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Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes¹

Department of General and Environmental Physiology and Center of Excellence in Comparative Genomics (CEGBA) and
^* Department of Biochemistry and Molecular Biology, University of Bari, I-70126 Bari, Italy

²Correspondence: Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Bari, via Amendola 165/A, I-70126 Bari, Italy. E-mail: a.frigeri{at}biologia.uniba.it

SPECIFIC AIM

Many brain pathologies such as stroke, head trauma, tumors, and infections result in brain edema, an important neurological problem associated with mortality and morbidity. It is well known that astrocytes are involved in regulating water and ion homeostasis to ensure an appropriate neuronal environment. In this study, we generated a 21 bp dsRNA oligonucleotide to suppress the expression of AQP4, the major water channel expressed in brain astrocytes, in order to analyze its physiopathological role.

PRINCIPAL FINDINGS

1. AQP4 expression in astrocyte primary cultures can be efficiently inhibited by RNA interference
To determine the efficiency and specificity of RNAi, AQP4 protein levels were determined 2, 4, and 6 days after inducing RNAi by a single initial (day 0) treatment (Fig. 1 A). The results show that AQP4 protein levels were progressively reduced by RNAi and reached maximal inhibition (77.9%±2.2) after 6 days. AQP4 protein expression levels were unaffected in control siRNA-treated compared with untreated cells, demonstrating the specificity of the results and the lack of toxic effects on the part of the lipid formulation and of the RNA duplexes.

View larger version (41K): [in this window] [in a new window]   Figure 1. AQP4 siRNA mediates gene silencing in astrocyte primary cultures. A, top) Western blot analysis of AQP4 protein expression 2, 4, and 6 days after transfection with RNA duplexes. A control siRNA (CTRL) was used in parallel to test potential nonspecific effects of the short RNA duplexes. The unaffected GFAP expression was used as internal standard of protein concentration in each lane. A, bottom) Quantification of AQP4 knockdown after Western blot analysis. Histogram shows the densitometric analysis of 3 to 5 independent experiments (*P<0.001). Levels of the AQP4 protein bands of astrocytes treated with specific (AQP4 siRNA, black bar) and nonspecific (CTRL siRNA, gray bar) RNA duplexes were normalized to the untreated astrocytes (CTRL, white bar). B, top) AQP4 mRNA levels were documented by semiquantitative RT-PCR experiments. Several controls (CTRL, untreated cells; Oligofect., oligofectamine-treated cells; CTRL siRNA, cells treated with control siRNA) were used to verify the specificity of the treatment. The arrow indicates a 410 bp AQP4-specific fragment coamplified with a 18S RNA fragment (324 bp) indicated by the arrowhead, used as internal standard. B, bottom) Densitometric analysis of 3 independent semiquantitative RT-PCR experiments. The histogram shows AQP4 mRNA knockdown data as AQP4/rRNA ratio (*P<0.001). Bars: CTRL, white; Oligofect, striped; CTRL siRNA, gray; AQP4 siRNA, black. C) Time course of scattered light intensity in response to a sudden increase in extracellular osmolality from 300 to 550 mOsm. The osmotic gradient causes water efflux, astrocyte shrinkage, and an increase in scattered light intensity. AQP4 silencing determines a significant decrease in the rate of the cell volume changes.

The levels of AQP4 mRNA were analyzed by semiquantitative RT-PCR. Figure 1B shows that AQP4 RNAi determined a significant and progressive decrease in AQP4 mRNA. The maximal effect was observed after 6 days with mRNA levels reduced by 75.6%.

Taken together, these results indicate that AQP4 protein is progressively reduced by RNAi as a consequence of a post-transcriptional siRNA-mediated degradation of AQP4 mRNA.

2. AQP4 knockdown (AQP4-KD) induces alteration in water transport properties of astrocytes
A stopped-flow light scattering method was used to examine the osmotic water permeability of AQP4-KD astrocytes. Figure 1C shows the time course of scattered light intensity in response to a 250 mOsm inwardly sucrose gradient. The osmotic gradient causes water efflux, astrocyte shrinkage, and an increase in scattered light intensity. By calculating the rate constant of shrinkage and the diameter of the astrocyte suspension, P_f was estimated to be 0.01 cm/s ± 0.002 (n=3) for CTRL siRNA-treated cells and 0.005 cm/s ± 0.001 (n=3) for AQP4-KD astrocytes. These results show that AQP4 gene silencing induces alteration in the water transport properties of the astrocyte plasma membrane. P_f reduction, as a consequence of AQP4 inhibition, represents the first direct evidence that the high water permeability of astrocytes in culture is AQP4 mediated.

3. AQP4 knockdown induces alteration in astrocyte morphology and growth
To determine the possible appearance of a cell phenotype associated with AQP4 gene silencing and to correlate AQP4 localization and expression levels together with changes in astrocyte morphology, double immunostaining experiments were performed (Fig. 2 ). AQP4 expression was analyzed together with the astrocyte marker, GFAP. Control siRNA-treated astrocytes strongly expressed AQP4 and no differences in AQP4 expression levels and distribution were found at low (day 2) and high (day 6) cell confluency. On day 2, AQP4 RNAi determined the appearance of a particular cell phenotype characterized by thin elongated astrocytes. Cell growth in the different cultures was largely comparable. Immunofluorescence analysis of AQP4 siRNA-treated astrocytes revealed that in these GFAP-positive cells AQP4 staining appeared to be clearly reduced; in many cells the AQP4 immunofluorescence signal appeared to be mainly intracellular. From days 4 to 6, the intensity of GFAP staining appeared to be unaffected, although dramatic morphological changes occurred. The AQP4 immunostaining signal was progressively reduced; on day 6, only a weak signal was detected in the perinuclear region of stellate astrocytes. AQP4 RNAi treatment determined a transformation of some elongated astrocytes into process-bearing stellate cells and a visible reduction in cell growth. When gene silencing came to an end, AQP4-KD astrocyte cultures were characterized almost totally by stellate astrocytes, which normally represent <10% in control cultures. Moreover, cell counts on day 6 revealed that the number of total cells in AQP4-KD astrocytes had reduced by 67.9% ± 3.7 (P<0.001, n=4) compared with controls.

View larger version (85K): [in this window] [in a new window]   Figure 2. AQP4 and GFAP expression during AQP4 gene silencing. Astrocytes were analyzed in parallel for AQP4 (red) and GFAP (green) expression 2, 4, and 6 days after treatment with CTRL siRNA and AQP4 siRNA. A weak diffuse AQP4 staining is observed after AQP4 post-translational gene silencing and no changes in GFAP expression level were found. Original magnification x400.

4. AQP4 gene silencing induces alteration in expression profile of edema-related genes
Gene expression microarray analysis was used to compare the gene expression profile of control astrocytes with AQP4 silenced astrocytes. We used a rat neurobiology array containing >800 known gene sequences relevant to the study of neurobiology. Bioinformatic analysis performed with a high level of stringency revealed the presence of 5 altered genes in AQP4 siRNA astrocytes compared with CTRL. Of these, glucose transporter (GLUT1), hexokinase, and metallothionein-1 were down-regulated while NGFI-B (immediate early gene transcription factor induced by NGF) and c-fos were up-regulated. All these genes, particularly GLUT1, are involved in ischemia-induced brain edema.

CONCLUSIONS AND SIGNIFICANCE

In this paper, we have demonstrated that AQP4 expression in astrocyte primary cultures can be efficiently inhibited using RNA interference technology.

Knockdown of AQP4 in astrocyte primary cultures results in a drastic reduction in membrane water permeability, impaired cell growth, and altered cell morphology. Changes in astrocyte morphology involved transformation of the astrocyte from a classical polygonal into an elongated/fibrous-like shape at the beginning, then into a stellate astrocyte. The morphological changes observed could be interpreted as a cell strategy to increase the surface to volume ratio and hence increase water flux in the absence of AQP4 water channels. AQP4 inhibition has a dramatic effect on cell growth, resulting in a 68% reduction in cells compared with the control at 6 days of treatment. These modifications suggest that cultured astrocytes require AQP4-mediated water transport to maintain the classical cell morphology and sustain optimal cell growth (Fig. 3 ).

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic drawing representing the consequences of AQP4 gene silencing in astrocyte primary cultures.

Evidence for the structural plasticity of astrocytes in the adult CNS has been extensively reported. Our data indicate that AQP4 is necessary for sustaining astrocyte cell morphology and that water channel activity and cell morphology are both strictly coupled to AQP4 function. This suggests a new functional role of AQP4 in astrocyte plasticity in vivo and supports our hypothesis that this aquaporin may be involved in cell plasticity phenomena.

AQP4 gene silencing determines the up-regulation of two genes, NGFI-B and c-fos, members of the immediate early gene family (IEGs) encoding transcription factors, also induced by ischemia. The three genes (GLUT1, hexokinase, and metallothionein-1) that were down-regulated as a consequence of AQP4 gene silencing have been demonstrated to be directly involved in cerebral ischemia.

It is believed that astrocytes represent the major cell type showing swelling after ischemia. Previous reports have demonstrated that astrocyte AQP4 expression, too, is up-regulated in edema induced by focal brain ischemia, hyponatremia, and in response to brain injuries and edematous tumors. Our data show that AQP4 gene silencing causes the down-regulation of ischemia-affected genes. Although the experimental design does not address the role of AQP4 in brain ischemia, our results suggest that AQP4 alteration may be a primary factor in ischemia-induced brain edema.

Recent studies suggest that AQP4 inhibition by pharmacological blockers might provide a new therapeutic alternative to current approaches for the treatment of some forms of cerebral edema. Although it is unlikely that RNA interference could be used as an efficient drug to promptly block astrocyte swelling, AQP4 inhibition studies by RNAi in animals will be useful to test the hypothesis that the inhibition of AQP4 activity is a potential target for treatment of brain edema. Since aquaporin inhibitors are not at present available, RNAi might be developed into a highly efficacious tool to study the roles of aquaporin genes in organ physiology, thus providing an alternative to the use of KO animals.

In conclusion, the results presented here indicate a direct involvement of AQP4 in physiological processes such as water transport and cell plasticity, as well as in the pathogenesis of brain edema. It is now possible to directly test the relationship between AQP4’s function and ion movements involved in cell excitability such as K⁺ and Cl^-. Finally, AQP4 RNAi can be envisaged as an in vivo tool to study the molecular mechanism of brain edema.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1183fje; doi: 10.1096/fj.02-1183fje

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