Aquaporin 4 Suppresses Neural Hyperactivity and Synaptic Fatigue and Fine-Tunes Neurotransmission to Regulate Visual Function in the Mouse Retina

Abstract

The bidirectional water channel aquaporin 4 (AQP4) is abundantly expressed in the neural tissue. The advantages and disadvantages of AQP4 neural tissue deficiency under pathological conditions, such as inflammation, and relationship with neural diseases, such as Alzheimer's disease, have been previously reported. However, the physiological functions of AQP4 are not fully understood. Here, we evaluated the role of AQP4 in the mouse retina using Aqp4 knockout (KO) mice. Aqp4 was expressed in Müller glial cells surrounding the synaptic area between photoreceptors and bipolar cells. Both scotopic and photopic electroretinograms showed hyperactive visual responses in KO mice, gradually progressing with age. Moreover, the amplitude reduction after frequent stimuli and synaptic fatigue was more severe in KO mice. Glutamine synthetase, glutamate aspartate transporter, synaptophysin, and the inward potassium channel Kir2.1, but not Kir4.1, were downregulated in KO retinas. KIR2.1 colocalized with AQP4 in Müller glial cells at the synaptic area, and its expression was affected by Aqp4 levels in primary Müller glial cell cultures. Intraocular injection of potassium in wild-type mice led to visual function hyperactivity, as observed in Aqp4 KO mice. Mitochondria molecules, such as Pgc1α and CoxIV, were downregulated, while apoptotic markers were upregulated in KO retinas. AQP4 may fine-tune synaptic activity, most likely by regulating potassium metabolism, at least in part, via collaborating with KIR2.1, and possibly indirectly regulating glutamate kinetics, to inhibit neural hyperactivity and synaptic fatigue which finally affect mitochondria and cause neurodegeneration.

Keywords: Aquaporin 4; Glutamate; Neural hyperactivity; Potassium; Retina; Synaptic fatigue.

Fig. 1

AQP4 localization at the synaptic area of the mouse retina. a–d Wild-type retinal sections immunostained with AQP4 and glutamine synthetase (GS; a), synaptophysin (Syp; b, b′), PSD95 (c, c′), or bassoon (d, d′). Arrowheads show the outer plexiform layer (OPL) where photoreceptor-bipolar synapses are distributed. b′, c′, and d′ are magnifications of b, c, and d, respectively. AQP4 is coexpressed with GS but not with Syp, PSD95, or bassoon. Bassoon signals are surrounded by AQP4 (d, d′). n = 8. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bars, 10 μm

Fig. 2

Visual function hyperactivity in Aqp4 knockout (KO) mice. Electroretinogram responses in wild-type (WT) and Aqp4 KO mice at 12 (a–e) and 16 (f–m) weeks of age. Scotopic (a–j) and photopic (k–m) responses. Representative wave forms from an individual mouse (a, f, k) and mean data of a-wave (b, d, g, i) and b-wave (c, e, h, j, l, m) are shown. The scotopic b-wave amplitude is higher in Aqp4 KO mice than in WT mice both at 12 (c) and 16 (h) weeks of age, while the scotopic a-wave (g) and photopic b-wave (l) amplitudes are higher in Aqp4 KO than WT mice at 16 weeks of age. There are no changes in the implicit time (d, e, i, j, m). Twelve-week-old mice, n = 5 (WT) and 5 (KO); 16-week-old mice, n = 4 (WT) and 5 (KO). *p < 0.05

Fig. 3

Decrement of visual responses after repeated stimuli in Aqp4 knockout (KO) mice. Scotopic electroretinogram responses using flicker stimuli from wild-type (WT) and Aqp4 KO mice at 10 (a–d) and 12 (e–h) weeks of age. Mice were stimulated with 0.5-Hz (a, b, e, f) or 1-Hz (c, d, g, h) flicker light. Representative wave forms from an individual mouse (a, c, e, g) and the mean b-wave amplitude ratio of the 20th to the 1st stimulus (b, d, g, i) are shown. The ratio is smaller in Aqp4 KO than WT mice at 10 weeks of age with 0.5-Hz stimuli (a, b) and at 12 weeks with 0.5-Hz (e, f) and 1-Hz stimuli (g, h). Ten-week-old mice, n = 5 (WT) and 6 (KO); 12-week-old mice, n = 4 (WT) and 6 (KO). *p < 0.05

Fig. 4

Alteration in mRNA levels related to synaptic transmission in the retina of Aqp4 knockout (KO) mice. Real-time RT-PCR in retinal samples derived from wild-type (WT) and Aqp4 KO mice at 16 weeks of age (a–f). The relative mRNA levels of glutamine synthetase (Gs; a), glutamate aspartate transporter (Glast; b), synaptophysin (Syp; c), and Kir2.1 (d) are downregulated in the retina of Aqp4 KO mice, compared with the respective levels in WT retinas. The levels of Kir4.1 (e) and Kcnv2 (f) are not changed. n = 5–10. *p < 0.05

Fig. 5

Modulation of intraocular potassium levels induces hyperactive photopic electroretinogram (ERG) responses in mice. a, b Double immunohistostaining for KIR2.1 and glutamine synthetase (GS; a, a′) or synaptophysin (Syp; b, b′) in wild-type (WT) retinal sections. a′ and b′ are magnifications of a and b, respectively. GS (a, a′; arrows) but not Syp (b, b′) colocalizes with KIR2.1. Arrowheads show the outer plexiform layer. n = 8. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bar, 10 μm. c–e Real-time RT-PCR of primary Müller glial cell culture samples transfected with control (si-cont) or Aqp4 siRNA (si-AQP4). Quantification of the relative mRNA levels confirms the knockdown (KD) of Aqp4 (c). Kir2.1 (d) but not Kir4.1 (e) is downregulated in Müller glial cells. n = 4 (control) and 5 (KD). f–h Photopic ERGs after intraocular injection of vehicle (PBS) or KCL. The b-wave amplitude is increased by the potassium load (f, g). The implicit time is not changed (H). n = 12 for each group; *p < 0.05; **p < 0.01

Fig. 6

Mitochondria changes in the retina of Aqp4 knockout (KO) mice. Real-time RT-PCR in retinal samples derived from wild-type (WT) and Aqp4 KO mice at 16 weeks of age (a–i). The relative mRNA levels of Pgc1α (a), CoxIV (b), CytC (c), Fis1 (e), Mfn1 (f), and Mfn2 (g) are downregulated in Aqp4 KO retinas, while Ho-1 (d) levels are not changed, compared with the respective levels in WT mice. The mRNA levels of Bcl2 (h) and Bax (i) are higher in Aqp4 KO than in WT retina. n = 5–10. *p < 0.05; **p < 0.01