Cellular distribution of brain aquaporins and their contribution to cerebrospinal fluid homeostasis and hydrocephalus
Abstract
Brain aquaporins facilitate the movement of water between the four water compartments: blood, cerebrospinal fluid, interstitial fluid, and intracellular fluid. This work analyzes the expression of the four most abundant aquaporins (AQPs) (AQP1, AQP4, AQP9, and AQP11) in the brains of mice and discuss their contribution to hydrocephalus. We analyzed available data from single-cell RNA sequencing of the central nervous system of mice to describe the expression of aquaporins and compare their distribution with that based on qPCR, western blot, and immunohistochemistry assays. Expression of AQP1 in the apical cell membrane of choroid plexus epithelial cells and of AQP4 in ependymal cells, glia limitans, and astrocyte processes in the pericapillary end foot is consistent with the involvement of both proteins in cerebrospinal fluid homeostasis. The expression of both aquaporins compensates for experimentally induced hydrocephalus in the animals. Recent data demonstrate that hypoxia in aged animals alters AQP4 expression in the choroidal plexus and cortex, increasing the ventricle size and intraventricular pressure. Cerebral distensibility is reduced in parallel with a reduction in cerebrospinal fluid drainage and cognitive deterioration. We propose that aged mice chronically exposed to hypoxia represent an excellent experimental model for studying the pathophysiological characteristics of idiopathic normal pressure hydrocephalus and roles for AQPs in such disease.
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Citation: Trillo-Contreras, J.L.; Ramírez-Lorca, R.; Villadiego, J.; Echevarría, M. Cellular Distribution of Brain Aquaporins and Their Contribution to Cerebrospinal Fluid Homeostasis and Hydrocephalus. Biomolecules 2022,12, 530. https:// doi.org/10.3390/biom12040530 Academic Editor: Mahmood Amiry-Moghaddam Received: 24 February 2022 Accepted: 30 March 2022 Published: 31 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Review Cellular Distribution of Brain Aquaporins and Their Contribution to Cerebrospinal Fluid Homeostasis and Hydrocephalus JoséLuis Trillo-Contreras 1,2 , Reposo Ramírez-Lorca 1,2 , Javier Villadiego 1,2,3,* and Miriam Echevarría1,2,* 1Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital, (HUVR)/Spanish National Research Council (CSIC)/University of Seville, 41013 Seville, Spain; [email protected] (J.L.T.-C.); reporamir[email protected] (R.R.-L.) 2Department of Physiology and Biophysics, University of Seville, 41009 Seville, Spain 3 Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), 28031 Madrid, Spain *Correspondence: [email protected] (J.V.); [email protected] (M.E.) Abstract: Brain aquaporins facilitate the movement of water between the four water compartments: blood, cerebrospinal fluid, interstitial fluid, and intracellular fluid. This work analyzes the expression of the four most abundant aquaporins (AQPs) (AQP1, AQP4, AQP9, and AQP11) in the brains of mice and discuss their contribution to hydrocephalus. We analyzed available data from single-cell RNA sequencing of the central nervous system of mice to describe the expression of aquaporins and compare their distribution with that based on qPCR, western blot, and immunohistochemistry assays. Expression of AQP1 in the apical cell membrane of choroid plexus epithelial cells and of AQP4 in ependymal cells, glia limitans, and astrocyte processes in the pericapillary end foot is consistent with the involvement of both proteins in cerebrospinal fluid homeostasis. The expression of both aquaporins compensates for experimentally induced hydrocephalus in the animals. Recent data demonstrate that hypoxia in aged animals alters AQP4 expression in the choroidal plexus and cortex, increasing the ventricle size and intraventricular pressure. Cerebral distensibility is reduced in parallel with a reduction in cerebrospinal fluid drainage and cognitive deterioration. We propose that aged mice chronically exposed to hypoxia represent an excellent experimental model for studying the pathophysiological characteristics of idiopathic normal pressure hydrocephalus and roles for AQPs in such disease. Keywords: aquaporins; cerebrospinal fluid; choroid plexus; ependyma; single-cell RNA; sequencing; hydrocephalus; aging; hypoxia 1. Introduction Water is the main compound of all living organisms and is essential for life. All organic ions and most macromolecules that form the cells are dissolved in water, and water is involved in most biological processes, such as homeostasis and the osmotic balance of cells. Little more than 30 years ago, hypotheses were debated and proposed as to the mechanism by which water crosses biological membranes [ 1 – 3 ]. The molecule’s small size, absence of electrical charge, low solubility in the membrane’s lipid phase, and its low activation energy to permeate in certain cells led to the consideration of the presence of preferential pathways for the passage of water across the plasma membrane [ 2 – 6 ]. In contrast, the presence of certain membranes with very low water permeability, such as in the renal cells of the ascending segment of the loop of Henle, showed that water permeability is not a constitutive property of the lipid bilayer [ 7 ]. The finding that the movement of water through the membranes of red blood cells could be blocked by mercury compounds derivatives [ 2 , 8 , 9 ] suggested the presence of specialized proteins in the formation of aqueous pores in the plasma membrane, whose expression and function could vary in different cell types. It was in such a scenario that Preston and Agre (1992) found a 28 kDa polypeptide, whose structural analysis revealed it as a new integral membrane protein not related to the rhesus Biomolecules 2022,12, 530. https://doi.org/10.3390/biom12040530 https://www.mdpi.com/journal/biomolecules
Biomolecules 2022,12, 530 2 of 18 (Rh) group of blood proteins [ 10 ]. This protein was called the channel-forming integral protein of 28 kDa (CHIP-28) and would later receive the name aquaporin 1 (AQP1) [ 11 , 12 ]. Since then, more than 450 different varieties of aquaporins have been identified, being present in all kingdoms of nature, from single-cell organisms such as archaea, bacteria, and yeast to multicellular organisms such as fungi, plants, and animals, with multiple genes that express different aquaporins, demonstrating the basic role of these proteins in maintaining the physiological functions of all organisms [13–15]. In humans, up to 13 different aquaporins have been cloned and characterized based on their homology to the sequence of the major intrinsic protein family (Figure 1). Based on their structure and permeability, three subfamilies of aquaporins are currently differentiated in mammals: (1) the classical or orthodox aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), which act as canonical water channels; (2) the aquaglyceroporins (AQP3, AQP7, AQP9, and AQP10) that are permeable to water, glycerol, and certain small solutes with neutral charge; and (3) the superaquaporins (AQP11 and AQP12), which are the subfamily with an amino acid sequence most distant to the rest of the major intrinsic protein members and are water-permeable; AQP11 can also facilitate the diffusion of glycerol and hydrogen peroxide through the membrane [ 16 – 18 ]. Recently, other AQPs besides AQP11 have been described as permeable to hydrogen peroxide (AQP1, AQP3, AQP5, AQP8, AQP9), and constitute the so called peroxiporins [19]. Biomolecules2022,12,xFORPEERREVIEW2of18 aqueousporesintheplasmamembrane,whoseexpressionandfunctioncouldvaryin differentcelltypes.ItwasinsuchascenariothatPrestonandAgre(1992)founda28kDa polypeptide,whosestructuralanalysisrevealeditasanewintegralmembraneprotein notrelatedtotherhesus(Rh)groupofbloodproteins[10].Thisproteinwascalledthe channel‐formingintegralproteinof28kDa(CHIP‐28)andwouldlaterreceivethename aquaporin1(AQP1)[11,12].Sincethen,morethan450differentvarietiesofaquaporins havebeenidentified,beingpresentinallkingdomsofnature,fromsingle‐cellorganisms suchasarchaea,bacteria,andyeasttomulticellularorganismssuchasfungi,plants,and animals,withmultiplegenesthatexpressdifferentaquaporins,demonstratingthebasic roleoftheseproteinsinmaintainingthephysiologicalfunctionsofallorganisms[13–15]. Inhumans,upto13differentaquaporinshavebeenclonedandcharacterizedbased ontheirhomologytothesequenceofthemajorintrinsicproteinfamily(Figure1).Based ontheirstructureandpermeability,threesubfamiliesofaquaporinsarecurrentlydifferentiatedinmammals:(1)theclassicalororthodoxaquaporins(AQP0,AQP1,AQP2, AQP4,AQP5,AQP6,andAQP8),whichactascanonicalwaterchannels;(2)theaquaglyceroporins(AQP3,AQP7,AQP9,andAQP10)thatarepermeabletowater,glycerol,and certainsmallsoluteswithneutralcharge;and(3)thesuperaquaporins(AQP11and AQP12),whicharethesubfamilywithanaminoacidsequencemostdistanttotherestof themajorintrinsicproteinmembersandarewater‐permeable;AQP11canalsofacilitate thediffusionofglycerolandhydrogenperoxidethroughthemembrane[16–18].Recently, otherAQPsbesidesAQP11havebeendescribedaspermeabletohydrogenperoxide (AQP1,AQP3,AQP5,AQP8,AQP9),andconstitutethesocalledperoxiporins[19]. Figure 1. Phylogenetic tree of human aquaporins and other known aquaporins. The 13 aquaporins known to date in humans are represented, according to the homology in their amino acid sequence
Biomolecules 2022,12, 530 3 of 18 for each member of the family, differentiating between “strict” aquaporins (water aquaporins, at the bottom) and aquaglyceroporins (at the top). Superaquaporins, the last aquaporins identified in mammals are also shown and are the most distant in their amino acid sequence homology from the two groups mentioned above but are closer to the aquaporins expressed in species of intracellular parasites such as Trypanosoma cruzi (TcAQP) and of nematodes such as Caenorhabditis elegans and Caenorhabditis briggsae . The tree also shows certain bacterial aquaporins such as GlpF, a facilitator of the passage of glycerol, which are similar in their sequence to the aquaglyceroporins, and AQPZ from E. coli, as well as the aquaporin from methanogen archaea, AQPM from Methanothermobacter marburgensis and the plant aquaporins from Arabidopsis thaliana (small basic intrinsic protein [SIP] and plasma membrane intrinsic protein [PIP], respectively). Modified from Gorelick et al., 2006 [17]. 2. Expression in the Cell Membrane Aquaporins are small proteins whose size ranges from 255 to 342 amino acids (approximately 30 kDa) and consist of six α -helices, joined by five connecting loops, with the terminal amino and carboxyl domains located on the intracellular side. The water permeability of aquaporins is very high: up to 3 × 10 9 water molecules are estimated to pass through each monomer per second [ 16 , 20 ]. Since they were discovered, aquaporins have attracted attention not only for their significant water transport capacity but also for their high selectivity and tetrameric arrangement in the cell membrane. Each monomer of an aquaporin constitutes an independent pore. Electronic crystallography and X-rays made it possible to determine that these monomers are assembled into a homotetrameric structure in the membrane [ 21 ]. The four monomers form a central pore whose function has not yet been clearly established [22,23]. Aquaporin-4 (AQP4) is the only known aquaporin to show a higher ordering level in the membrane. Tetramers are assembled in supramolecular structures called orthogonal arrays of particles (OAP) [ 24 ], which are stabilized by the interaction between aminoterminal residues of AQP4 monomers. AQP4 presents various isoforms that are generated by alternative splicing [ 25 , 26 ], the two best known of which are AQP4-M1 and AQP4-M23. The two isoforms differ by 22 amino acids, due to the presence of two translation initiation sites, one in methionine Met1 (AQP4-M1) and the other in Met23 (AQP4-M23) [ 27 ]. The shorter N-terminal end allows the formation of orthogonal structures or matrices of the isoform homotetramers AQP4-M23. Although the longer and voluminous N-terminal of AQP4-M1 hinders the stabilization of the crystalline structure due to steric obstacles [ 28 , 29 ], no differences in the water permeability of biological membranes have been detected for either of these isoforms [ 30 ]. The only differences in biological function among both isoforms have been observed regarding the capacity that AQP4-M23 has to form OAP in the membrane respect to AQP4-M1 that seems to be less efficient forming those structural arrays. Although the function of OAPs is still unknown, it has been speculated that alterations in this organization could contribute to explain certain pathologies, as neuromyelitis optica. In this autoimmune disease, the presence of serum IgG autoantibodies against AQP4 is an effective marker for the accurate diagnosis of the disease [ 31 ]. The AQP4-M23 isoform appears to activate the immune system, in fact, neuromyelitis optica autoantibodies show greater reactivity against AQP4-M23 than AQP4-M1 [ 32 ]. Moreover, it has also been suggested that differences in the M1-M23 ratio might lead to changes in cell migration and adhesion in astrocytes [27]. 3. Aquaporin Expression in the Central Nervous System Aquaporin expression is not restricted to a single organ or tissue (Table 1); aquaporins presentin the central nervous system arethereforenotexclusiveto itandcan befound elsewhere.
Biomolecules 2022,12, 530 4 of 18 Table 1. Distribution of aquaporins in humans and the diseases associated with their dysfunction or absence. Modified from Verkman et al., 2014. [28]. Protein Tissue Expression Associated Diseases Functions References AQP0 Fiber cells of the crystalline lens Congenital cataracts Cell adhesion Verkman et al., 2002 [29] AQP1 Erythrocytes, eye, choroid plexuses, kidneys, heart, lungs, vascular endothelium, skin, gastrointestinal tract, salivary gland, liver, testes, ovaries, and uterus, among others Pulmonary, cerebral and peripheral edema, glaucoma, skin cancer Urinary concentration, migration, proliferation Ma et al., 1998 [33]; Osorio et al. [34]; Galan et al. [35–37]. AQP2 Renal collecting duct, ears, ductus deferens Nephrogenic diabetes insipidus ADH-Hormone dependent water reabsorption Deen et al., 1994 [38] AQP3 Kidneys, leukocytes, erythrocytes, skin, eyes, respiratory tract, gastrointestinal tract, ovaries Skin and breast cancer Sweating and skin elasticity. Glycerol homeostasis. Echevarría et al., 1994; Ma et al., 2002; Levine and Verkman., 2006. [39–41]; Marlar et al. [42]; Osorio et al. [34]; Zhu et al. [43]; Azad et al. [44] AQP4 Brain, retinas, salivary and lacrimal glands, olfactory and auditory epithelium, gastrointestinal tract, kidneys, lungs, and skeletal muscle Cerebral edema, deafness, anosmia Control of brain water balance Manley et al., 2000; Jarius et al., 2010 [31,45] AQP5 Epithelia of lacrimal, sweat, and salivary glands, alveolar, bronchial, and tracheal mucosa, ovaries, kidneys, and skin Bronchial asthma, chronic bronchitis, Sjögren’s syndrome, palmoplantar keratoderma Absorption and secretion of water in pulmonary and salivary mucosa Tsubota et al., 2001 [46] AQP6 Intracellular vesicles of the renal collector tubule and brain Unidentified Unidentified Yasui et al., 1999 [47] AQP7 Adipose tissue, carotid body, testes, kidneys, heart, skeletal muscle, and ovaries Hyperglycemia, platelet defect, obesity, inflammation Lipid metabolism from adipocytes Skowronki et al., 2007; Muñoz-Cabello et al., 2010 [48,49]; Da Silva et al. [50] AQP8 Liver, pancreas, gastrointestinal tract, salivary glands, kidneys, lungs and trachea, testes, and ovaries Cancer Bile production Huebert et al., 2002; Saparov et al., 2007 [51,52]; Zhu et al. [53] AQP9 Liver, leukocytes, erythrocytes, testes, ovaries, brain, kidneys, and spleen Hyperglycemia Hepatic lipid metabolism Amiry-Moghaddam et al., 2005 [54] AQP10 Small intestine, specifically in the duodenum and jejunum Psoriasis, skin dermatitis Lipid metabolism Ishibashi et al., 2002 [55]; Soler et al. [56] AQP11 Testes, heart, kidneys, ovaries, muscles, gastrointestinal tract, leukocytes, liver, brain Obesity and Inflammation RE stress Morishita et al., 2005; Koike et al., 2016 [57,58]; Frühbeck et al. [59]; Ishibashi et al. [60] AQP12 Acinar cells of the pancreas Inflammation Pancreatic function Boone and Deen., 2009 [61]; Da Silva et al. [50] In the brain, AQPs mediate the movement of water between the different fluid compartments (intracellular fluid, interstitial fluid [ISF], cerebrospinal fluid [CSF], and blood) [62–66] . Currently, 9 AQPs have been identified in different sites in the central nervous system (CNS): AQP1 [ 67 – 69 ], AQP3 [ 70 , 71 ], AQP4 [ 72 – 74 ], AQP5 [ 75 ], AQP6 [ 76 , 77 ], AQP7 [ 71 , 74 , 78 ], AQP8 [ 53 , 70 , 79 , 80 ], AQP9 [ 30 , 31 , 71 , 74 ], and AQP11 [ 17 , 58 ] (Figures 1and 2) . Table 1de- scribes the location and certain properties of the aquaporins expressed in the CNS, as well as a number of conditions associated with their dysfunction in humans.
Biomolecules 2022,12, 530 5 of 18 Biomolecules2022,12,xFORPEERREVIEW6of18 Figure2.Molecularstudyofthemousenervoussystemusingsingle‐cellRNAsequencingtechnology.The265distinctcellclustersidentifiedbasedontheanalysisof160,796transcriptomesofthe mousenervoussystem:brain,spinalcord,dorsalandperipheralsympatheticganglia,andenteric nervoussystemarerepresentedandclassifiedinthetreediagrams;(A)TreediagramforNeuron clusters,and(B)Treediagramfornon‐neuronclusters.Therepresenteddendrogramhasbeenconstructedusingthedigitalresource(http://mousebrain.org;accessedon23October2019);prepared inthelaboratoryofStenLinnarsson,obtainedfromZeiseletal.(2018)[81],andshowstherelationshipbetweencelltypesandthegeneexpressionofAQP1,AQP4,AQP9,andAQP11.Thecolorgradedbandsatthebottomofthefigureshowtheexpressionpatternofeachgeneinthe265identifiedcelltypes.Eachbandrepresentstherelativeexpressionofatranscriptinacelltypewithrespecttothemaximumexpressiondetectedforthatsametranscriptinanycelltype.Giventhatthey arerelativedata,therichnessofexpressionofthedifferenttranscriptscannotbecomparedwith eachother,buttheydohelplocatewheretheexpressionoccurs.Thus,AQP4andAQP9appearto showaverysimilarexpressionpattern,clearlypredominantintheastroependymalline,although theirexpressionlevelsareverydifferent.AQP1expressioniswelldefinedinthedorsalgangliaand ependymalcellsofthechoroidplexusandcontrastswiththehighdelocalizationofAQP11.The dataonabsoluteexpressionarerepresentedinTable2. Figure 2. Molecular study of the mouse nervous system using single-cell RNA sequencing technology. The 265 distinct cell clusters identified based on the analysis of 160,796 transcriptomes of the mouse nervous system: brain, spinal cord, dorsal and peripheral sympathetic ganglia, and enteric nervous system are represented and classified in the tree diagrams; ( A ) Tree diagram for Neuron clusters, and ( B ) Tree diagram for non-neuron clusters. The represented dendrogram has been constructed using the digital resource (http://mousebrain.org; accessed on 23 October 2019); prepared in the laboratory of Sten Linnarsson, obtained from Zeisel et al. (2018) [ 81 ], and shows the relationship between cell types and the gene expression of AQP1, AQP4, AQP9, and AQP11. The color-graded bands at the bottom of the figure show the expression pattern of each gene in the 265 identified cell types. Each band represents the relative expression of a transcript in a cell type with respect to the maximum expression detected for that same transcript in any cell type. Given that they are relative data, the richness of expression of the different transcripts cannot be compared with each other, but they do help locate where the expression occurs. Thus, AQP4 and AQP9 appear to show a very similar expression pattern, clearly predominant in the astroependymal line, although their expression levels are very different. AQP1 expression is well defined in the dorsal ganglia and ependymal cells of the choroid plexus and contrasts with the high delocalization of AQP11. The data on absolute expression are represented in Table 2.
Biomolecules 2022,12, 530 6 of 18 Table 2. Gene expression pattern of brain aquaporins according to cell type analysis using single-cell RNA sequencing technology. Each row represents a cell type and the expression richness of the AQP1, AQP4, AQP9 and AQP11 genes in that cluster. The pattern is based on the calculation of the frequency of expression in a cell population, using a binomial β -Bayesian model (trinarization) of the raw data of the expression of each gene in each cell of all clusters. Therefore, the values represented are defined as the frequency of gene expression in a percentage of cells in that cluster. Values > 0.2 are considered positive, with an expression error <0.05. The data presented in the table were obtained with the digital resource (http://mousebrain.org; accesed on 23 October 2019) from Zeisel et al., 2018 [81] . #, represents a specific cell cluster, out of the 265 distinct cell clusters identified. Index Cluster # Name Description Cell Types Gene Expression Aqp1 Aqp4 Aqp9 Aqp11 7 TEGLU10 Excitatory neurons, cerebral cortex - 0.02 - 0.26 9 TEGLU8 Excitatory neurons, cerebral cortex - 0.01 - 0.27 14 TEGLU5 Excitatory neurons, cerebral cortex - 0.06 - 0.21 15 TEGLU16 Excitatory neurons, cerebral cortex - 0.08 - 0.26 16 TEGLU15 Excitatory neurons, cerebral cortex - 0.05 - 0.35 17 TEGLU17 Excitatory neurons, cerebral cortex - 0.02 - 0.27 21 TEGLU21 Excitatory neurons, hippocampus CA1 - 0.02 - 0.22 24 TEGLU23 Excitatory neurons, hippocampus CA3 - 0.01 - 0.77 57 TEINH13 Trilaminar cells, hippocampus - 0.02 - 0.22 69 HBCHO4 Afferent nuclei of cranial nerves III-V 0.39 0.10 - 0.21 73 HYPEP7 Pmch neurons, hypothalamus - - - 1.23 77 MBDOP2 Dopaminergic neurons, ventral midbrain (SNc, VTA) 0.03 0.02 0.01 0.06 131 MEGLU6 Excitatory neurons, midbrain - 0.05 - 0.36 160 HBINH2 Inhibitory neurons, hindbrain - 0.50 -0.23 161 HBCHO1 Cholinergic neurons, hindbrain 0.10 0.10 - 0.20 162 HBCHO2 Cholinergic neurons, hindbrain - 0.05 - 0.23 165 HBGLU6 Excitatory neurons, hindbrain - 0.04 - 0.27 167 HBGLU8 Excitatory neurons, hindbrain - 0.09 - 0.73 185 ENT4 Cholinergic enteric neurons 0.56 0.01 - 0.01 186 ENT5 Cholinergic enteric neurons 0.68 - - 0.03 188 ENT7 Cholinergic enteric neurons, VGLUT2 0.03 - - 0.23 194 SYNOR4 Noradrenergic erector muscle neurons 0.84 - - 0.22 195 SYNOR5 Noradrenergic erector muscle neurons 0.81 - - 0.02 200 PSPEP6 Peptidergic (TrpM8), DRG 0.63 - - 0.07 201 PSPEP5 Peptidergic (PEP1.2), DRG 2.93 - - 0.10 202 PSPEP2 Peptidergic (PEP1.3), DRG 5.70 0.03 - 0.28 203 PSPEP4 Peptidergic (PEP1.1), DRG 2.93 - - 0.04 204 PSPEP3 Peptidergic (PEP1.4), DRG 4.02 0.01 0.01 0.14 205 PSPEP1 Peptidergicv (PEP2), DRG 2.22 0.01 - 0.35 206 PSNF3 Neurofilament (NF2/3), DRG 0.03 0.03 - 0.42 209 PSNP1 Non-peptidergic (TH), DRG 3.82 0.02 - 0.07 210 PSNP2 Non-peptidergic (NP1.1), DRG 1.49 - - 0.38 211 PSNP3 Non-peptidergic (NP1.2), DRG 4.19 0.01 - 0.32 212 PSNP4 Non-peptidergic (NP2.1), DRG 0.47 - - 0.11 214 PSNP6 Non-peptidergic (NP3), DRG 1.43 0.03 - 0.27 224 CHOR Chorid plexus epithelial cells 1.93 0.05 - 0.07 227 EPEN Ependymal cells - 3.61 0.01 0.03
Biomolecules 2022,12, 530 7 of 18 Table 2. Cont. Index Cluster # Name Description Cell Types Gene Expression Aqp1 Aqp4 Aqp9 Aqp11 228 EPMB Ependymal cells, midbrain - 1.29 0.01 0.04 230 RGSZ Subventricular zone radial glia-like cells - 0.43 0.02 0.01 231 ACTE1 Telencephalon astrocytes, fibrous - 2.94 0.03 0.04 232 ACTE2 Telencephalon astrocytes, protoplasmic - 1.09 0.04 0.04 233 ACOB Olfactory astrocytes - 3.25 0.04 0.12 234 ACNT1 Non-telencephalon astrocytes, protoplasmic - 2.10 0.03 0.03 235 ACNT2 Non-telencephalon astrocytes, fibrous - 3.80 0.02 0.04 236 ACMB Dorsal midbrain Myoc-expressing astrocyte-like - 10.5 0.04 0.07 237 ACBG Bergmann glia - 2.49 0.04 0.01 254 VECA Vascular endothelial cells, arterial - 0.02 - 0.21 257 PER1 Pericytes - 0.01 - 0.23 260 VECV Vascular endothelial cells, venous - 0.01 - 0.29 Using recently available data generated from single-cell RNA sequencing technology in the CNS of mice [ 81 ], we analyzed the expression of the three most widely expressed brain AQPs, namely AQP1, AQP4, and AQP9. The first two (AQP1 and AQP4) are aquaporins strictly permeable to water and extensively associated with CSF homeostasis [ 63 – 66 ], while AQP9, an aquaglyceroporin, might play a role in brain energy metabolism [ 63 – 66 ]. Data on AQP11, the most recent aquaporin detected in the brain, were also included in the analysis [ 17 , 58 ] (Table 1), allowing us to compare the findings based on classical reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunostaining, with this novel and powerful tool for detecting gene expression at the unique cell level. The transcriptome analysis performed by Zeisel et al. [ 81 ] of half a million single cells obtained from the mouse nervous system helped identify 265 clusters of distinct cell types and determine the specific repertoire of genes expressed in them. The classification of each cell cluster was organized based on three aspects: (1) cell type (e.g., neurons, astrocytes), (2) developmental/anatomical region (e.g., telencephalon, midbrain, diencephalon), and (3) neurotransmitter type (e.g., dopamine, gamma aminobutyric acid, glutamate). Lastly, the gene expression pattern for each cell cluster was obtained based on the expression frequency of a given gene in each cell of a cell cluster with respect to the expression in all the analyzed cells. Expression levels for AQP1, AQP4, AQP9, and AQP11 in each of the 265 identified cell clusters were obtained by using the tool provided by Zeisel et al. (http://mousebrain.org; accessed on 23 October 2019) (Figure 2, Table 2). As shown in Table 2, the data analyzed by single-cell RNA sequencing validate the high AQP1 expression in cell cluster #224, corresponding to choroid plexus epithelial cells (#224-CP epithelial cells: 1.93), thereby corroborating the AQP1 expression previously reported in the plasma membrane of epithelial cells of the choroid plexus in the brain of primates and rodents. A number of studies have also identified the presence of AQP1 in the dorsal horn of the spinal cord and in the sensory ganglion of the trigeminal nerve associated with nociception [ 73 ]. In fact, the single-cell RNA sequencing analysis shows abundant AQP1 expression in cell groups #200–214, representative of the dorsal root ganglia of the spinal cord (Table 2). Moreover, single-cell RNA sequencing data supported the presence of AQP1 in the afferent pathway of the fifth cranial nerve, of the trigeminal nerve (#69 -Afferent nuclei of cranial nerves III–V: 0.39), as well as in the ganglia of motor neurons (clusters# 194, 195: 0.81–0.84). AQP4, the most abundant aquaporin in the CNS, is mainly located in the ependymalglial-limiting membranes, including the processes of the subpial astrocytes in the cortex, the subependymal glia and the ependymal cells that border the brain ventricles, and in the feet of the perivascular astrocytes that surround the blood vessels forming the blood-
Biomolecules 2022,12, 530 8 of 18 brain barrier [ 53 , 74 ]. This AQP4 distribution has been corroborated with the expression data by single-cell RNA sequencing (Table 2). We observed that AQP4 presents a highly marked expression pattern in ependymal cells (cluster #227-Ependymal cells: 3.61; cluster #228-Ependymal cells in the midbrain: 1.29), which are cylindrical-shaped ciliated cells bordering the brain ventricles that participate in CSF circulation and homeostasis. AQP4 is also highly expressed in subventricular zone radial glia-like cells (cluster #230: 0.43) and in various astrocytes (clusters #231–236: 1.09–10.5). In general, the data reflected in Table 2confirm that AQP4 is the cerebral aquaporin with the broadest and highest expression in the mouse CNS. Although AQP4 is also detected at extremely low levels in neurons, its expression is mainly localized in ependymal cells and astrocytes, with significant differences in expression levels between various types of astrocytes. For example, there is a relative abundance of AQP4 expression in fibrous astrocytes in the cerebral cortex compared with protoplasmic cortical astrocytes (cluster #231—Telencephalon astrocytes, fibrous: 2.94; vs. cluster #232—Telencephalon astrocytes, protoplasmic: 1.09). Additionally, differential AQP4 expression can be observed in fibrous versus protoplasmic astrocytes from non-tencephalic regions, such as those of the thalamus, medulla oblongata, and spinal cord (cluster #235—Non-telencephalon astrocytes, fibrous: 3.80; vs. cluster #234—Non-telencephalon astrocytes, protoplasmic: 2.10). Notable expression was observed in other glial cells, such as astrocytes in the olfactory bulb and Bergmann glia (cluster #233—Olfactory astrocytes: 3.25; cluster #237—Bergmann glia: 2.49). Interestingly, the cell type with the highest AQP4 expression is a specific cell cluster of astrocytes located in the dorsal midbrain, with relative expression levels above 10 (cluster #236—Dorsal midbrain Myoc-expressing astrocyte-like: 10.5). Consistent with this individual cell RNA expression, previous findings have already reported AQP4 expression in sensory organs such as Müller cells in the retina [ 82 ], in the olfactory epithelium, and in Claudius and Hensen cells in the inner ear [82,83]. AQP9 expression in the CNS has been reported in astrocytes, ependymocytes, tanycytes (specific ependymocytes in the third and fourth ventricles that send processes to the hypothalamus), endothelial cells of pial vessels, and dopaminergic neurons of the midbrain (ventral tegmental area and substantia nigra) [ 84 ]. The similarities in expression patterns between AQP4 and AQP9 in the brain has led to the proposal that both proteins could act synergistically in the transfer of water between the CSF and brain parenchyma. An analysis of the expression data by single-cell RNA sequencing effectively reflects that AQP9 is predominantly distributed in the astroependymal line. However, AQP9 expression is much lower than that observed for AQP4, being below the threshold value (0.2), which considers that the gene expression is positive in a group of cells (Table 2). Therefore, AQP9 expression, although very minor, was found in the dopaminergic neurons of the ventral tegmental area and the substantia nigra (cluster #77—Dopaminergic neurons: Substantia nigra and Ventral tegmental area: 0.0146) and in the same groups (clusters #227–237: 0.01–0.12) of the astroependymal type, where the AQP4 expression was indicated above. Of the various aquaporins identified in the CNS, AQP11 is the least known and has been proposed to play an important role in the cellular response to oxidative stress, as well as in the translation and protein-folding processes, given its location in the endoplasmic reticulum [ 19 , 58 , 85 ]. The presence of AQP11 has been reported in the cerebellum, specifically in the dendrites of Purkinje cells, in neurons from the CA1 and CA3 regions of the hippocampus, in neurons from the cerebral cortex (layers II–VI), and in endothelial cells from the brain parenchyma and choroidal plexus epithelial cells [ 17 , 58 ]. The single-cell RNA sequencing analysis revealed the broad AQP11 distribution in almost all cell clusters identified in this study, although AQP11 expression is very minor and in many cases below the threshold. Specifically, nonsignificant AQP11 expression was observed in the neurons in the cerebellum, in Purkinje cells (clusters #172–178: <0.2; data not shown), and in choroidal plexus epithelial cells (cluster #224—Choroid plexus epithelial cells: 0.07). In other regions such as the hippocampus (cluster #21—Excitatory neurons, hippocampus CA1: 0.22 ; cluster #24—Excitatory neurons, hippocampus CA3: 0.77;
Biomolecules 2022,12, 530 9 of 18 cluster #57—Trilaminar cells , hippocampus: 0.22), cerebral cortex (clusters #7–17: Excitatory neurons, Cerebral Cortex: >0.2 ), and vascular endothelial cells (cluster #254—Vascular endothelial cells, arterial: 0.21; cluster #257—Pericytes : 0.23; cluster #260—Vascular endothelial cells, venous: 0.29), the data show a greater presence of AQP11, validating previous reports. Lastly, it is worth highlighting the strong expression observed in hypothalamic neurons (cluster #73—Pmch neurons Hypothalamus: 1.23), which could be associated with the endocrine function. 4. The Role of Aquaporins in Cerebrospinal Fluid Homeostasis Since the beginning of the twentieth century, physiologists interested in understanding CSF homeostasis have accepted that the circulation of cerebrospinal liquid has a unidirectional path [ 86 , 87 ]. Consequently, with the “classic view”, CSF would be produced primarily by the choroid plexus to fill the ventricles and circulate unidirectionally toward the subarachnoid space and the central canal of the spinal cord, ultimately draining through arachnoid granulations into the meningeal sinus or through lymphatic vessels of the nasal mucosa after crossing the cribriform plate [ 85 , 88 ]. Regarding the role of aquaporins in this circuit, AQP1 was classically associated with CSF production, given its exclusive presence in the apical membrane of choroidal plexus epithelial cells. AQP4, abundantly present in ependymal cells, glia limitans, and astrocyte processes in the pericapillary end foot, was mainly associated with ISF/CSF exchange and CSF absorption. However, several findings led to an updating of the classic concept to a more complex hypothesis (but one surely closer to reality) for understanding CSF production and circulation [ 89 ]. Experiments with aquaporin knock-out animals have definitively contributed to clarifying the role of aquaporins in the CSF. In 2005, the study by Oshio et al. [ 90 ] using AQP1 −/− mice essentially demonstrated the prominent role of AQP1 in CSF formation. In these knock-out animals, a decrease in CSF production of approximately 20% and reduced intraventricular pressure (IVP) demonstrated the participation of AQP1 in CSF production. However, experiments in which 17 O-labeled water (H 2 O 17 ) was administered intravenously in AQP1 −/− and AQP4 −/− mice showed that water entry into the lateral ventricles was significantly reduced in AQP4 −/− mice but not in AQP1 −/− mice, indicating the important role of AQP4 in CSF formation [ 91 ]. Concomitantly, the finding also supports the important and constant formation of large amounts of CSF distributed in the brain as a consequence of water exchange between the brain capillaries and ISF [ 92 ], and between ISF and CSF through ependymal cells. Furthermore, the description of AQP4-facilitated perivascular flow regulating the CSF/ISF exchange between periarterial and perivenous Virchow-Robin spaces [ 93 – 95 ] that could respond to a so-called “glymphatic” system for the drainage of CSF and proteins [ 93 ] provided additional support for the important role of AQP4 in CSF homeostasis. Using single AQP1 −/− , AQP4 −/− , and double-AQP1 −/− :AQP4 −/− knock-out mice, our recent study strongly reinforced the clear role of both aquaporins in CSF homeostasis. When we evaluated the ventricular volume by magnetic resonance imaging, as a readout of CSF formation, we confirmed that CSF production is reduced by the lack of not only AQP1 but also AQP4 and to a similar degree [ 96 ]. The results confirm that both choroidal-expressed AQP1 and extrachoroidal-expressed AQP4 contribute comparably to CSF production and IVP. Furthermore, both aquaporins also play a role in the CSF evacuation process and in ventricular compliance [ 96 ]. We found that although the absence of either of the two AQPs did not significantly affect CSF output at low perfusion values, the simultaneous lack of both proteins significantly reduced CSF efflux and distensibility of the cerebral ventricular system. However, it is still unclear how the lack of these cerebral aquaporins could modify the structural properties of the CSF drainage routes, the ventricle ependyma, or the cerebral parenchyma so as to modify these processes. In addition to AQP1 and AQP4, other aquaporins are also expressed in the brain, as previously indicated. Of these, AQP9 and AQP11 appear to be the next most abundant, but their specific contribution to the various aspects of CSF homeostasis and dynamics
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