governance via institutional and national budgetary commitments, which often led to swift adoption of globally recommended programs and standards. The integration of HIV testing into all points of care fostered a sense that testing was part of routine care, which reduced stigma. Challenges, however, centred on social norms that disadvantage certain groups with high ongoing HIV risk (such as key populations, adolescent girls and young women), a heavy reliance on external donor funding, and stigma that had subsided but nevertheless persisted. Amid concerns about whether the 90-90-90 targets could be achieved by 2020, the experience of Eswatini provides tangible insights into factors that have successfully influenced HIV testing uptake and may thus prove informative for other countries.

Inhibitors of Mammalian Aquaporin Water Channels.

Aquaporins (AQPs) are water channel proteins that are essential to life, being expressed in all kingdoms. In humans, there are 13 AQPs, at least one of which is found in every organ system. The structural biology of the AQP family is well-established and many functions for AQPs have been reported in health and disease. AQP expression is linked to numerous pathologies including tumor metastasis, fluid dysregulation, and traumatic injury. The targeted modulation of AQPs therefore presents an opportunity to develop novel treatments for diverse conditions. Various techniques such as video microscopy, light scattering and fluorescence quenching have been used to test putative AQP inhibitors in both AQP-expressing mammalian cells and heterologous expression systems. The inherent variability within these methods has caused discrepancy and many molecules that are inhibitory in one experimental system (such as tetraethylammonium, acetazolamide, and anti-epileptic drugs) have no activity in others. Some heavy metal ions (that would not be suitable for therapeutic use) and the compound, TGN-020, have been shown to inhibit some AQPs. Clinical trials for neuromyelitis optica treatments using anti-AQP4 IgG are in progress. However, these antibodies have no effect on water transport. More research to standardize high-throughput assays is required to identify AQP modulators for which there is an urgent and unmet clinical need.

Structural Determinants of Oligomerization of the Aquaporin-4 Channel.

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.

Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism.

Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.

Transcriptome analysis suggests a role for the differential expression of cerebral aquaporins and the MAPK signalling pathway in human temporal lobe epilepsy.

Epilepsies are common disorders of the central nervous system (CNS), affecting up to 2% of the global population. Pharmaco-resistance is a major clinical challenge affecting about 30% of temporal lobe epilepsy (TLE) patients. Water homeostasis has been shown crucial for regulation of neuronal excitability. The control of water movement is achieved through a family of small integral membrane channel proteins called aquaporins (AQPs). Despite the fact that changes in water homeostasis occur in sclerotic hippocampi of people with TLE, the expression of AQPs in the epileptic brain is not fully characterised. This study uses microarray and ELISA methods to analyse the mRNA and protein expression of the human cerebral AQPs in sclerotic hippocampi (TLE-HS) and adjacent neocortex tissue (TLE-NC) of TLE patients. The expression of AQP1 and AQP4 transcripts was significantly increased, while that of the AQP9 transcript was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while expression of AQP1 protein remained unchanged, and AQP9 was undetected. Microarray data analysis identified 3333 differentially regulated genes and suggested the involvement of the MAPK signalling pathway in TLE pathogenesis. Proteome array data validated the translational profile for 26 genes and within the MAPK pathway (e.g. p38, JNK) that were identified as differentially expressed from microarray analysis. ELISA data showed that p38 and JNK inhibitors decrease AQP4 protein levels in cultured human primary cortical astrocytes. Elucidating the mechanism of selective regulation of different AQPs and associated regulatory proteins may provide a new therapeutic approach to epilepsy treatment.

Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel.

The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema.

Beyond water homeostasis: Diverse functional roles of mammalian aquaporins.

BACKGROUND: Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. SCOPE OF REVIEW: AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. MAJOR CONCLUSIONS: As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. GENERAL SIGNIFICANCE: Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.

Control of the Aquaporin-4 Channel Water Permeability by Structural Dynamics of Aromatic/Arginine Selectivity Filter Residues.

The aquaporins (AQPs) make up a family of integral membrane proteins that control cellular water flow. Gating of the water channel by conformational changes induced by phosphorylation or protein-protein interactions is an established regulatory mechanism for AQPs. Recent in silico and crystallographic analyses of the structural biology of AQPs suggest that the rate of water flow can also be controlled by small movements of single-amino acid side chains lining the water pore. Here we use measurements of the membrane water permeability of mammalian cells expressing AQP4 mutants to provide the first in vitro evidence in support of this hypothesis.

Human aquaporins: regulators of transcellular water flow.

BACKGROUND: Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistent with their expression in most tissues, AQPs are associated with diverse physiological and pathophysiological processes. SCOPE OF REVIEW: AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volume regulation (CVR) is particularly notable. This review examines the regulatory role of AQPs in transcellular water flow, especially in CVR. We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. MAJOR CONCLUSIONS: AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapid movement of water across diverse cell membranes and playing regulatory roles in CVR. Gating mechanisms have been proposed for human AQPs, but have only been reported for plant and microbial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. GENERAL SIGNIFICANCE: Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.

Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers.

Extraction of proteins from the membrane using styrene maleic acid co-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and N-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg2+ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.

Global prevalence of hepatitis B or hepatitis C infection among patients with tuberculosis disease: systematic review and meta-analysis.

Background: There is a substantial overlap in the epidemiology of chronic hepatitis B (HBV), hepatitis C (HCV) and tuberculosis (TB) due to overlapping risk factors. Testing for viral hepatitis is not widely recommended for patients with TB. The aim of this systematic review was to evaluate the global prevalence of chronic viral hepatitis infection among patients with TB. Methods: MEDLINE, EMBASE, Web of Science, Cochrane Library, African Journals Online, LILACS, and country TB reports were searched for studies published between January 1st, 2011 and June 17th 2021. Random-effects meta-analyses for proportions were conducted to obtain pooled prevalences. The prevalence of chronic HBV/HCV infection among patients with TB was also compared to that in the general population. The protocol was registered on PROSPERO (CRD42021276468). Findings: This analysis included 127 studies (83 for both HBV and HCV, 28 for HBV only, and 25 for HCV only) and data from 94,936 patients. The global pooled seroprevalence was 5.8% (95% CI 5.0-6.8) for HBs-antigen and 10.3% (95% CI 8.4-12.3) for HCV-antibodies. Pooled prevalence was highest in the WHO African Region for HBV at 7.8% (95% CI 5.2-10.9) and in the WHO European Region at 17.5% (95% CI 12.2-23.5) for HCV. In studies among TB patients who inject drugs, HCV prevalence was 92.5% (95% CI 80.8-99.0). Pooled HCV-antibody seroprevalence among patients with TB was higher than in the general population in all six WHO regions while HBs-antigen seroprevalence was higher in 3/6 regions. Interpretation: This review highlights the syndemicity of chronic viral hepatitis and TB and suggests that routine testing for hepatitis upon TB diagnosis may be justified. The prevalence of chronic HBV and HCV infections was higher among patients with TB than in the general population. Funding: This study was study was funded by the Global Tuberculosis Programme, World Health Organization.