Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid β (Aβ) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aβ deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

J. Neurochem. (2012) 120, 899–912. Abstract Aquaporin‐4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin‐4 gene, respectively. To identify cis‐acting elements responsible for the astrocyte‐specific transcription of M1 mRNA, the promoter activity of the 5′‐flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte‐specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286‐bp region were found to function as a novel astrocyte‐specific enhancer. Among the five elements, a consensus sequence of Pit‐1/Oct/Unc‐86 (POU) transcription factors was indispensable to the astrocyte‐specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity.

Aquaporin‐4 is a transmembrane water channel protein, the C‐terminal domain of which is facing the cytosol. In the process of investigating the role of the C‐terminal domain of aquaporin‐4 with regard to intracellular trafficking, we observed that a derivative of aquaporin‐4, in which the C‐terminal 53 amino acids had been removed (Δ271‐323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin‐4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C‐terminal domain of aquaporin‐4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53‐amino acid deletion on the subcellular localization of aquaporin‐4 could be attributed to the formation of a signal at the C terminus that retained aquaporin‐4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271‐323‐associated retention in the cytosol, suggesting that the C‐terminal sequence of the mutant served as a signal similar to a di‐lysine motif.

Aquaporin-4, a predominant water channel in the central nervous system, has two isoforms, M1 and M23, whose transcripts are driven by distinct promoters. Using a reporter assay, we found that a fragment located between exons 0 and 1 of the mouse aquaporin-4 gene, which had been thought to be the promoter for M23, lacked enhancers functioning in astrocytes. When the astrocyte-specific enhancer (ASE) of the M1 promoter is connected to the putative M23 core promoter, it also works in astrocytes. Importantly, the ASE inhibits downstream promoter activity in NIH3T3 cells, indicating that the ASE also functions as a silencer in cells lacking aquaporin-4.

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Aquaporin-4 (AQP4) is a dominant water channel in the brain and is expressed on astrocytic end-feet, mediating water homeostasis in the brain. AQP4 is a target of an inflammatory autoimmune disease, neuromyelitis optica spectrum disorders (NMOSD), that causes demyelination. An autoantibody recognizing the extracellular domains of AQP4, called NMO-IgG, is critically implicated in the pathogenesis of the disease. Complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in astrocytes are the primary causes of the disease, preceding demyelination and neuronal damage. Additionally, some cytotoxic effects of binding of NMO-IgG to AQP4, independent of CDC/ADCC, have been proposed. Antibody-induced endocytosis of AQP4 is thought to be involved in CDC/ADCC-independent cytotoxicity induced by the binding of NMO-IgG to AQP4. To clarify the mechanism responsible for antibody-induced endocytosis of AQP4, we investigated the subcellular localization and trafficking of AQP4, focusing on its C-terminal domain, by making a variety of deletion and substitution mutants of mouse AQP4. We found that a tyrosine-based YXXΦ motif in the C-terminal domain of AQP4 plays a critical role in the steady-state subcellular localization/turnover and antibody-induced endocytosis/lysosomal degradation of AQP4. Our results indicate that the YXXΦ motif has to escape the inhibitory effect of the C-terminal 10-amino-acid sequence and be located at an appropriate distance from the plasma membrane to act as a signal for lysosomal degradation of AQP4. In addition to lysosomal degradation, we demonstrated that the YXXΦ motif also functions as a signal to degrade AQP4 using proteasomes under specific conditions.

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Aquaporin-4 (AQP4) is a water cannel playing a role in water transport and homeostasis of the brain. Previously, we observed that deletion of the C-terminal domain causes degradation of AQP4. In this report, we identified three amino-acid sequences that regulate the expression level of AQP4. First, deleting C-terminal 10 amino acids (Asp 314 -Val 323 ) greatly reduced the level of AQP4. Substitutions of Ala for Asp 314 and/or Glu 318 mimicked this effect, suggesting that two acidic amino acids in this region is important to prevent AQP4 from degradation. Second, this reduction of AQP4 was rescued when the C-terminal domain was deleted more than 43 amino acids, suggesting that the region between Val 280 and Lys 313 contains a signal for the degradation. Substitution of Phe for Tyr 277 or Arg for Val 280 increased the level of AQP4 lacking C-terminal 42 amino acids (Δ282-323), suggesting that a tyrosine-based endocytic motif (YXXΦ) is involved in the degradation of AQP4. Finally, deletion between Lys 259 and Ala 270 increased the level of AQP4. In contrast to the disruption of the putative YXXΦmotif, the 12-amino-acid deletion could not rescue AQP4 Δ282-323 from degradation, indicating that the deletion increased the level of AQP4 with a different mechanism from YXXΦmotif mutants.