Written by - Zhu Dandan, Huang Yuelin, Li Shao, Ma Tonghui Editor-in-Charge - Wang Sizhen Editor - Yang Binwei
AQP4 (aquaporin-4) is a highly expressed aquaporin-subtype of the central nervous system, the main polarity is distributed in the blood-brain barrier astrocyte endapeupsy, and plays an important role in the formation of cytocerebral edema caused by various factors [1-3
].

Firstly, the research team constructed a transgenic knock-in mouse model of the 25th alanine → glutamine mutation (A25Q) in AQP4, and demonstrated for the first time the key role
of AQP4-OAP formation in polarized expression at the end of the astrocytes that constitute the blood-brain barrier.

By comparing the expression levels of mRNA and protein in AQP4^+/+ and AQP4 A25Q^/A25Q mouse brain tissue AQP4, and the OAP structure of AQP4 by BN-PAGE non-denaturing glue electrophoresis, serum IgG to AQP4 in patients with optic nerve myelitis (NMO) formed by the AQP4-M23 subtype was used to specifically identify the OAP structure formed by the AQP4-M23 subtype ^+/+ and AQP4^A25Q/A25Q mouse brain sheets were immunofluorescence stained to observe the distribution of AQP4 at the end of astrocytes that make up the blood-brain barrier
.

The results showed that the AQP4-A25Q mutation did not affect the overall AQP4 mRNA and protein expression, but significantly reduced the OAP structure expression and the polarization distribution of the AQP4 protein at the end of astrocytes that constitute the blood-brain barrier (Figure 1).

Figure 1 Effect of A25Q mutation on AQP4 expression and OAP formation in the brain of AQP4 A25Q/A25Q mice (Source: Zhu DD, et al.
, J Neurosci, 2022)
To further observe the ultramolecular structure of^AQP4 in mice with AQP4-A25Q gene mutations, The researchers used g-STED ultra-high-resolution microscopy to assess the AQP4 distribution
at the terminal foot of astrocytes that make up the blood-brain barrier.

This technique can determine the size and distribution of individual AQP4 (OAPs) at nanoscale resolution [6
].

They found that the structure of AQP4 OAPs at the end of the perivascular astrocyte of AQP4 A25Q/A25Q mice was significantly reduced compared with AQP4+/+, and the average OAP size of AQP4^A25Q/^A25Q mice was 3.
5 times smaller than that of AQP4^+/+ mice
。
OAP size distributions also vary, with an average measurement of 291.
7±14.
6 nm2 in AQP4^A25Q/A25Q mice and 998.
7±50.
0^nm2 in AQP4^+/+ mice (Figure 2).


This indicates that AQP4 A25Q^/A25Q mouse brain tissue AQP4 OAP structure has been depolymerized
.
Figure 2 AQP4-A25Q mutation regulates supramolecular aggregation of AQP4 (Source: Zhu DD, et al.
, J Neurosci, 2022)
To further clarify the distribution of AQP4 in the brains of AQP4^A25Q/A25Q mice, the researchers observed the distribution of AQP4 at the astrocyte terminal foot at the blood-brain barrier by laser confocal and immunoelectron microscopy-colloidal gold technology.
Laser confocal found that AQP4 expression decreased significantly at the end of the astrocytes of AQP4 A25Q^/A25Q mice that constitute the blood-brain barrier.
Colloidal gold immunoelectron microscopy also showed a significant decrease in AQP4^A25Q/A25Q mice expressed in the astrocyte terminal AQP4 colloidal gold particles at the end of astrocytes, while the glial gold particles expressed in other parts of astrocytes were significantly increased
compared with wild-type mice.

This suggests that A25Q mutations lead to a significant decrease in AQP4 expression at the astrocyte end (Figure 3
).


Figure 3 AQP4-A25Q mutation reduces the polarized expression of AQP4 in glial tetrapodes (Source: Zhu DD, et al.
, J Neurosci, 2022)
Previous studies have shown that AQP4 knockout mice significantly reduce brain astrocyte endoedema caused by various factors[7].


In order to evaluate the effect of AQP4-A25Q mutation on the occurrence and development of cerebral edema, the researchers first adopted a recognized cerebral edema model of acute intoxication-induced cytocerebral edema, and found that the AQP4-A25Q point mutation could improve cerebral edema in water-poisoned mice and significantly reduce the activation
of brain astrocytes and microglia in mice in water-poisoning models.

To improve the structural function of AQP4-OAP, the researchers used another MCAO/R model of cerebral edema to mimic human ischemic stroke [8].


The MCAO/R model leads to cellular and angiogenic edema, as well as severe brain damage, and can lead to nerve damage and even death [9
].

By analyzing the survival rate, neurological function score, cerebral water content, cerebral infarction volume and neuronal damage of mice for ischemia for 2 h and reperfusion for 24 h, the results showed that AQP4 A25Q/^A25Q mice had higher postoperative survival rate, lower cerebral water content, smaller cerebral infarction volume and lower neuronal damage than AQP4^+/+ mice (Figure 4 left).
Otema decreases at the astrocyte end-foot
.

Further observation of astrocyte and microglia activation by immunofluorescence showed that the AQP4-A25Q point mutation significantly reduced astrocyte activation and microglia activation in mouse mice in MCAO/R model (Figure 4 right).


This suggests that AQP4-A25Q point mutations are able to improve cerebral edema and its secondary neuroinflammatory response
in mice in the MCAO/R model.
Figure 4 AQP4 A25Q/^A25Q mice exhibit cerebral edema, improved neural function recovery and survival rate, astrocyte swelling, and decreased activation after MCAO/R (Source: Zhu DD, et al.
, J Neurosci, 2022).

Figure 5 Work summary figure: Neuroprotective mechanisms associated with AQP4-A25Q mutations in cerebral edema (Source: Zhu DD, et al.
, J Neurosci, 2022) Conclusion and discussion of the article inspires and prospects that under normal circumstances, astrocytes that constitute the blood-brain barrier play an important role
in maintaining the homeostasis of ions and water in the brain.

In cerebral edema, AQP4 transports water from within the cerebral microvessels to astrocytes, causing astrocytes to swell and activate
.

AQP4-A25Q point mutations lead to OAP structure depolymerization, which in turn reduces astrocyte end-foot AQP4 expression, thereby reducing water entering astrocytes from cerebral microvessels, reducing astrocyte edema and activation, and reducing neuronal damage (Figure 5).

In summary, the AQP4-A25Q point mutant mice constructed by the researchers led to the depolymerization of the AQP4 OAP structure, but did not affect the overall expression level
of the AQP4 protein.

The A25Q point mutation reduces the polar distribution of AQP4 at the end of the blood-brain barrier astrocyte and changes the water transport characteristics of astrocytes in the brain, further demonstrating that the polarized expression of AQP4 OAP is closely related
to the edema formation of astrocytes during cerebral ischemic injury.

Thus, the study may provide new perspectives
on intervention in cerebral edema by reducing the formation of AQP4 OAP.

The downside is that there are multiple subtypes of AQP4 (such as M1, M23, M1ex, AQP4e, etc.
), and the researchers did not observe whether different AQP4 subtypes are affected
by A25Q mutations.

Original link: _mstmutation="1" _istranslated="1"> This paper is supported
by the Xingliao Talents Program of Liaoning Province (XLYC1902044, XLYC1808031), the Key R&D Program of Liaoning Province (2019020048-JH2/103), the National Natural Science Foundation of China (81571061, U1908208, 81671111), and the Natural Science Foundation of Liaoning Province (2021-MS-280).

[1] Nielsen, S.

[2] Papadopoulos, M.

[3] Nagelhus, E.

[4] Crane, J.

[5] Smith, A.

[6] Rosito, S.

[7] Manley, G.

[8] Hossmann, K.

[9]   Li, Q.

End of article