The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice.

Purpose: Earlier reports highlighted the predominant presence of aquaporin 4 (AQP4) in the duct cells of rabbit lacrimal glands (LGs). Whereas significant alterations in AQP4 mRNA levels have been observed in experimental dry eye and during pregnancy, the impact of AQP4 in LG ductal fluid production remains unclear. In our recent work, the role of AQP4 in LG ductal fluid secretion was investigated utilizing wild type (WT) and AQP4 knock out (KO) mice. Methods: Tear production was assessed in both WT and KO animals. Immunostaining was used to identify AQP4 protein. Duct segments were harvested from LGs of WT and KO mice. Fluid secretion and filtration permeability (Pf) were quantified using video-microscopy. Ductal tear production, elicited by a cell-permeable cAMP analogue (8-bromo cAMP), carbachol, vasoactive intestinal peptide (VIP), and phenylephrine (PHE), were assessed in both WT and KO ducts. Results: A higher expression of AQP4 protein was noted in the duct cells from WT mice when compared to acinar cells. Pf did not show notable alterations between WT and AQP4 KO ducts. Carbachol elicited comparable secretory responses in ducts from both WT and KO animals. However, 8-bromo cAMP, VIP, and PHE stimulation resulted in decreased secretion in ducts from AQP4 KO LGs. Conclusions: Our findings underscore the functional relevance of AQP4 in the fluid production of mouse LG ducts. AQP4 seems to play different roles in fluid secretions elicited by different secretagogues. Specifically, cAMP-mediated, and adrenergic agonist-related secretions were reduced in AQP4 KO ducts.

Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy.

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke.

BACKGROUND: Notch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. METHODS: Sprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). RESULTS: Compared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. CONCLUSIONS: The present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Voluntary wheel exercise improves glymphatic clearance and ameliorates colitis-associated cognitive impairment in aged mice by inhibiting TRPV4-induced astrocytic calcium activity.

BACKGROUND AND OBJECTIVES: Chronic colitis exacerbates neuroinflammation, contributing to cognitive impairment during aging, but the mechanism remains unclear. The polarity distribution of astrocytic aquaporin 4 (AQP4) is crucial for the glymphatic system, which is responsible for metabolite clearance in the brain. Physical exercise (PE) improves cognition in the aged. This study aims to investigate the protective mechanism of exercise in colitis-associated cognitive impairment. METHODS: To establish a chronic colitis model, 18-month-old C57BL/6 J female mice received periodic oral administration of 1% wt/vol dextran sodium sulfate (DSS) in drinking water. The mice in the exercise group received four weeks of voluntary wheel exercise. High-throughput sequencing was conducted to screen for differentially expressed genes. Two-photon imaging was performed to investigate the function of the astrocytic calcium activity and in vivo intervention with TRPV4 inhibitor HC-067047. Further, GSK1016790A (GSK1), a TRPV4 agonist, was daily intraperitoneally injected during the exercise period to study the involvement of TRPV4 in PE protection. Colitis pathology was confirmed by histopathology. The novel object recognition (NOR) test, Morris water maze test (MWM), and open field test were performed to measure colitis-induced cognition and anxiety-like behavior. In vivo two-photon imaging and ex vivo imaging of fluorescent CSF tracers to evaluate the function of the glymphatic system. Immunofluorescence staining was used to detect the Aß deposition, polarity distribution of astrocytic AQP4, and astrocytic phenotype. Serum and brain levels of the inflammatory cytokines were tested by Enzyme-linked immunosorbent assay (ELISA). The brain TUNEL assay was used to assess DNA damage. Expression of critical molecules was detected using Western blotting. RESULTS: Voluntary exercise alleviates cognitive impairment and anxiety-like behavior in aged mice with chronic colitis, providing neuroprotection against neuronal damage and apoptosis. Additionally, voluntary exercise promotes the brain clearance of Aß via increased glymphatic clearance. Mechanistically, exercise-induced beneficial effects may be attributed, in part, to the inhibition of TRPV4 expression and TRPV4-related calcium hyperactivity, subsequent promotion of AQP4 polarization, and modulation of astrocyte phenotype. CONCLUSION: The present study reveals a novel role of voluntary exercise in alleviating colitis-related cognitive impairment and anxiety disorder, which is mediated by the promotion of AQP4 polarization and glymphatic clearance of Aß via inhibition of TRPV4-induced astrocytic calcium hyperactivity.

Single-molecule imaging of aquaporin-4 array dynamics in astrocytes.

Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in regulating cerebrospinal fluid (CSF) circulation and facilitating the clearance of solutes from the extracellular space of the brain. Adrenergic signaling has been shown to modulate the volume of the extracellular space of the brain via AQP4 localized at the end-feet of astrocytes, but the mechanisms by which AQP4 regulates CSF inflow and outflow in the brain remain elusive. Using advanced imaging techniques, including super-resolution microscopy and single-molecule tracking, we investigated the hypothesis that ß-adrenergic receptor activation induces cellular changes that regulate AQP4 array size and mobility, thus influencing water transport in the brain. We report that the ß-adrenergic agonist, isoproterenol hydrochloride, decreases AQP4 array size and enhances its membrane mobility, while hyperosmotic conditions induce the formation of larger, less mobile arrays. These findings reveal that AQP4 arrays are dynamic structures, responsive to adrenergic signals and osmotic changes, highlighting a novel regulatory mechanism of water transport in the brain. Our results provide insights into the molecular control of CSF circulation and extracellular brain space volume, laying the groundwork for understanding the relationship between astrocyte water transport, sleep physiology, and neurodegeneration.

Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover.

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury in diabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascular changes in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visual cycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18). Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved in the visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposure alleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascular Aqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantly up-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantly higher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via its anti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression.

AAV-Mediated Restoration of Dystrophin-Dp71 in the Brain of Dp71-Null Mice: Molecular, Cellular and Behavioral Outcomes.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

Ginkgolide B can alleviate spinal cord glymphatic system dysfunction and provide neuroprotection in painful diabetic neuropathy rats by inhibiting matrix metalloproteinase-9.

The glymphatic system plays a crucial role in maintaining optimal central nervous system (CNS) function by facilitating the removal of metabolic wastes. Aquaporin-4 (AQP4) protein, predominantly located on astrocyte end-feet, is a key pathway for metabolic waste excretion. ß-Dystroglycan (ß-DG) can anchor AQP4 protein to the end-feet membrane of astrocytes and can be cleaved by matrix metalloproteinase (MMP)-9 protein. Studies have demonstrated that hyperglycemia upregulates MMP-9 expression in the nervous system, leading to neuropathic pain. Ginkgolide B (GB) exerts an inhibitory effect on the MMP-9 protein. In this study, we investigated whether inhibition of MMP-9-mediated ß-DG cleavage by GB is involved in the regulation of AQP4 polarity within the glymphatic system in painful diabetic neuropathy (PDN) and exerts neuroprotective effects. The PDN model was established by injecting streptozotocin (STZ). Functional changes in the glymphatic system were observed using magnetic resonance imaging (MRI). The paw withdrawal threshold (PWT) was measured to assess mechanical allodynia. The protein expressions of MMP-9, ß-DG, and AQP4 were detected by Western blotting and immunofluorescence. Our findings revealed significant decreases in the efficiency of contrast agent clearance within the spinal glymphatic system of the rats, accompanied by decreased PWT, increased MMP-9 protein expression, decreased ß-DG protein expression, and loss of AQP4 polarity. Notably, GB treatment demonstrated the capacity to ameliorate spinal cord glymphatic function by modulating AQP4 polarity through MMP-9 inhibition, offering a promising therapeutic avenue for PDN.

Regulation of renal aquaporin water channels in acute pyelonephritis.

Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.

Role of aquaporin-4 polarization in extracellular solute clearance.

Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4-/-) or lacking the endfoot pool of AQP4 (Snta1-/-). We confirm that Aqp4-/- mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1-/- mice have reduced clearance only for the 500 kDa [3H]dextran, but not 0.18 kDa [3H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [3H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.

MAIN POINTS: Solute clearance is reduced in mice lacking AQP4 Polarization of AQP4 to the endfeet may have a greater impact on clearance of large versus small molecules Clearance of large but not small solutes is correlated with age within adult age.

Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice.

Glymphatic system denotes a brain-wide pathway that eliminates extracellular solutes from brain. It is driven by the flow of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) via perivascular spaces. Glymphatic convective flow is driven by cerebral arterial pulsation, which is facilitated by a water channel, aquaporin-4 (AQP4) expressed in astrocytic end-foot processes. Since its discovery, the glymphatic system receives a considerable scientific attention due to its pivotal role in clearing metabolic waste as well as neurotoxic substances such as amyloid b peptide. Tau is a microtubule binding protein, however it is also physiologically released into extracellular fluids. The presence of tau in the blood stream indicates that it is eventually cleared from the brain to the periphery, however, the detailed mechanisms that eliminate extracellular tau from the central nervous system remained to be elucidated. Recently, we and others have reported that extracellular tau is eliminated from the brain to CSF by an AQP4 dependent mechanism, suggesting the involvement of the glymphatic system. In this chapter, we describe the detailed protocol of how we can assess glymphatic outflow of tau protein from brain to CSF in mice.

The Expression and Role of Aquaporin 4 in Colon Cancer.

BACKGROUND/AIM: Aquaporins (AQPs) were initially discovered as water channel proteins that facilitate transcellular water movements. Recent studies have shown that AQPs are expressed and play an oncogenic role in various cancers. However, the expression and role of Aquaporin 4 (AQP4) in colon cancer have not been investigated. This study aimed to examine the clinical and pathophysiologic significance of AQP4 in colon cancer. PATIENTS AND METHODS: Immunohistochemistry (IHC) of AQP4 for 145 primary tumor samples obtained from patients with stage II or III colon cancer was performed, and the relationship between AQP4 expression and patients' prognoses was analyzed. Knockdown experiments with AQP4 small interfering RNA using human colon cancer cells were conducted to analyze the effects on cell invasiveness. RESULTS: IHC revealed that AQP4 was scarcely expressed in the noncancerous colonic mucosa. Of the 145 patients who enrolled in this study, 109 (75.2%) and 36 (24.8%) patients were classified as negative and positive for AQP4 expression, respectively. A high level of AQP4 expression is significantly associated with deeper tumors with lymph node metastasis and venous invasion. A 5-year progression-free survival rate of AQP4-positive patients was significantly worse than that of AQP-4 negative patients (70.7% vs. 87.0%, p=0.049). Furthermore, AQP4 knockdown significantly inhibited cell migration and invasion in HCT116 cells. CONCLUSION: AQP4 may be a novel biomarker and therapeutic target for colon cancer.

Multisensory gamma stimulation promotes glymphatic clearance of amyloid.

The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Glymphatic fluid transport is suppressed by the aquaporin-4 inhibitor AER-271.

The glymphatic system transports cerebrospinal fluid (CSF) into the brain via arterial perivascular spaces and removes interstitial fluid from the brain along perivenous spaces and white matter tracts. This directional fluid flow supports the clearance of metabolic wastes produced by the brain. Glymphatic fluid transport is facilitated by aquaporin-4 (AQP4) water channels, which are enriched in the astrocytic vascular endfeet comprising the outer boundary of the perivascular space. Yet, prior studies of AQP4 function have relied on genetic models, or correlated altered AQP4 expression with glymphatic flow in disease states. Herein, we sought to pharmacologically manipulate AQP4 function with the inhibitor AER-271 to assess the contribution of AQP4 to glymphatic fluid transport in mouse brain. Administration of AER-271 inhibited glymphatic influx as measured by CSF tracer infused into the cisterna magna and inhibited increases in the interstitial fluid volume as measured by diffusion-weighted MRI. Furthermore, AER-271 inhibited glymphatic efflux as assessed by an in vivo clearance assay. Importantly, AER-271 did not affect AQP4 localization to the astrocytic endfeet, nor have any effect in AQP4 deficient mice. Since acute pharmacological inhibition of AQP4 directly decreased glymphatic flow in wild-type but not in AQP4 deficient mice, we foresee AER-271 as a new tool for manipulation of the glymphatic system in rodent brain.

Aquaporin-4 and Parkinson's Disease.

The water-selective channel aquaporin-4 (AQP4) is implicated in water homeostasis and the functioning of the glymphatic system, which eliminates various metabolites from the brain tissue, including amyloidogenic proteins. Misfolding of the α-synuclein protein and its post-translational modifications play a crucial role in the development of Parkinson's disease (PD) and other synucleopathies, leading to the formation of cytotoxic oligomers and aggregates that cause neurodegeneration. Human and animal studies have shown an interconnection between AQP4 dysfunction and α-synuclein accumulation; however, the specific role of AQP4 in these mechanisms remains unclear. This review summarizes the current knowledge on the role of AQP4 dysfunction in the progression of α-synuclein pathology, considering the possible effects of AQP4 dysregulation on brain molecular mechanisms that can impact α-synuclein modification, accumulation and aggregation. It also highlights future directions that can help study the role of AQP4 in the functioning of the protective mechanisms of the brain during the development of PD and other neurodegenerative diseases.

Early involvement of peripherally derived monocytes in inflammation in an NMO-like mouse model.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease that primarily affects the optic nerve and spinal cord within the central nervous system (CNS). Acute astrocyte injury caused by autoantibodies against aquaporin 4 (NMO-IgG) is a well-established key factor in the pathogenesis, ultimately leading to neuronal damage and patient disability. In addition to these humoral immune processes, numerous innate immune cells were found in the acute lesions of NMO patients. However, the origin and function of these innate immune cells remain unclear in NMO pathogenesis. Therefore, this study aims to analyze the origin and functions of these innate immune cells in an NMO-like mouse model and evaluate their role in the pathophysiology of NMO. The expression of Tmem119 on Iba1 + cells in brain tissue disappeared immediately after the injection of NMO-IgG + human complement mixture, while the expression of P2ry12 remained well-maintained at 1 day after injection. Based on these observations, it was demonstrated that monocytes infiltrate the brain during the early stages of the pathological process and are closely associated with the inflammatory response through the expression of the proinflammatory cytokine IL-1ß. Understanding the variations in the expression patterns of P2ry12, Tmem119, and other markers could be helpful in distinguishing between these cell types and further analyzing their functions. Therefore, this research may contribute to a better understanding of the mechanisms and potential treatments for NMO.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

Large molecules from the cerebrospinal fluid enter the optic nerve but not the retina of mice.

It has been proposed that cerebrospinal fluid (CSF) can enter and leave the retina and optic nerve along perivascular spaces surrounding the central retinal vessels as part of an aquaporin-4 (AQP4) dependent ocular 'glymphatic' system. Here, we injected fluorescent dextrans and antibodies into the CSF of mice at the cisterna magna and measured their distribution in the optic nerve and retina. We found that uptake of dextrans in the perivascular spaces and parenchyma of the optic nerve is highly sensitive to the cisternal injection rate, where high injection rates, in which dextran disperses fully in the sub-arachnoid space, led to uptake along the full length of the optic nerve. Accumulation of dextrans in the optic nerve did not differ significantly in wild-type and AQP4 knockout mice. Dextrans did not enter the retina, even when intracranial pressure was greatly increased over intraocular pressure. However, elevation of intraocular pressure reduced accumulation of fluorescent dextrans in the optic nerve head, and intravitreally injected dextrans left the retina via perivascular spaces surrounding the central retinal vessels. Human IgG distributed throughout the perivascular and parenchymal areas of the optic nerve to a similar extent as dextran following cisternal injection. However, uptake of a cisternally injected AQP4-IgG antibody, derived from a seropositive neuromyelitis optica spectrum disorder subject, was limited by AQP4 binding. We conclude that large molecules injected in the CSF can accumulate along the length of the optic nerve if they are fully dispersed in the optic nerve sub-arachnoid space but that they do not enter the retina.