Blood Brain Barrier
Brain and Nerves

AQUAPORINS FUNCTIONS IN BRAIN

About 80% of the brain is water. Aquaporins (AQP1, AQP4, AQP9) are brain water-channel proteins serving in the transfer of water and small solutes across cellular membranes. The AQPs are proteins that assemble in cell membranes as tetramers. Each monomer is 30 kDa and has six membrane spanning domains surrounding a water pore that can transport water in both directions.

The molecular basis of water transport in the brain 2003

• 1. Expressed in a number of tissues including erythrocytes, renal tubules, retinal pigment epithelium, heart, lung, skeletal muscle, kidney and pancreas. Weakly expressed in brain, placenta and liver.
• 4. Brain - muscle >> heart, kidney, lung, and trachea.
• 9. Forms a channel with a broad specificity. Mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl-, and deprotonated monocarboxylates are excluded. Also permeable to urea and glycerol. Highly expressed in peripheral leukocytes. Also expressed in liver, lung, and spleen.

AQP1 is expressed in the apical membrane of the choroids plexus, and plays a role in forming CSF. AQP1 is upregulated in choroid plexus tumours, which are associated with increased CSF production, again supporting a role for AQP1 in CSF secretion. Interestingly, AQP1 is not found in normal brain capillary endothelium, although highly expressed in peripheral endothelial cells.

AQP9 may play a role in controlling cerebral energy metabolism because AQP9 transports energy substrates (glycerol, lactate). Further support for this idea comes from reports of AQP9 immunoreactivity in glucose-sensitive catecholaminergic neurons and in mitochondria. In general, AQP9 protein expression becomes upregulated in several brain diseases. It has been suggested that AQP9 facilitates the clearance of lactate during reperfusion.
Lactate transport across astrocyte cell membranes, facilitated by AQP9, is consistent with the shuttle hypothesis whereby lactate, produced by astrocytes in a neuron-activity-dependent manner, diffuses to neurons for energy consumption.

AQP4 is the primary water channel found in the brain and it is much more abundantly expressed in brain than is AQP1 or AQP9.
AQP4 was found in electrically excitable tissues including brain, spinal cord, retina, inner ear and skeletal muscle. AQP4 is not expressed in excitable cells, but is found in supporting cells (astrocytes and ependyma in the central nervous system; Muller glia in the retina; Hensen’s, Claudius and inner sulcus cells in the ear). Brain AQP4 is strongly expressed at the borders between brain parenchyma and major fluid compartments including astrocyte foot processes (brain–blood), glia limitans (brain–subarachnoid cerebrospinal fluid – CSF), as well as ependymal cells and subependymal astrocytes (brain–ventricular CSF) .

There are several reports of altered AQP4 expression in different diseases. In general, AQP4 expression is upregulated in astrocytes associated with brain edema. AQP4 plays an important role in the formation and/or absorption of brain edema fluid. AQP4 expression also becomes upregulated in reactive astrocytes and reactive microglia, suggesting a possible role in glial scar formation.

Recently, AQP4 autoantibodies were discovered in patients with neuromyelitis optica, a demyelinating disease, and are now being used to diagnose this condition. Some publications have suggested that AQP4 autoantibodies cause NMO, probably by inhibiting AQP4.

AQP4 1. controls water movements into and out of the brain 2. facilitates astrocyte migration 3. alters neuronal activity

1. Water movements into and out of brain

Water molecules moving from the blood, through intact blood–brain barrier (BBB), into brain cross three cell membranes: luminal and basal endothelial membranes that lack AQPs, and astrocyte foot process membrane that contains AQP4. Water moves across the endothelium by simple diffusion and vesicular transport, and across the astrocyte foot process primarily through AQP4 channels.
In the normal adult human intracranial cavity, water flows between blood, CSF and brain parenchyma intracellular and interstitial spaces, in response to osmotic and hydrostatic forces. In several diseases, the brain swells because excess water accumulates in the brain parenchyma.

For example:

(a)Cytotoxic oedema is cell swelling, associated with a reduced ECS volume and intact BBB. Water flows from the vasculature into the intracellular brain compartment when the Na+/K+ ATPase fails or

extracellular [Na+] falls. Cytotoxic oedema is produced by early phase cerebral ischaemia, hypoxia and hyponatraemia.

(b)In vasogenic edema, hydrostatic forces cause extravasation of a protein-rich exudate from plasma, through leaky BBB into brain ECS, causing ECS expansion. Brain tumour and brain abscess produce vasogenic edema.

©Hydrocephalic edema arises when CSF pressure is high, causing CSF extravasation through ependyma into periventricular brain. As the brain swells inside the noncompliant skull, intracranial pressure rises, causing brain ischaemia, herniation and eventually death.

The routes for water entry into brain are:

a) In cytotoxic edema, water moves from the blood through endothelial cells and astrocyte foot process membrane (through AQP4) into brain.

b) In vasogenic edema, water moves from blood down a hydrostatic pressure gradient through a leaky BBB into brain ECS. Water entry is not through AQP4.

The routes of water exit from the brain are:

a) Excess water moves from brain, through several layers of astrocyte processes comprising the glia limitans externa and pia into subarachnoid CSF.

b) Excess water also moves from brain, through layers of subependymal astrocyte processes (glia limitans interna) and ependyma into ventricular CSF.

c) Some excess water moves from brain through astrocyte foot processes and endothelial cells into the blood. All exit routes strongly express AQP4.

AQP4 inhibitors may reduce cytotoxic brain swelling in humans, whereas AQP4 activators or upregulators may reduce vasogenic edema and hydrocephalus.

2. Astrocyte migration

Studies showed that AQPs enhance cell migration independent of which AQP or cell type is involved. In general, lack of AQP slows migration speed two to threefold. Molecular mechanisms responsible for AQP involvement in cell migration are unknown. Two theories have recently been suggested:
First theory: actin depolymerisation and ion influx at the leading edge of migrating cells increase local cytoplasm osmolality, thus promoting water influx. Water entry through the cell membrane, facilitated by AQPs, causes a local rise in hydrostatic pressure that expands the cell membrane. This is followed by actin repolymerisation, which stabilizes the membrane protrusion. This theory accounts for the increased cell membrane ruffling observed at the front end of AQP-expressing migrating astrocytes, and for the enhanced migration of astrocytes toward hypo-osmolality when placed in osmotic gradients.

Second theory: migrating cells undergo rapid changes in cell shape and cell volume as they move through the irregularly shaped ECS between stationary cells. AQPs accelerate cell migration by facilitating the transmembrane water fluxes that mediate such cell volume changes. This theory explains why differences in cell migration between wild-type and AQP4-null astrocytes are more marked in brain than in culture and why wild-type astrocytes migrating through brain ECS have more elongated shapes than migrating AQP4-null astrocytes.

The involvement of AQPs in cell migration has therapeutic implications. First, because glial scarring is a major obstacle to neuronal regeneration, AQP4 inhibitors may enhance axonal sprouting and synaptogenesis after CNS injury by delaying glial scarring. Second, AQP4 overexpression is a feature of astrocytomas, which are highly infiltrative tumours. AQP upregulation has also been detected in other aggressive human tumours, and a recent study found more metastases in mice after intravenous injection with AQP1-expressing versus non-AQP-expressing tumour cells. AQPs may facilitate cancer spread, and thus AQP inhibitors may slow tumour growth.

3. Neuronal activity

In the 1990s morphological studies showed colocalisation of AQP4 with the inward rectifying channel Kir4.1 in the retina, leading to the idea that water and K+ fluxes are coupled. Several lines of evidence show reduced neural excitability in AQP4 deletion: AQP4-null mice have a higher seizure threshold and prolonged seizure duration than do wild-type mice, and they have sensorineural deafness and mild retinal impairment.
There is also evidence of altered neurotransmitter release after high K+ depolarisation in AQP4-null mouse brain. It has been proposed that altered K+ kinetics in brain ECS account for the altered neuronal activity in AQP4 deficiency. To explain the altered K+ kinetics in AQP4 deficiency, a functional association between AQP4-facilitated water movement and K+ movement through the Kir4.1 channel has been proposed.

An alternative mechanism (ECS expansion) has been proposed to account for higher seizure threshold and prolonged seizure duration in AQP4 deficiency. According to this idea, increased ECS volume increases the buffering capacity for K+ released into the ECS during neuronal excitation, preventing large changes in ECS [K+].

Recent experiments revealed increased diffusion of fluorescently labelled macromolecules in mouse superficial cerebral cortex and deep in brain, consistent with an expanded ECS in AQP4 deficiency. It is unclear, however, why the ECS volume is increased in AQP4-null mice, and whether ECS expansion can quantitatively account for the differences in K+ fluxes into and out of the ECS.

Water movements in the brain:
role of aquaporins

Aquaporin-4 and brain edema

Three distinct roles of aquaporin-4 in brain function revealed
by knockout mice

Glial Cell Aquaporin-4 Overexpression in Transgenic Mice
Accelerates Cytotoxic Brain Swelling</p

Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice 2008

Drugs and BBB

Endothelial histamine H1 receptor signaling reduces blood-brain barrier permeability and susceptibility to autoimmune encephalomyelitis. 2010

Drug and gene targeting to the brain with molecular trojan horses 2002

Three classes of BBB transport systems

Microbiology of the Nervous System

Aquaporin-4, homeostasis, and neurologic disease 2007

BBB opening

J Cereb Blood Flow Metab. 2010 Mar 24. [Epub ahead of print]
Reorganization of gap junctions after focused ultrasound blood-brain barrier opening in the rat brain. 2010

Alonso A, Reinz E, Jenne JW, Fatar M, Schmidt-Glenewinkel H, Hennerici MG, Meairs S.

Department of Neurology, Universitätsklinikum Mannheim, University of Heidelberg, Mannheim, Germany.

Ultrasound-induced opening of the blood-brain barrier (BBB) is an emerging technique for targeted drug delivery to the central nervous system. Gap junctions allow transfer of information between adjacent cells and are responsible for tissue homeostasis. We examined the effect of ultrasound-induced BBB opening on the structure of gap junctions in cortical neurons, expressing Connexin 36, and astrocytes, expressing Connexin 43, after focused 1-MHz ultrasound exposure at 1.25 MPa of one hemisphere together with intravenous microbubble (Optison, Oslo, Norway) application. Quantification of immunofluorescence signals revealed that, compared with noninsonicated hemispheres, small-sized Connexin 43 and 36 gap-junctional plaques were markedly reduced in areas with BBB breakdown after 3 to 6 hours (34.02+/-6.04% versus 66.49+/-2.16%, P=0.02 for Connexin 43; 33.80+/-1.24% versus 36.77+/-3.43%, P=0.07 for Connexin 36). Complementing this finding, we found significant increases in large-sized gap-junctional plaques (5.76+/-0.96% versus 1.02+/-0.84%, P=0.05 for Connexin 43; 5.62+/-0.22% versus 4.65+/-0.80%, P=0.02 for Connexin 36). This effect was reversible at 24 hours after ultrasound exposure. Western blot analyses did not show any change in the total connexin amount. These results indicate that ultrasound-induced BBB opening leads to a reorganization of gap-junctional plaques in both neurons and astrocytes. The plaque-size increase may be a cellular response to imbalances in extracellular homeostasis after BBB leakage.Journal of Cerebral Blood Flow & Metabolism advance online publication, 24 March 2010; doi:10.1038/jcbfm.2010.41.