Ole Petter Ottersen

Aquaporin water channels – from atomic structure to clinical medicine

The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single‐file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes ‐ ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

Anatomical organization of excitatory amino acid receptors and their pathways

Abstract Synapses that employ excitatory amino acids (EAA) as their neurotransmitter use multiple combinations of receptors, apparently yielding different functional properties. The most well-characterized receptor class, the N-methyl-d-aspartate (NMDA) receptor, is found throughout the brain but primarily in telencephalic regions. Phencyclidine and glycine binding sites, which interact allosterically with NMDA receptors, have almost identical distributions. NMDA sites also extensively overlap with the quisqualate excitatory amino acid receptor class, but are less often found co-localized with the kainate receptor class. Evidence obtained with presynaptic markers - high affinity glutamate or aspartate uptake, Ca 2+ -dependent release, and glutamate and aspartate contents - each indicate that EAA are major transmitters of corticocortical, corticofugal, and sensory systems. Recent advances in the histological analysis of these markers are now providing a more detailed map of the excitatory amino acid system and this anatomical map appears to correspond to the distribution of the sum of the receptors. Thus the receptor systems may represent distinct, anatomically-organized, subsystems of excitatory amino acid-mediated neurotransmission.

GLUTAMATE TRANSPORTERS IN GLIAL PLASMA MEMBRANES : HIGHLY DIFFERENTIATED LOCALIZATIONS REVEALED BY QUANTITATIVE ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY

The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer approximately hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerve terminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsin on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.

The molecular basis of water transport in the brain

Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.

Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.

Postembedding immunogold labeling was used to examine the subcellular distribution of the inwardly rectifying K+ channel Kir4.1 in rat retinal Müller cells and to compare this with the distribution of the water channel aquaporin‐4 (AQP4). The quantitative analysis suggested that both molecules are enriched in those plasma membrane domains that face the vitreous body and blood vessels. In addition, Kir4.1, but not AQP4, was concentrated in the basal ∼300–400 nm of the Müller cell microvilli. These data indicate that AQP4 may mediate the water flux known to be associated with K+ siphoning in the retina. By its highly differentiated distribution of AQP4, the Müller cell may be able to direct the water flux to select extracellular compartments while protecting others (the subretinal space) from inappropriate volume changes. The identification of specialized membrane domains with high Kir4.1 expression provides a morphological correlate for the heterogeneous K+ conductance along the Müller cell surface. GLIA 26:47–54, 1999.

An α-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to α-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding α-syntrophin (α-Syn−/−). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of α-Syn−/− mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in α-Syn−/− mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but α-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus α-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain–blood interface. The anchoring of AQP4 to α-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, β-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking α-syntrophin (α-Syn−/−). The total level of AQP4 expression appears normal in brains of α-Syn−/− mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of α-Syn−/− mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in α-Syn−/− mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse–chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires α-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Injections of kainic acid into the amygdaloid complex of the rat: An electrographic, clinical and histological study in relation to the pathology of epilepsy

Abstract Kainic acid has been injected unilaterally into the amygdaloid complex of rats. Electro-encephalographic and clinical changes have been studied in relation to subsequently demonstrated neuropathology using Fink-Heimer and Nissl stainings. Epileptiform electroencephalographic activity began (after 5–60 min) at the site of the injection and spread to the ipsilateral hippocampus, contralaterally and to the cortex. Motor signs of epilepsy occurred repetitively for 2–6 h; subsequently, irregular or regular spikes occurred continuously (for 4–30 h) without positive motor signs. Neuronal loss and gliosis was invariably noted at the injection site. In addition, neuronal loss and degenerative changes were present at other sites where lesions are found after status epilepticus; these included various hippocampal fields, the contralateral amygdala and claustrum and, bilaterally, the midline thalamic nuclei, lateral septum and various cortical areas. The first damage to appear (after 2 h of epileptiform activity) was in the ipsilateral CA3a hippocampal subfield. A correlation was found between the severity of the epileptiform activity in the ipsilateral hippocampus and the severity of pathological alterations. This, as well as other observations suggest that the distant brain damage is not a consequence of the diffusion (or intra-axonal transport) of kainic acid but is causally related to the epileptiform activity induced by the toxin. Intra-amygdaloid injections of kainic acid thus provide a particularly suitable model for investigating the relationship between seizure activity and epileptic brain damage.

The Perivascular Astroglial Sheath Provides a Complete Covering of the Brain Microvessels: An Electron Microscopic 3D Reconstruction

The unravelling of the polarized distribution of AQP4 in perivascular astrocytic endfeet has revitalized the interest in the role of astrocytes in controlling water and ion exchange at the brain–blood interface. The importance of the endfeet is based on the premise that they constitute a complete coverage of the vessel wall. Despite a number of studies based on different microscopic techniques this question has yet to be resolved. We have made an electron microscopic 3D reconstruction of perivascular endfeet in CA1 (stratum moleculare) of rat hippocampus. The endfeet interdigitate and overlap, leaving no slits between them. Only in a few sites do processes—tentatively classified as processes of microglia—extend through the perivascular glial sheath to establish direct contact with the endothelial basal lamina. In contrast to the endfoot covering of the endothelial tube, the endfoot covering of the pericyte is incomplete, allowing neuropil elements to touch the basal lamina that enwraps this type of cell. The 3D reconstruction also revealed large bundles of mitochondria in the endfoot processes that came in close apposition to the perivascular endfoot membrane. Our data support the idea that in pathophysiological conditions, the perivascular astrocytic covering may control the exchange of water and solutes between blood and brain and that free diffusion is limited to narrow clefts between overlapping endfeet.

Delayed K+ clearance associated with aquaporin-4 mislocalization: Phenotypic defects in brains of α-syntrophin-null mice

Recovery from neuronal activation requires rapid clearance of potassium ions (K+) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with α-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and α-syntrophin-null mice (α-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the α-Syn-/- mice, whereas labeling of the inwardly rectifying K+ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K+ clearance were studied in hippocampal slices to test whether AQP4 and K+ channels work in concert to achieve isosmotic clearance of K+ after neuronal activation. Microelectrode recordings of extracellular K+ ([K+]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K+ clearance was prolonged up to 2-fold in α-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the α-Syn-/-mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K+ after neuronal activation.