Inhibition of AQP4 expression in astrocytes by RNAi determines alterations in cell morphology, growth, and water transport and induces changes in ischemia related genes

Abstract

Recent studies indicate a key role of aquaporin (AQP) 4 in astrocyte swelling and brain edema and suggest that AQP4 inhibition may be a new therapeutic way for reducing cerebral water accumulation. To understand the physiological role of AQP4-mediated astroglial swelling, we used 21-nucleotide small interfering RNA duplexes (siRNA) to specifically suppress AQP4 expression in astrocyte primary cultures. Semiquantitative RT-PCR experiments and Western blot analysis showed that AQP4 silencing determined a progressive and parallel reduction in AQP4 mRNA and protein. AQP4 gene suppression determined the appearance of a new morphological cell phenotype associated with a strong reduction in cell growth. Water transport measurements showed that the rate of shrinkage of AQP4 knockdown astrocytes was one-half of that of controls. Finally, cDNA microarray analysis revealed that the gene expression pattern perturbed by AQP4 gene silencing concerned ischemia-related genes, such as GLUT1 and hexokinase. Taken together, these results indicate that 1) AQP4 seems to be the major factor responsible for the fast water transport of cultured astrocytes; 2) as in skeletal muscle, AQP4 is a protein involved in cell plasticity; 3) AQP4 alteration may be a primary factor in ischemia-induced cerebral edema; and 4) RNA interference could be a new potent tool for studying AQP pathophysiology in those organs and tissues where they are expressed.

Aquaporin‐4 and epilepsy

“…Also, water permeability was seven-fold reduced in primary astrocyte cultures from Aqp4 2/2 mice as measured by a calcein fluorescence quenching method (Solenov et al, 2004), and similar results were obtained with Aqp4 RNAi knockdown experiments in wild-type astrocytes (Nicchia et al, 2003). These data directly demonstrated that AQP4 provides the predominant pathway for transmembrane water movement in astrocytes.…”

New advances on glial activation in health and disease

“…Furthermore, studies have shown that aquaporin-4 (AQP4) is important for sustaining astrocyte morphology, indicating a functional role of AQP4 in astrocyte plasticity. Knockdown of AQP4 in primary cultures resulted in a drastic reduction in membrane water permeability, impaired cell growth, and altered cell morphology [115] as well as the downregulation of three genes (glucose transporter 1, hexokinase, and metallothionein-1) involved in brain edema. Furthermore, changes in astrocyte morphology may not necessarily be permanent and can change with amelioration of CNS insult [116] and/or the administration of therapeutic medication (Lee et al, under review).…”

Cytoprotective effects of growth factors: BDNF more potent than GDNF in an organotypic culture model of Parkinson's disease

“…In the context of transgene brain aquaporin models where Aqp4 or Aqp9 are knocked out, it cannot be excluded that other brain aquaporins are upregulated to compensate for the loss of function or that other channels are upregulated in a similar way as formation of astrocytic connexins are increased following Aqp4 knockout, as shown by Katoozi et al (369). Another possibility is that other proteins may be codownregulated with the knockout of the target proteins (370). Indeed, siAQP4 treatment in astrocyte cultures has been shown to induce a slight but not significant decrease in AQP9 expression (277).…”