In this study we investigated the relevance of AQP4ex in brain water homeostasis and solute clearance. AQP4ex is critical for the proper localization and polarization of AQP4 channels at astrocytic endfeet, Using the water intoxication model and the solute brain injection together with the unique opportunity of the AQP4ex-KO mice, we provide evidence on the crucial role of AQP4ex on brain water balance and amyloid-β (Aβ) clearance. The latter we considered here as a measure of the AQP4ex functional role in the waste clearance system.

The regulation of brain water content is fundamental for cerebral homeostasis under both normal and pathological condition. Our data reveals significant differences in basal brain water content between WT and AQP4ex-KO mice with AQP4ex-KO mice exhibiting lower brain water content. This suggests that AQP4ex’s critical role in maintaining normal brain water levels.

Following acute water intoxication, brain water content increased in all genotypes, confirming the model’s effectiveness in inducing cytotoxic brain edema.

OAP-null mice, in which AQP4 protein is drastically reduced, exhibited a delayed response, in line with a reduced edema formation reported in AQP4-KO mice [2]. AQP4ex-KO mice showed the highest increase in brain water content at 20 min, suggesting AQP4ex’s importance under acute osmotic stress likely through its interactions with other cellular components.

By 30 min post-AWI, brain water content plateaued across all genotypes indicating compromised water regulation beyond this threshold where increased intracranial pressure (ICP) severely restricts cerebral perfusion, leading to irreversible brain damage [30].

These findings highlight the important role of AQP4ex in both normal and pathological states, underscoring its importance in both immediate and sustained responses to brain edema.

Our immunoblot analyses reveal significant insights into the modulation of AQP4ex and p-AQP4ex during AWI. Total AQP4 levels remained relatively stable at early AWI stages but increased significantly at 30 min, indicating a delayed response to prolonged osmotic stress.

Conversely, AQP4ex and p-AQP4ex levels were upregulated at all time points examined, indicating a dynamic and responsive modulation to osmotic stress. The ratio of p-AQP4ex to AQP4ex was similar to control at 10 and 30 min, indicating that the newly produced AQP4ex is phosphorylated at these time points. However, at 20 min, this ratio was significantly reduced. This reduction in phosphorylation at 20 min could be due to several factors, including the presence of regulatory mechanisms that control the timing and extent of phosphorylation, possibly to balance between immediate functional demands and long-term protein stability. Additionally, the phosphorylation process itself may be influenced by the availability of kinases and other modifying enzymes, as well as substrate accessibility.

Interestingly, the time point of 20 min also corresponds to the peak in brain water content observed in AQP4ex-KO mice. The lack of full phosphorylation at this critical time may impair the structural role of AQP4ex in anchoring AQP4 channels at the astrocytic endfeet, thereby affecting the overall water dynamics. This temporal mismatch between AQP4ex production and phosphorylation may result in a transient inability to effectively manage water accumulation, leading to the observed peak in brain water content.

Previous research has demonstrated that phosphorylation of AQP4ex reduces the water permeability of AQP4 channels [15]. However, given that AQP4ex constitutes only a small fraction of the total AQP4 pool, it is unlikely that this phosphorylation plays a significant functional role in water transport. Instead, phosphorylation of AQP4ex likely serves a more structural role, such as anchoring AQP4 at the astrocytic endfeet. This structural role is crucial for maintaining the polarized distribution of AQP4, which is essential for the efficient functioning of the waste clearance system. The enhanced phosphorylation observed at other time points is posited to facilitate interactions with scaffolding proteins and cytoskeletal elements, thereby anchoring AQP4 at the astrocytic endfeet and ensuring efficient water and solute transport.

In AQP4ex-KO mice, the absence of AQP4ex led to a compensatory increase in canonical AQP4 isoforms at 20 min post-AWI. This transient upregulation may reflect an early-stage adaptive mechanism in response to acute osmotic stress, where the brain attempts to compensate for the loss of AQP4ex by increasing the expression of other AQP4 isoforms. However, the brain’s inability to sustain this compensation over time may result in the attenuation of this expression by 30 min. The phosphorylation state of AQP4ex may play an essential role in modulating the efficiency of these compensatory mechanisms.

Despite its minor proportion, AQP4ex appears to be essential for maintaining the polarized expression and functionality of AQP4, ensuring efficient water clearance during acute cytotoxic stress. These findings highlight AQP4ex’s non-redundant role in regulating brain water homeostasis, particularly in conditions of rapid intracranial pressure changes.

It is reasonable to speculate that the short-term regulation of AQP4ex expression can be influenced by translational mechanisms, especially considering the high levels of AQP4 mRNA [31]. Brain edema can significantly influence these translational mechanisms [32]. The increased osmotic stress and cellular signaling changes associated with brain edema could enhance the efficiency of translational readthrough, leading to an upregulation of AQP4ex production. Factors such as the availability of specific tRNAs, readthrough-promoting sequences in the mRNA, and the presence of translation initiation factors may be modulated under edematous conditions. These modifications may enable the cell to quickly adjust AQP4ex levels in response to the immediate demands of water homeostasis and edema management.

Effective clearance of interstitial solutes such as Aβ is critical for preventing neurodegenerative diseases like Alzheimer’s. Our data show that AQP4ex is important for this clearance process. In WT mice, the drainage system operates efficiently, as evidenced by the rapid decline in Aβ fluorescence intensity within a short distance from the injection site facilitated by polarized AQP4 at astrocytic endfeet [4]. In contrast, AQP4ex-KO mice exhibited significantly extended Aβ diffusion distances and a larger space constant, indicating impaired clearance due to the loss of AQP4 polarization. The absence of AQP4ex disrupts the structural organization required for effective flow, resulting in a reliance on a slower, less efficient clearance mechanism. This impaired clearance was further validated by reduced Aβ fluorescence in the cervical lymph nodes of AQP4ex-KO mice, indicating compromised drainage from the brain parenchyma. These data are in line with those reported by Sapkota [21].

Recent findings by Mueller et al. [33] suggest that the absence of AQP4ex (AQP4x) may lead to changes in blood-brain barrier (BBB) permeability or impaired efflux mechanisms, though the study could not definitively conclude whether these effects were due to increased leakage or reduced clearance, highlighting the need for further investigation.

In our own study [17] we used high-resolution immunogold cytochemistry to examine the CNS of AQP4ex-KO mice and found no major alterations in key structures, such as capillaries, endothelial cells, or pericytes. This aligns with our current findings, where we observed impaired Aβ clearance without significant changes in BBB permeability. Together, these results suggest that altered clearance mechanisms, rather than direct BBB dysfunction, may be the primary consequence of AQP4ex loss. However, we cannot exclude that subtle BBB alterations may also occur in specific brain areas and contribute to the observed defect.

Our data suggest that the impaired clearance in AQP4ex-KO mice aligns more closely with the diffusion model rather than the glymphatic model. The extended diffusion distances and reduced space constants observed indicate that in the absence of AQP4ex, the brain relies more on passive diffusion for solute movement, which is significantly less efficient than the convective flow seen in the glymphatic system of WT mice. This reliance on diffusion results in slower and less effective clearance of Aβ, highlighting the critical role of AQP4ex in maintaining the efficiency of the brain’s waste removal system.

An important aspect to consider is the impact of differences in basal water content on solute clearance. AQP4ex-KO mice, which have lower basal brain water content compared to WT mice, may experience altered extracellular space dynamics. Since the BBB is not altered in the AQP4ex-KO mouse, we can conclude that the observed variations are not due to increased vascular permeability. This lower water content could result in a denser extracellular matrix, potentially hindering the movement of solutes such as Aβ. Consequently, the compromised drainage system in AQP4ex-KO mice could be further impaired by the unfavorable conditions for solute diffusion.

The existence and significance of the glymphatic system have been subjects of debate. Proposed in 2017 [6], the glymphatic system describes a waste clearance mechanism dependent on AQP4 polarization at astrocytic endfeet, believed to function predominantly during sleep to facilitate CSF movement through brain parenchyma. However, an alternative diffusion-based theory challenges the robustness of the glymphatic system, suggesting that waste clearance in the brain may not rely heavily on AQP4 channels [34]. Recent research [35] adds to this debate by reporting that brain clearance is reduced during sleep and anesthesia, contrasting with earlier studies. This discrepancy may be due to the mechanisms of waste clearance varying under different physiological states, indicating that the efficiency of waste clearance pathways may be context-dependent and more complex than previously thought.

Our findings contribute to this debate by providing evidence that supports the critical role of AQP4ex in facilitating efficient solute clearance, aligning with the glymphatic model. However, the impaired clearance observed in AQP4ex-KO mice also highlights the complexity of the system and suggests that multiple mechanisms, including passive diffusion, may be involved in brain waste clearance. Further research is necessary to fully understand the mechanisms underlying cerebral waste removal and the potential interplay between different pathways.