Regulation of aquaporin‐4 expression in the central nervous system investigated using M23‐AQP4 null mouse

Abstract

In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin‐4 (M1‐AQP4 and M23‐AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23‐null mouse model (M23‐null). In the M23‐null spinal cord OAP assembly, the perivascular localization of AQP4 and M1‐AQP4 protein were drastically reduced. In heterozygous, M1‐AQP4 was proportionally reduced with M23‐AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23‐AQP4 in the regulation of M1‐AQP4 expression. M1‐AQP4 transcription, splicing and M1‐AQP4 protein degradation were found to be unaffected in M23‐null spinal cord and in M23‐null astrocyte primary culture. The translational control was investigated by mRNA‐protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4‐RBPs). Compared to WT, in M23‐null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4‐RBPs and provide evidence that in mouse spinal cord M23‐AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.

Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

“…In addition to preferential adhesion to scaffolding proteins as a mechanism that enhances endfoot expression of AQP4 and especially the AQP4ext isoform, it has recently been reported that local translation of certain astrocyte proteins occurs in endfeet 42 . Regulatory proteins that direct translation of individual AQP4 isoforms have been discovered (see 43 ), raising the possibility that local translation may bias expression in specific cellular compartments. Combined with selective retention by cytoskeleton-linked scaffolds, this could lead to both stable and dynamic polarization of the endfoot to respond to local demands for water flux.…”

Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis

“… 19 A better understanding of OAPs may be possible in the future with the development of a novel mouse lacking the OAP-forming AQP4-M23 isoform. 20 , 21 An AQP4 isoform (AQPex) has also been reported that has an extended carboxy-terminus containing a conserved perivascular localization signal generated by translational read-through. 22 , 23 The consequences of this carboxy-terminal extension are yet to be established.…”

Rho-Kinase inhibition decreases focal cerebral ischemia-induced glial activation in rats

“…Although we did not explore the reason for decreased M1 isoform, the possible cause could be altered translational regulation by Rho-kinase. 48 Such altered ratio of M1 and M23 isoform might be important for the formation of water channels and regulation of edema.…”

“…Although we did not explore the reason for decreased M1 isoform, the possible cause could be altered translational regulation by Rho-kinase. 48 Such altered ratio of M1 and M23 isoform might be important for the formation of water channels and regulation of edema. After cerebral ischemia, activated microglia and astrocytes had been observed to take part in acute neuroinflammatory processes, and thereby neuronal cell death.…”