One of the tasks of modern medicine is to address the many diseases affecting the brain. These maladies come in various forms – including neurodegenerative complications, tumors, vascular constriction, and buildup of intracranial pressure.1,2,3 Several of these disease classes are caused, in part or in full, by a faulty waste clearance or water flux. Although a pervasive system of slow cerebrospinal fluid (CSF) movement in the brain’s ventricles is present, a rapid method for clearing solutes from the cortex’s interstitial space, which contains neural tissue and the surrounding extracellular matrix, was unknown.4,5 Recently, Iliff et al discovered a new mechanism for the flow of CSF in the mouse brain – the “glymphatic” system.5,6 This pathway provides an accelerated mechanism to clear dissolved materials from the interstitial space, preventing a buildup of solutes and toxins.5,6 At the center of this system is the water transport protein aquaporin-4 (AQP4), and the extent of this water channel’s various roles are only now being identified. New perspectives on the mechanism by which the brain is “cleansed” may lead to breakthroughs in therapeutics for brain disorders such as Alzheimer’s Disease (AD), which is the sixth leading cause of death in the US each year.7

The Infrastructure

To understand AQP4 and its role in the brain, the environment in which it operates must be examined. As seen in Figure 1, surrounding the brain are three meninges, protective layers between the skull and the cortex.8 Between these layers – the dura, arachnoid, and pia maters – are cavities, including the subarachnoid space that lies just below the arachnoid layer.9 As the figure shows, directly underneath the pia mater are the cortex and interstitial space. Within the cortex, CSF flows through a system of chambers called the ventricles, illustrated in Figure 2.10 CSF suffuses the brain and has several vital functions, namely shock absorption, nutrient provision, and waste clearance.11 CSF is produced in a mass of capillaries called the choroid plexus and flows through the ventricles into the subarachnoid space, bathing the brain and never crossing the blood-brain barrier.10 After circulating through the brain’s interstitial space and ventricles, the CSF then leaves the brain through aquaporin channels surrounding the cephalic veins.6

A New Plumbing System

A team of researchers has discovered an alternate pathway for CSF that clears water-soluble materials from the interstitial space.6 CSF in this so-called “glymphatic pathway” starts in the subarachnoid cavity and then seeps into the cortex, as seen in Figure 3.6 This fluid eventually leaves the brain, carrying with it the waste generated by cells. CSF enters the parenchyma from the subarachnoid cavity and travels immediately alongside the blood vessels.6 This route, which forms a sheath around the blood vessels, is dubbed the “paravascular” pathway, and CSF enters and exits the interstitial space through these avenues.6 The pia membrane guides this pathway until the artery penetrates the cortex, as seen in Figure 3. From there, the endfeet of astrocytes bind the outer wall.6 Astrocytes are glial cells that play structural roles in the nervous system, and endfeet are the enlarged endings of the astrocytes that contact other cell bodies and contain AQP4 proteins.6,12

Iliff et al found that AQP4 are highly polarized at the endfeet of astrocytes, which suggested that these proteins provide a pathway for CSF into the parenchyma.6 To test this hypothesis, they compared wild type and Aqp4-null mice on the basis of CSF influx into the parenchyma.6 They injected tracers such as radiolabeled TR-d3 intracisternally, finding that that tracer influx into the parenchyma was significantly reduced in Aqp4-null mice.4 According to their model, AQP4 facilitates CSF flow into the parenchyma. There, CSF mixes with the interstitial fluid in the parenchyma; AQP4 then drives these fluids out and into the paravenous pathway by bulk flow.6 The rapid clearance of tracer in wild type mice and the significantly reduced clearance in Aqp4-null mice demonstrated the pathway’s ability to clear solutes from the brain. This finding is important because the build-up of Aβ is often associated with the onset and progression of AD.

AQP4 and Aβ

To facilitate Aβ removal, astrocytes become activated at a threshold Aβ, but undergo apoptosis at high concentrations.13,14,15 Thus, the concentration of astrocytes has to be in a narrow window. A study by Yang et al further explored the role of AQP4 in the removal of Aβ.16 They found that AQP4 deficiency reduced the astrocytic activation in response to Aβ in mice, and Aqp4-knockout reduced astrocyte death at high Aβ levels.16 Furthermore, AQP4 expression increased as Aβ concentration increased, likely due to a protein synthesis mechanism. Further investigation demonstrated that lipoprotein receptor-related protein-1 (LRP1) is directly involved in the uptake of Aβ, and knockout of Aqp4 reduced up-regulation of LRP1 in response to Aβ.15,16 Finally, AQP4 deficiency was found to alter the levels and time-course of MAPKs, a family of protein kinases involved in the response to astrocyte stressors.16 The role of AQP4 in cleansing the parenchyma as well as modulating astrocytic responses to Aβ thus pinpoint it as a major target for the following potential therapies: repairing defects in toxin clearance from the interstitial space, increasing expression in AQP4-deficient patients to increase astrocyte response, and knocking out Aqp4 in patients with high levels of Aβ to prevent astrocyte damage.

Sleep and Aβ Clearance

Interestingly, there is a link between Aβ clearance and sleep. Xie et al. studied Aβ clearance from the parenchyma in sleeping, anesthetized, and wakeful rodents, obtaining evidence that sleep plays a role in solute clearance from the brain. The researchers found that glymphatic CSF influx was suppressed in wakeful rodents compared to sleeping rodents.17 Glymphatic CSF influx is vital because it clears solutes from the brain in a way somewhat analogous to the way kidneys filer the blood. Real-time measurements showed that the parenchymal space was reduced in wakeful rodents, which led to increased resistance to fluid influx.17 Moreover, Aβ clearance was faster in sleeping rodents. Adrenergic signaling was hypothesized as the cause of volume reduction, implicating hormones such as norepinephrine.17 AQP4 is implicated in this phenomenon, as constricted interstitial space resists the CSF influx that this protein enables.

Aquaporin Therapy

If future treatment will target AQP4 function, then researchers must learn to manipulate its expression. However such regulatory mechanisms are not well understood. It is well-known that cells can ingest proteins in the plasma membrane and thus modulate the membrane protein landscape. Huang et al studied this phenomenon with AQP4, utilizing the fact that occluding the middle cerebral artery mimics ischemia and alters AQP4 expression in astrocyte membranes.18 They found that artery occlusion down-regulates AQP4 expression and discovered various mechanisms behind this response.18 Specifically, they determined that AQP4 co-localized in the cytoplasm with several proteins involved in membrane protein endocytosis, after the onset of ischemia.18 They posited that this co-localization indicates the internalization of AQP4.18 These correlations indicate that AQP4 is intimately connected with fluctuations in brain oxygen and nutrient levels, which are limited when blood flow is restricted.

Future Research

Aquaporin-4 is vital to many processes in the brain, but the range and details of these roles are not yet fully understood. As demonstrated, this protein is the central actor in the newly defined glymphatic system responsible for clearing solutes from CSF in the interstitial space. This function implicates AQP4 in the progression of AD and suggests other brain states and neurological conditions may have links to the protein’s function. Studies have demonstrated that AQP4 expression is dynamic, indicating that it can be regulated. The hope is that modulation of aquaporin expression or function could be used in brain therapy. Future research will no doubt focus on these mechanisms, and discoveries will aid in developing a treatment for various brain disorders.


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