Aquaporin-4 Levels Research Articles

Single-cell analysis of osmoregulation reveals heterogeneity of aquaporin 4 functionality in human astrocytes.

The water channel aquaporin 4 (AQP4) contributes to water flow and waste removal across the blood-brain barrier and its levels, organization and localization are perturbed in various neurological diseases, including Alzheimer's Disease. This renders AQP4 a potentially valuable therapeutic target. However, most functional assays aimed at identifying modulators of AQP4 function are performed with primary rodent cells and do not consider inter-cellular variations in AQP4 abundance and presentation. To address this, we have established and applied a robust live cell microscopy assay that captures the contribution of AQP4 in the osmotically driven (de-)quenching of the vital dye Calcein-AM with single-cell resolution. Using human astrocytoma cells, we found that performing measurements on cellular regions instead of whole fields of view yielded a more sensitive readout of the osmotic response, which correlated with AQP4 abundance. Stable co-expression of the two major AQP4 isoforms, but not of the individual isoforms, provoked a faster adaptation to osmotic changes, while siRNA-mediated knockdown of AQP4 had the opposite effect. Post-hoc correlation with the canonical membrane marker CD44 revealed that the speed of the osmotic response scaled with AQP4 membrane enrichment. Coating the substrate with laminin promoted AQP4 membrane enrichment, while cell confinement with fixed-size micropatterns further increased the speed of osmoregulation, underscoring the influence of extracellular factors. The osmotic response of primary fetal astrocytes and human iPSC-derived astrocyte models was comparable to AQP4-deficient astrocytoma cells, in line with their low AQP4 levels and indicative of their immature state. In conclusion, a correlative single-cell approach based on the quantification of Calcein-AM quenching capacity, AQP4 abundance and AQP4 membrane enrichment, allows resolving osmoregulation in a more sensitive manner and reveals heterogeneity between and within human astrocyte (-like) cultures, which could prove crucial for future screens aimed at identifying AQP4 modulators.

Exploring the reduction in aquaporin-4 and increased expression of ciliary neurotrophic factor with the frontal-striatal gliosis induced by chronic high-fat dietary stress.

High-fat diet (HFD)-induced obesity induces peripheral inflammation and hypothalamic pathogenesis linking the activation of astrocytes and microglia. Clinical evidence indicates a positive correlation between obesity and psychiatric disorders, such as depression. The connectivity of the frontal-striatal (FS) circuit, involving the caudate putamen (CPu) and anterior cingulate cortex (ACC) within the prefrontal cortex (PFC), is known for its role in stress-induced depression. Thus, there is a need for a thorough investigation into whether chronic obesity-induced gliosis, characterized by the activation of astrocytes and microglia, in these brain regions of individuals with chronic obesity. The results revealed increased S100β+ astrocytes and Iba1+ microglia in the CPu and ACC of male obese mice, along with immune cell accumulation in meningeal lymphatic drainage. Activated GFAP+ astrocytes and Iba1+ microglia were observed in the corpus callosum of obese mice. Gliosis in the CPu and ACC was linked to elevated cleaved caspase-3 levels, indicating potential neural cell death by chronic HFD feeding. There was a loss of myelin and adenomatous polyposis coli (APC)+ oligodendrocytes (OLs) in the corpus callosum, an area known to be linked with injury to the CPu. Additionally, reduced levels of aquaporin-4 (AQP4), a protein associated within the glymphatic systems, were noted in the CPu and ACC, while ciliary neurotrophic factor (CNTF) gene expression was upregulated in these brain regions of obese mice. The invitro study revealed thathigh-dose CNTF causing a trend of reduced astrocytic AQP4 expression, but it significantly impaired OL maturation. This pathological evidence highlights that prolonged HFD consumption induces persistent FS gliosis and demyelination in the corpus callosum. An elevated level of CNTF appears to act as a potential regulator, leading to AQP4 downregulation in the FS areas and demyelination in the corpus callosum. This cascade of events might contribute to neural cell damage within these regions and disrupt the glymphatic flow.

Melatonin Improves Vasogenic Edema via Inhibition to Water Channel Aquaporin-4 (AQP4) and Metalloproteinase-9 (MMP-9) Following Permanent Focal Cerebral Ischemia.

Background: The efficacy of melatonin in reducing vasogenic and cytotoxic edema was investigated using a model of permanent middle cerebral artery occlusion (pMCAO). Methods: Rats underwent pMCAO, followed by intravenous administration of either melatonin (5 mg/kg) or a vehicle 10 min post-insult. Brain infarction and edema were assessed, and Western blot analyses were conducted to examine the expression levels of aquaporin-4 (AQP4), metalloproteinase-9 (MMP-9), and the neurovascular tight-junction protein ZO-1 upon sacrifice. The permeability of the blood-brain barrier (BBB) was measured using spectrophotometric quantification of Evans blue dye leakage. Results: Compared to controls, melatonin-treated rats exhibited a significant reduction in infarct volume by 26.9% and showed improved neurobehavioral outcomes (p < 0.05 for both). Melatonin treatment also led to decreased Evans blue dye extravasation and brain edema (p < 0.05 for both), along with lower expression levels of AQP4 and MMP-9 proteins and better preservation of ZO-1 protein (p < 0.05 for all). Conclusions: Therefore, melatonin offers neuroprotection against brain swelling induced by ischemia, possibly through its modulation of AQP4 and MMP-9 activities in glial cells and the extracellular matrix (ECM) during the early phase of ischemic injury.

A "glympse" into neurodegeneration: Diffusion MRI and cerebrospinal fluid aquaporin-4 for the assessment of glymphatic system in Alzheimer's disease and other dementias.

The glymphatic system (GS) is a whole-brain perivascular network, consisting of three compartments: the periarterial and perivenous spaces and the interposed brain parenchyma. GS dysfunction has been implicated in neurodegenerative diseases, particularly Alzheimer's disease (AD). So far, comprehensive research on GS in humans has been limited by the absence of easily accessible biomarkers. Recently, promising non-invasive methods based on magnetic resonance imaging (MRI) along with aquaporin-4 (AQP4) quantification in the cerebrospinal fluid (CSF) were introduced for an indirect assessment of each of the three GS compartments. We recruited 111 consecutive subjects presenting with symptoms suggestive of degenerative cognitive decline, who underwent 3 T MRI scanning including multi-shell diffusion-weighted images. Forty nine out of 111 also underwent CSF examination with quantification of CSF-AQP4. CSF-AQP4 levels and MRI measures-including perivascular spaces (PVS) counts and volume fraction (PVSVF), white matter free water fraction (FW-WM) and mean kurtosis (MK-WM), diffusion tensor imaging analysis along the perivascular spaces (DTI-ALPS) (mean, left and right)-were compared among patients with AD (n = 47) and other neurodegenerative diseases (nAD = 24), patients with stable mild cognitive impairment (MCI = 17) and cognitively unimpaired (CU = 23) elderly people. Two runs of analysis were conducted, the first including all patients; the second after dividing both nAD and AD patients into two subgroups based on gray matter atrophy as a proxy of disease stage. Age, sex, years of education, and scanning time were included as confounding factors in the analyses. Considering the whole cohort, patients with AD showed significantly higher levels of CSF-AQP4 (exp(b) = 2.05, p = .005) and FW-WM FW-WM (exp(b) = 1.06, p = .043) than CU. AQP4 levels were also significantly higher in nAD in respect to CU (exp(b) = 2.98, p < .001). CSF-AQP4 and FW-WM were significantly higher in both less atrophic AD (exp(b) = 2.20, p = .006; exp(b) = 1.08, p = .019, respectively) and nAD patients (exp(b) = 2.66, p = .002; exp(b) = 1.10, p = .019, respectively) compared to CU subjects. Higher total (exp(b) = 1.59, p = .013) and centrum semiovale PVS counts (exp(b) = 1.89, p = .016), total (exp(b) = 1.50, p = .036) and WM PVSVF (exp(b) = 1.89, p = .005) together with lower MK-WM (exp(b) = 0.94, p = .006), mean and left ALPS (exp(b) = 0.91, p = .043; exp(b) = 0.88, p = .010 respectively) were observed in more atrophic AD patients in respect to CU. In addition, more atrophic nAD patients exhibited higher levels of AQP4 (exp(b) = 3.39, p = .002) than CU. Our results indicate significant changes in putative MRI biomarkers of GS and CSF-AQP4 levels in AD and in other neurodegenerative dementias, suggesting a close interaction between glymphatic dysfunction and neurodegeneration, particularly in the case of AD. However, the usefulness of some of these biomarkers as indirect and standalone indices of glymphatic activity may be hindered by their dependence on disease stage and structural brain damage.

Chemical Composition and Neuroprotective Properties of Indonesian Stingless Bee (Geniotrigona thoracica) Propolis Extract in an In-Vivo Model of Intracerebral Hemorrhage (ICH).

Stroke is the world's second-leading cause of death. Current treatments for cerebral edema following intracerebral hemorrhage (ICH) mainly involve hyperosmolar fluids, but this approach is often inadequate. Propolis, known for its various beneficial properties, especially antioxidant and anti-inflammatory properties, could potentially act as an adjunctive therapy and help alleviate stroke-associated injuries. The chemical composition of Geniotrigona thoracica propolis extract was analyzed by GC-MS after derivatization for its total phenolic and total flavonoid content. The total phenolic content and total flavonoid content of the propolis extract were 1037.31 ± 24.10 μg GAE/mL and 374.02 ± 3.36 μg QE/mL, respectively. By GC-MS analysis, its major constituents were found to be triterpenoids (22.4% of TIC). Minor compounds, such as phenolic lipids (6.7% of TIC, GC-MS) and diterpenic acids (2.3% of TIC, GC-MS), were also found. Ninety-six Sprague Dawley rats were divided into six groups; namely, the control group, the ICH group, and four ICH groups that received the following therapies: mannitol, propolis extract (daily oral propolis administration after the ICH induction), propolis-M (propolis and mannitol), and propolis-B+A (daily oral propolis administration 7 days prior to and 72 h after the ICH induction). Neurocognitive functions of the rats were analyzed using the rotarod challenge and Morris water maze. In addition, the expression of NF-κB, SUR1-TRPM4, MMP-9, and Aquaporin-4 was analyzed using immunohistochemical methods. A TUNEL assay was used to assess the percentage of apoptotic cells. Mannitol significantly improved cognitive-motor functions in the ICH group, evidenced by improved rotarod and Morris water maze completion times, and lowered SUR-1 and Aquaporin-4 levels. It also significantly decreased cerebral edema by day 3. Similarly, propolis treatments (propolis-A and propolis-B+A) showed comparable improvements in these tests and reduced edema. Moreover, combining propolis with mannitol (propolis-M) further enhanced these effects, particularly in reducing edema and the Virchow-Robin space. These findings highlight the potential of propolis from the Indonesian stingless bee, Geniotrigona thoracica, from the Central Tapanuli region as a neuroprotective, adjunctive therapy.

Improvement of neuronal and cognitive functions following treatment with 3′,4′ dihydroxyflavonol in experimental focal cerebral ischemia-reperfusion injury in rats

IntroductionIschemia/reperfusion is a pathological condition by the restoration of perfusion and oxygenation following a period of restricted blood flow to an organ. To address existing uncertainty in the literature regarding the effects of 3′, 4′-dihydroxy flavonol (DiOHF) on cerebral ischemia/reperfusion injury, our study aims to investigate the impact of DiOHF on neurological parameters, apoptosis (Caspase-3), aquaporin 4 (AQP4), and interleukin-10 (IL-10) levels in an experimental rat model of brain ischemia-reperfusion injury. Materials/methodsA total of 28 Wistar-albino male rats were used in this study. Experimental groups were formed as 1-Control, 2-Sham, 3-Ischemia-reperfusion, 4-Ischemia-reperfusion + DiOHF (10 mg/kg). The animals were anaesthetized, and the carotid arteries were ligated (ischemia) for 30 min, followed by reperfusion for 30 min. Following reperfusion, DiOHF was administered intraperitoneally to the animals at a dose of 10 mg/kg for 1 week. During the one-week period neurological scores and new object recognition tests were performed. Then, caspase 3 and AQP4 levels were determined by PCR method and IL-10 by ELISA method in hippocampus tissue samples taken from animals sacrificed under anaesthesia. ResultsBrain ischemia reperfusion significantly increased both caspase 3 and AQP4 values in the hippocampus tissue, while decreasing IL-10 levels. However, 1-week DiOHF supplementation significantly suppressed increased caspase 3 and AQP4 levels and increased IL-10 values. While I/R also increased neurological score values, it suppressed the ability to recognize new objects, and the administered treatment effectively ameliorated the adverse effects observed, resulting in a positive outcome. ConclusionsThe results of the study show that brain ischemia caused by bilateral carotid occlusion in rats and subsequent reperfusion causes tissue damage, but 1-week DiOHF application has a healing effect on both hippocampus tissue and neurological parameters.

Changes in Astroglial Water Flow in the Pre-amyloid Phase of the STZ Model of AD Dementia.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease that is typically sporadic and has a high social and economic cost. We utilized the intracerebroventricular administration of streptozotocin (STZ), an established preclinical model for sporadic AD, to investigate hippocampal astroglial changes during the first 4 weeks post-STZ, a period during which amyloid deposition has yet to occur. Astroglial proteins aquaporin 4 (AQP-4) and connexin-43 (Cx-43) were evaluated, as well as claudins, which are tight junction (TJ) proteins in brain barriers, to try to identify changes in the glymphatic system and brain barrier during the pre-amyloid phase. Glial commitment, glucose hypometabolism and cognitive impairment were characterized during this phase. Astroglial involvement was confirmed by an increase in glial fibrillary acidic protein (GFAP); concurrent proteolysis was also observed, possibly mediated by calpain. Levels of AQP-4 and Cx-43 were elevated in the fourth week post-STZ, possibly accelerating the clearance of extracellular proteins, since these proteins actively participate in the glymphatic system. Moreover, although we did not see a functional disruption of the blood-brain barrier (BBB) at this time, claudin 5 (present in the TJ of the BBB) and claudin 2 (present in the TJ of the blood-cerebrospinal fluid barrier) were reduced. Taken together, data support a role for astrocytes in STZ brain damage, and suggest that astroglial dysfunction accompanies or precedes neuronal damage in AD.

AQP4 regulates ferroptosis and oxidative stress of Muller cells in diabetic retinopathy by regulating TRPV4

Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.

Effects of angiopoietin (Ang) 1/endothelial-enriched Tunica interna endothelial cell kinase 2 (Tie2) signaling pathway on autophagy-based neuroprotection and expression of aquaporin 4

We investigated Ang-1/Tie2 signaling’s role in cellular autophagy-based neuroprotection and aquaporins expression. 40 mice were randomized into sham surgery group, model group, Tie2-antagonist group, and Ang-1+Tie2 agonist group, followed by observing brain pathological changes, and mRNA levels of LC3-I, LC3-II and aquaporin 4 (AQP4), together with levels of TNF-α and IL-1β. A certain regularity of brain tissue structure was observed in sham surgery group but not in model group. In Ang-1+Tie2 agonist group, the brain tissue structure was more disordered, which was ameliorated in Tie2-antagonist group, along with a proportion of intact cells. The neurological score of mice in Tie2 antagonist group was lower than Ang-1+Tie2 agonist and model groups. Levels of LC3-I and AQP4 in Tie2 antagonist group were also lower, along with higher levels of LC3-I and AQP4. LC3-II levels were higher in Tie2 antagonist group compared with those in Ang-1+Tie2 agonist and model groups. Whereas LC3-II and IL-1β/TNF-α levels in Ang-1+Tie2 agonist group were lower with higher levels of IL-1β and TNF-α. Inhibition of Ang-1/Tie2 signaling could have a restorative effect on neural function in stroke mice.

Norepinephrine-Activated p38 MAPK Pathway Mediates Stress-Induced Cytotoxic Edema of Basolateral Amygdala Astrocytes.

The amygdala is a core region in the limbic system that is highly sensitive to stress. Astrocytes are key players in stress disorders such as anxiety and depression. However, the effects of stress on the morphology and function of amygdala astrocytes and its potential mechanisms remain largely unknown. Hence, we performed in vivo and in vitro experiments using a restraint stress (RS) rat model and stress-induced astrocyte culture, respectively. Our data show that norepinephrine (NE) content increased, cytotoxic edema occurred, and aquaporin-4 (AQP4) expression was up-regulated in the basolateral amygdala (BLA) obtained from RS rats. Additionally, the p38 mitogen-activated protein kinase (MAPK) pathway was also observed to be significantly activated in the BLA of rats subjected to RS. The administration of NE to in vitro astrocytes increased the AQP4 level and induced cell edema. Furthermore, p38 MAPK signaling was activated. The NE inhibitor alpha-methyl-p-tyrosine (AMPT) alleviated cytotoxic edema in astrocytes, inhibited AQP4 expression, and inactivated the p38 MAPK pathway in RS rats. Meanwhile, in the in vitro experiment, the p38 MAPK signaling inhibitor SB203580 reversed NE-induced cytotoxic edema and down-regulated the expression of AQP4 in astrocytes. Briefly, NE-induced activation of the p38 MAPK pathway mediated cytotoxic edema in BLA astrocytes from RS rats. Thus, our data provide novel evidence that NE-induced p38 MAPK pathway activation may be one of the mechanisms leading to cytotoxic edema in BLA under stress conditions, which also could enable the development of an effective therapeutic strategy against cytotoxic edema in BLA under stress and provide new ideas for the treatment of neuropsychiatric diseases.

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.

Association of novel CSF biomarker candidates with cortical thickness in genetic frontotemporal dementia

AbstractBackgroundA novel panel of 14 proteins measured in the CSF could separate individuals with genetic frontotemporal dementia (FTD) from controls, with most significant findings observed for neurofilament medium (NEFM), neuronal pentraxin 2 (NPTX2), neurosecretory protein VGF (VGF) and aquaporin 4 (AQP4) [1]. However, it is currently unknown whether these altered protein levels in the CSF reflect neurodegenerative changes in the brain. The aim of this study was to explore the cross‐sectional associations between the previously identified CSF biomarker candidates and cortical thickness in presymptomatic and symptomatic mutation carriers, and whether those associations differ by FTD mutation type.MethodWe have analyzed T1 MRI scans alongside concurrent CSF samples from 202 individuals from the GENFI cohort, belonging to families that carry FTD mutations in either C9orf72, GRN or MAPT genes. The study sample included symptomatic mutation carriers, presymptomatic mutation carriers and non‐carrier controls. Cortical thickness was estimated with FreeSurfer and CSF protein levels were measured via a multiplexed antibody‐based suspension bead array. The correlations between regional cortical thickness and protein levels were calculated via linear regression.ResultAltered levels of NEFM, AQP4, APOA1, PTPRN2, CTSS, SERPINA3, C4, AMPH and CD14 were all correlated with increased atrophy of at least one cortical region. Some effects were mutation specific, but NEFM, AQP4 and APOA1 correlated with atrophy in all mutation groups. We also observed mutation specific effects for 10 of the proteins. CTSS levels were only correlated with cortical atrophy in C9orf72 mutation carriers while NPTX2, VGF and PTPRN2 correlated with atrophy in GRN mutation carriers. In MAPT mutation carriers, 6 different proteins correlated with atrophy in the right temporal pole.ConclusionThe proposed fluid biomarker candidates continue to show promise and further longitudinal studies will contribute to elucidate their relationship to cortical atrophy and their prognostic value in genetic FTD.[1] Bergström et al. Mol Neurodegener. 2021; 16(1):79

Protective mechanisms of tea polyphenols regulating the PI3K/Akt pathway on early brain injury after subarachnoid hemorrhage in rats.

In recent years, numerous studies have demonstrated that tea polyphenols (TPPs) can exert neuroprotective effects through the regulation of the PI3K/Akt pathway. The objective of this work was to verify whether TPPs could protect against early brain injury in rats after subarachnoid hemorrhage (SAH) by modulating the PI3K/Akt pathway. A total of 150 rats were randomly rolled into control (C), TPP, and SAH groups. The TPP and SAH groups underwent endovascular perforation to induce SAH, while C group received only endovascular needle puncture and saline injection. Brain water content, Evans Blue (EB) extravasation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Western blot, and RT-PCR analyses were performed. Relative to SAH group, TPP treatment considerably improved neurological function scores following SAH, reduced brain edema, cortical neuronal apoptosis, and blood-brain barrier damage. Levels of aquaporin-4 (AQP4) and apoptosis-related protein Bax were considerably lower in the TPP group than in SAH group. Conversely, levels of anti-apoptotic protein Bcl-2 and tight junction protein Zona occludens 1 (ZO-1) were considerably higher in the TPP group. Furthermore, TPP treatment was found to activate the PI3K/Akt signaling. TPPs can mitigate early brain injury caused by SAH in rats by reducing AQP4 levels, alleviating cortical damage, and attenuating neuronal apoptosis. These findings elucidate the protective mechanisms of TPPs against early brain injury following SAH through the regulation of the PI3K/Akt signaling.

Reduction of claudin-5 and aquaporin-4 in the rat hippocampal CA-1 and CA-3 regions of a learned helplessness model of depression

BackgroundAlthough findings from both animal and clinical research indicate that the blood-brain barrier (BBB) contributes to the pathogenesis of various psychiatric disorders (including depression), the underlying mechanisms are unknown. We investigated the levels of the tight-junction proteins claudin-5 and aquaporin-4 (AQP-4) in astrocytes of learned helplessness (LH) rats (an animal model of depression) and non-LH rats (a model of resilience). MethodsWe administered inescapable mild electric shock to rats and then identified the LH and non-LH rats by a post-shock test. The expressions of claudin-5 and AQP-4 in several brain regions of the LH and non-LH rats were then evaluated by a western blot analysis. ResultsThe levels of both claudin-5 and AQP-4 in the CA-1 and CA-3 hippocampal areas of the LH group were significantly lower than those of the control group, whereas those of the non-LH rats were not significantly different from those of the control and LH rats. ConclusionsThese results suggest that LH rats but not non-LH rats experienced down-regulations of claudin-5 and AQP-4 in the CA-1 and CA-3. It is possible that a region-specific modulation of claudin-5 and AQP-4 is involved in the mechanisms of vulnerability but not resilience in depression.

TMIC-27. THE ROLE OF AQUAPORIN-4 IN GLIOBLASTOMA-ASSOCIATED VASOGENIC CEREBRAL EDEMA

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Vasogenic cerebral edema is a severe complication that occurs in a majority of GBM patients, leading to a significant increase in intracranial pressure, accompanying neurological deficits, and increased mortality rates. Edema in GBM patients is almost exclusively managed by the corticosteroid dexamethasone (DEX), which has recently been found to have several drawbacks, including immunosuppression and interference with radiation therapy. Thus, there is an urgent need to better understand the mechanisms of edema resolution in GBM in order to identify alternative treatment strategies. The water channel aquaporin-4 (AQP4) is a critical player in the brain's water homeostasis, with increased levels in GBM compared to the normal brain. To gain more insight into the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO), heterozygous (HET), and wild-type (WT) mice. Multiplex IF staining combined with RNAScope of tumors showed that AQP4 expression co-localized with GFAP-positive reactive astrocytes in perivascular areas. Our results indicated that tumor-bearing mice in an Aqp4-deficient background had increased edema content and decreased survival, suggesting that AQP4 is important for edema resolution from tumors. Loss of Aqp4 also significantly decreased astrocyte coverage in the tumor core, but not in the tumor periphery. Moreover, anti-VEGFA treatment only improved vessel leakage and reduced edema in WT tumor-bearing mice. Ongoing studies are focused on characterizing the impact of Aqp4 loss on the gene expression profile of tumor core-specific astrocytes using single-cell RNA sequencing and spatial transcriptomics. We are also studying the cellular distribution of AQP4 in GBM and the effect of DEX treatment on AQP4 levels.

Inhibition of NADPH oxidase 2 improves cognitive abilities by modulating aquaporin-4 after traumatic brain injury in mice

Traumatic brain injury (TBI) is caused by acquired damage that includes cerebral edema after a mechanical injury and may cause cognitive impairment. We explored the role of nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2; NOX2) and aquaporin-4 (AQP4) in the process of edema and cognitive abilities after TBI in NOX2−/− and AQP4−/− mice by using the Morris water maze test (MWM), step-down test (STD), novel object recognition test (NOR) and western blotting. Knockout of NOX2 in mice decreased the AQP4 and reduce edema in the hippocampus and cortex after TBI in mice. Moreover, inhibiting AQP4 by 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) or genetic deletion of AQP4 could attenuate neurological deficits without changing reactive oxygen species (ROS) levels after TBI in mice. Taken together, we suspected that inhibiting NOX2 could improve cognitive abilities by modulating ROS levels, then affecting AQP4 levels and brain edema after in TBI mice. Our study demonstrated that NOX2 play a key role in decreasing edema in brain and improving cognitive abilities by modulating AQP4 after TBI.

Association between enlarged perivascular spaces and cerebrospinal fluid aquaporin-4 and tau levels: report from a memory clinic.

Perivascular spaces (PVS) are fluid-filled compartments that dilate in response to many different conditions. A high burden of enlarged PVS (EPVS) in the centrum semiovale (CSO) has been linked to neurodegeneration. Moreover, an increase in cerebrospinal fluid (CSF) levels of aquaporin-4 (AQP4), a water channel expressed on PVS-bounding astrocytes, has been described in patients with neurodegenerative dementia. Our aim was to investigate the relationship between neurodegenerative diseases and two putative glymphatic system biomarkers: AQP4 and EPVS. We included 70 individuals, 54 patients with neurodegenerative diseases and 16 subjects with non-degenerative conditions. EPVS were visually quantified on MRI-scans applying Paradise's scale. All subjects underwent lumbar puncture for the measurement of AQP4 levels in the cerebrospinal fluid (CSF). CSF levels of amyloid-β-1-42, phosphorylated and total tau (tTau) were also measured. Linear regression analyses were adjusted for age, sex, education and disease duration, after excluding outliers. Cerebrospinal fluid (CSF)-AQP4 levels were independent predictors of total (β = 0.28, standard error [SE] = 0.08, p = 0.001), basal ganglia (β = 0.20, SE = 0.08, p = 0.009) and centrum semiovale EPVS (β = 0.37, SE = 0.12, p = 0.003). tTau levels predicted CSO-EPVS (β = 0.30, SE = 0.15, p = 0.046). Moreover, increased levels of AQP4 were strongly associated with higher levels of tTau in the CSF (β = 0.35, SE = 0.13, p = 0.008). We provide evidence that CSO-EPVS and CSF-AQP4 might be clinically meaningful biomarkers of glymphatic dysfunction and associated neurodegeneration.

The link between SARS-CoV-2 related microglial reactivity and astrocyte pathology in the inferior olivary nucleus.

The pathological involvement of the central nervous system in SARS-CoV2 (COVID-19) patients is established. The burden of pathology is most pronounced in the brain stem including the medulla oblongata. Hypoxic/ischemic damage is the most frequent neuropathologic abnormality. Other neuropathologic features include neuronophagia, microglial nodules, and hallmarks of neurodegenerative diseases: astrogliosis and microglial reactivity. It is still unknown if these pathologies are secondary to hypoxia versus a combination of inflammatory response combined with hypoxia. It is also unknown how astrocytes react to neuroinflammation in COVID-19, especially considering evidence supporting the neurotoxicity of certain astrocytic phenotypes. This study aims to define the link between astrocytic and microglial pathology in COVID-19 victims in the inferior olivary nucleus, which is one of the most severely affected brain regions in COVID-19, and establish whether COVID-19 pathology is driven by hypoxic damage. Here, we conducted neuropathologic assessments and multiplex-immunofluorescence studies on the medulla oblongata of 18 COVID-19, 10 pre-pandemic patients who died of acute respiratory distress syndrome (ARDS), and 7-8 control patients with no ARDS or COVID-19. The comparison of ARDS and COVID-19 allows us to identify whether the pathology in COVID-19 can be explained by hypoxia alone, which is common to both conditions. Our results showed increased olivary astrogliosis in ARDS and COVID-19. However, microglial density and microglial reactivity were increased only in COVID-19, in a region-specific manner. Also, olivary hilar astrocytes increased YKL-40 (CHI3L1) in COVID-19, but to a lesser extent than ARDS astrocytes. COVID-19 astrocytes also showed lower levels of Aquaporin-4 (AQP4), and Metallothionein-3 in subsets of COVID-19 brain regions. Cluster analysis on immunohistochemical attributes of astrocytes and microglia identified ARDS and COVID-19 clusters with correlations to clinical history and disease course. Our results indicate that olivary glial pathology and neuroinflammation in the COVID-19 cannot be explained solely by hypoxia and suggest that failure of astrocytes to upregulate the anti-inflammatory YKL-40 may contribute to the neuroinflammation. Notwithstanding the limitations of retrospective studies in establishing causality, our experimental design cannot adequately control for factors external to our design. Perturbative studies are needed to confirm the role of the above-described astrocytic phenotypes in neuroinflammation.

Neuroprotective Effects of Delayed TGF-β1 Receptor Antagonist Administration on Perinatal Hypoxic-Ischemic Brain Injury

Hypoxic-ischemic (HI) brain injury in neonatal encephalopathy triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the primary damage. The cellular processes mediating this prolonged neurodegeneration in HI injury are not sufficiently understood. Consequently, current therapies are not fully protective. In a recent study, we found significant improvements in neurologic outcomes when a small molecule antagonist for activin-like kinase 5 (ALK5), a transforming growth factor beta (TGF-β) receptor was used as a therapeutic in a rat model of moderate term HI. Here, we have extended those studies to a mouse preterm pup model of HI. For these studies, postnatal day 7 CD1 mice of both sexes were exposed to 35–40 min of HI. Beginning 3 days later, SB505124, the ALK5 receptor antagonist, was administered systemically through intraperitoneal injections performed every 12 h for 5 days. When evaluated 23 days later, SB505124-treated mice had ∼2.5-fold more hippocampal area and ∼2-fold more thalamic tissue. Approximately 90% of the ipsilateral hemisphere (ILH) was preserved in the SB505124-treated HI mice compared to the vehicle-treated HI mice, where the ILH was ∼60% of its normal size. SB505124 also preserved the subcortical white matter. SB505124 treatment preserved levels of aquaporin-4 and n-cadherin, key proteins associated with blood-brain barrier function. Importantly, SB505124 administration improved sensorimotor function as assessed by a battery of behavioral tests. Altogether, these data lend additional support to the conclusion that SB505124 is a candidate neuroprotective molecule that could be an effective treatment for HI-related encephalopathy in moderately injured preterm infants.

Klotho gene might antagonize ischemic injury in stroke rats by reducing the expression of AQP4 via P38MAPK pathway

ObjectivesThis study was aimed at exploring whether klotho improved neurologic function in rats with cerebral infarction by inhibiting P38 mitogen-activated protein kinase (MAPK) activation and thus down-regulating aquaporin 4 (AQP4). MethodsIn this study, we induced intracerebral Klotho overexpression in 6-week-old Sprague Dawley rats by injecting lentivirus carrying full-length rat Klotho cDNA into the lateral ventricle of the brain, followed by middle cerebral artery occlusion (MCAO) surgery after three days. Neurologic function was evaluated by neurological deficit scores. Infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The expressions of Klotho, AQP4, and P38 MAPK were detected by Western blot and Immunofluorescence. Resultswhen rats were subjected to cerebral ischemia, their neurologic function was impaired, the protein expressions of klotho downregulated, the protein expressions of AQP4 and P38 MAPK increased, and the ratios of AQP4 and P-P38-positive area were significantly increased compared with the sham group rats. LV-KL-induced Klotho overexpression greatly improved neurobehavioral deficits and reduced infarct volume in MCAO rats. Klotho overexpression significantly reduced AQP4 and P38 MAPK pathway-related protein expression levels and the ratios of P-P38 and AQP4-positive area in MCAO rats. In addition, SB203580, a P38 MAPK signal pathway inhibitor, improved neurobehavioral deficits, reduced infarct volume, downregulated the expressions levels of AQP4 and P38 MAPK, and reduced the size of P-P38 and AQP4-positive area in MCAO rats. ConclusionKlotho could alleviate the infraction volume and neurological dysfunction in MCAO rats, and its mechanism may involve AQP4 expression downregulation by suppressing P38-MAPK activation.