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Ole P Ottersen

Publications and source records attributed to Ole P Ottersen.

16 recordsLinked to original sources
PubMed2006

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice.

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.

Animals↗
PubMed2006

Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb.

Odorant/receptor binding and initial olfactory information processing occurs in olfactory receptor neurons (ORNs) within the olfactory epithelium. Subsequent information coding involves high-frequency spike synchronization of paired mitral/tufted cell dendrites within olfactory bulb (OB) glomeruli via positive feedback between glutamate receptors and closely-associated gap junctions. With mRNA for connexins Cx36, Cx43 and Cx45 detected within ORN somata and Cx36 and Cx43 proteins reported in ORN somata and axons, abundant gap junctions were proposed to couple ORNs. We used freeze-fracture replica immunogold labeling (FRIL) and confocal immunofluorescence microscopy to examine Cx36, Cx43 and Cx45 protein in gap junctions in olfactory mucosa, olfactory nerve and OB in adult rats and mice and early postnatal rats. In olfactory mucosa, Cx43 was detected in gap junctions between virtually all intrinsic cell types except ORNs and basal cells; whereas Cx45 was restricted to gap junctions in sustentacular cells. ORN axons contained neither gap junctions nor any of the three connexins. In OB, Cx43 was detected in homologous gap junctions between almost all cell types except neurons and oligodendrocytes. Cx36 and, less abundantly, Cx45 were present in neuronal gap junctions, primarily at "mixed" glutamatergic/electrical synapses between presumptive mitral/tufted cell dendrites. Genomic analysis revealed multiple miRNA (micro interfering RNA) binding sequences in 3'-untranslated regions of Cx36, Cx43 and Cx45 genes, consistent with cell-type-specific post-transcriptional regulation of connexin synthesis. Our data confirm absence of gap junctions between ORNs, and support Cx36- and Cx45-containing gap junctions at glutamatergic mixed synapses between mitral/tufted cells as contributing to higher-order information coding within OB glomeruli.

Animals↗
PubMed2006

Localization of LRRK2 to membranous and vesicular structures in mammalian brain.

OBJECTIVE: The PARK8 gene responsible for late-onset autosomal dominant Parkinson's disease encodes a large novel protein of unknown biological function termed leucine-rich repeat kinase 2 (LRRK2). The studies herein explore the localization of LRRK2 in the mammalian brain. METHODS: Polyclonal antibodies generated against the amino or carboxy termini of LRRK2 were used to examine the biochemical, subcellular, and immunohistochemical distribution of LRRK2. RESULTS: LRRK2 is detected in rat brain as an approximate 280kDa protein by Western blot analysis. Subcellular fractionation demonstrates the presence of LRRK2 in microsomal, synaptic vesicle-enriched and synaptosomal cytosolic fractions from rat brain, as well as the mitochondrial outer membrane. Immunohistochemical analysis of rat and human brain tissue and primary rat cortical neurons, with LRRK2-specific antibodies, shows widespread neuronal-specific labeling localized exclusively to punctate structures within perikarya, dendrites, and axons. Confocal colocalization analysis of primary cortical neurons shows partial yet significant overlap of LRRK2 immunoreactivity with markers specific for mitochondria and lysosomes. Furthermore, ultrastructural analysis in rodent basal ganglia detects LRRK2 immunoreactivity associated with membranous and vesicular intracellular structures, including lysosomes, endosomes, transport vesicles, and mitochondria. INTERPRETATION: The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain.

Animals↗
PubMed2006

Assembly of a perivascular astrocyte protein scaffold at the mammalian blood-brain barrier is dependent on alpha-syntrophin.

alpha-Syntrophin, a member of the dystrophin-associated protein complex, is required for proper localization of the water channel aquaporin-4 at the blood-brain barrier. Mice lacking alpha-syntrophin have reduced levels of aquaporin-4 in perivascular astroglial endfeet. Consequently, they exhibit reduced edema and infarct volume in brain trauma models and reduced K+ clearance from the neuropil, leading to increased seizure susceptibility. We have used the alpha-syntrophin null mice to investigate whether alpha-syntrophin is required for proper localization of other components of the dystrophin complex at the blood-brain barrier. We find that alpha-syntrophin is required for the full recruitment of gamma2-syntrophin and alpha-dystrobrevin-2 to glial endfeet in adult cerebellum. In contrast, the localization of beta1- and beta2-syntrophin and alpha-dystrobrevin-1 at the blood-brain barrier is not dependent on the presence of alpha-syntrophin. The localization patterns of alpha-dystrobrevin-1 and -2 in wild type cerebellum are strikingly different; while alpha-dystrobrevin-1 is present in glial endfeet throughout the cerebellum, alpha-dystrobrevin-2 is restricted to glial endfeet in the granular layer alone. Finally, we show that the enrichment of dystrophin in glial endfeet depends on the presence of alpha-syntrophin. This finding is the first demonstration that dystrophin localization is dependent on syntrophin. Since the localization of gamma2-syntrophin, alpha-dystrobrevin-2, and dystrophin is contingent on alpha-syntrophin, we conclude that alpha-syntrophin is a central organizer of the astrocyte dystrophin complex, an important molecular scaffold for localization of aquaporin-4 at the blood-brain barrier.

Animals↗
PubMed2005

Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis.

Both homozygous (L166P, M26I, deletion) and heterozygous mutations (D149A, A104T) in the DJ-1 gene have been identified in Parkinson's disease (PD) patients. The biochemical function and subcellular localization of DJ-1 protein have not been clarified. To date the localization of DJ-1 protein has largely been described in studies over-expressing tagged DJ-1 protein in vitro. It is not known whether the subcellular localization of over-expressed DJ-1 protein is identical to that of endogenously expressed DJ-1 protein both in vitro and in vivo. To clarify the subcellular localization and function of DJ-1, we generated three highly specific antibodies to DJ-1 protein and investigated the subcellular localization of endogenous DJ-1 protein in both mouse brain tissues and human neuroblastoma cells. We have found that DJ-1 is widely distributed and is highly expressed in the brain. By cell fractionation and immunogold electron microscopy, we have identified an endogenous pool of DJ-1 in mitochondrial matrix and inter-membrane space. To further investigate whether pathogenic mutations might prevent the distribution of DJ-1 to mitochondria, we generated human neuroblastoma cells stably transfected with wild-type (WT) or mutant (M26I, L166P, A104T, D149A) DJ-1 and performed mitochondrial fractionation and confocal co-localization imaging studies. When compared with WT and other mutants, L166P mutant exhibits largely reduced protein level. However, the pathogenic mutations do not alter the distribution of DJ-1 to mitochondria. Thus, DJ-1 is an integral mitochondrial protein that may have important functions in regulating mitochondrial physiology. Our findings of DJ-1's mitochondrial localization may have important implications for understanding the pathogenesis of PD.

Blotting, Western↗
PubMed2005

Carbonic anhydrase XIV is enriched in specific membrane domains of retinal pigment epithelium, Muller cells, and astrocytes.

Carbonic anhydrases (CAs) are ubiquitous enzymes important to many cell types throughout the body. They help determine levels of H(+) and HCO(-)(3) and thereby regulate intracellular and extracellular pH and volume. CA XIV, an extracellular membrane-bound CA, was recently shown to be present in brain and retina. Here, we analyze the subcellular distribution of CA XIV in retina by high-resolution immunogold cytochemistry and show that the distribution in retina (on glial cells but not neurons) is different from that reported for brain (on neurons but not glia). In addition, CA XIV is strongly expressed on retinal pigment epithelium (RPE). The specific membrane domains that express CA XIV were endfoot and nonendfoot membranes on Muller cells and astrocytes and apical and basolateral membranes of RPE. Gold particle density was highest on microvilli plasma membranes of RPE, where it was twice that of glial endfoot and Muller microvilli membranes and four times that of other glial membrane domains. Neither neurons nor capillary endothelial cells showed detectable labeling for CA XIV. This enrichment of CA XIV on specific membrane domains of glial cells and RPE suggests specialization for buffering pH and volume in retinal neurons and their surrounding extracellular spaces. We suggest that CA XIV is the target of CA inhibitors that enhance subretinal fluid absorption in macular edema. In addition, CA XIV may facilitate CO(2) removal from neural retina and modulate photoreceptor function.

Animals↗
PubMed2005

Loss of perivascular aquaporin 4 may underlie deficient water and K+ homeostasis in the human epileptogenic hippocampus.

An abnormal accumulation of extracellular K+ in the brain has been implicated in the generation of seizures in patients with mesial temporal lobe epilepsy (MTLE) and hippocampal sclerosis. Experimental studies have shown that clearance of extracellular K+ is compromised by removal of the perivascular pool of the water channel aquaporin 4 (AQP4), suggesting that an efficient clearance of K+ depends on a concomitant water flux through astrocyte membranes. Therefore, we hypothesized that loss of perivascular AQP4 might be involved in the pathogenesis of MTLE. Whereas Western blot analysis showed an overall increase in AQP4 levels in MTLE compared with non-MTLE hippocampi, quantitative ImmunoGold electron microscopy revealed that the density of AQP4 along the perivascular membrane domain of astrocytes was reduced by 44% in area CA1 of MTLE vs. non-MTLE hippocampi. There was no difference in the density of AQP4 on the astrocyte membrane facing the neuropil. Because anchoring of AQP4 to the perivascular astrocyte endfoot membrane depends on the dystrophin complex, the localization of the 71-kDa brain-specific isoform of dystrophin was assessed by immunohistochemistry. In non-MTLE hippocampus, dystrophin was preferentially localized near blood vessels. However, in the MTLE hippocampus, the perivascular dystrophin was absent in sclerotic areas, suggesting that the loss of perivascular AQP4 is secondary to a disruption of the dystrophin complex. We postulate that the loss of perivascular AQP4 in MTLE is likely to result in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K+ and an increased propensity for seizures.

Adolescent↗
PubMed2005

Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane.

Aquaporins are a family of water channels found in animals, plants, and microorganisms. A subfamily of aquaporins, the aquaglyceroporins, are permeable for water as well as certain solutes such as glycerol, lactate, and urea. Here we show that the brain contains two isoforms of AQP9--an aquaglyceroporin with a particularly broad substrate specificity--and that the more prevalent of these isoforms is expressed in brain mitochondria. The mitochondrial AQP9 isoform is detected as an approximately 25 kDa band in immunoblots. This isoform is likely to correspond to a new AQP9 mRNA that is obtained by alternative splicing and has a shorter ORF than the liver isoform. Subfractionation experiments and high-resolution immunogold analyses revealed that this novel AQP9 isoform is enriched in mitochondrial inner membranes. AQP9 immunopositive mitochondria occurred in astrocytes throughout the brain and in a subpopulation of neurons in the substantia nigra, ventral tegmental area, and arcuate nucleus. In the latter structures, the AQP9 immunopositive mitochondria were located in neurons that were also immunopositive for tyrosine hydroxylase, as demonstrated by double labeling immunogold electron microscopy. Our findings suggest that mitochondrial AQP9 is a hallmark of astrocytes and midbrain dopaminergic neurons. In physiological conditions, the flux of lactate and other metabolites through AQP9 may confer an advantage by allowing the mitochondria to adjust to the metabolic status of the extramitochondrial cytoplasm. We hypothesize that the complement of mitochondrial AQP9 in dopaminergic neurons may relate to the vulnerability of these neurons in Parkinson's disease.

Amino Acid Sequence↗
PubMed2004

Alpha-syntrophin deletion removes the perivascular but not endothelial pool of aquaporin-4 at the blood-brain barrier and delays the development of brain edema in an experimental model of acute hyponatremia.

The formation of brain edema, commonly occurring as a potentially lethal complication of acute hyponatremia, is delayed following knockout of the water channel aquaporin-4 (AQP4). Here we show by high-resolution immunogold analysis of the blood-brain-barrier that AQP4 is expressed in brain endothelial cells as well as in the perivascular membranes of astrocyte endfeet. A selective removal of perivascular AQP4 by alpha-syntrophin deletion delays the buildup of brain edema (assessed by Diffusion-weighted MRI) following water intoxication, despite the presence of a normal complement of endothelial AQP4. This indicates that the perivascular membrane domain, which is peripheral to the endothelial blood-brain barrier, may control the rate of osmotically driven water entry. This study is also the first to demonstrate that the time course of edema development differs among brain regions, probably reflecting differences in aquaporin-4 distribution. The resolution of the molecular basis and subcellular site of osmotically driven brain water uptake should help design new therapies for acute brain edema.

Animals↗
PubMed2004

Increased expression of erythropoietin receptor on blood vessels in the human epileptogenic hippocampus with sclerosis.

Microvascular (capillary) proliferation is a readily observed, but largely ignored phenomenon of the mesial temporal lobe epilepsy (MTLE) hippocampus. Here, we report that the proliferated capillaries in surgically resected MTLE hippocampi were strongly immunoreactive for erythropoietin receptor (EPO-r). Further, we found that these capillaries were most prominent in areas of the MTLE hippocampus with extensive neuronal loss and gliosis, i.e. the CA3, CA1, and dentate hilus. High-resolution immunogold electron microscopy revealed that the capillary EPO-r was localized to the luminal and abluminal plasma membrane of endothelial cells, to endosome-like structures of these cells, and to pericapillary astrocytic end-feet. Previous studies have shown that systemically administered EPO appears in the cerebrospinal fluid in experimental animals, and the present results are consistent with the idea that EPO enters the brain via receptor-mediated endocytosis. The enrichment of EPO-r shown here suggests a highly efficient uptake of plasma EPO into the MTLE hippocampus and a possible role for this cytokine in epileptogenesis. Moreover, the presence of EPO-r in the MTLE hippocampus may provide a new vehicle for highly efficient delivery of hitherto impermeable drugs into the epileptic brain.

Adult↗
PubMed2003

Intracellular domains of NMDA receptor subtypes are determinants for long-term potentiation induction.

NMDA receptors (NMDARs) are essential for modulating synaptic strength at central synapses. At hippocampal CA3-to-CA1 synapses of adult mice, different NMDAR subtypes with distinct functionality assemble from NR1 with NR2A and/or NR2B subunits. Here we investigated the role of these NMDA receptor subtypes in long-term potentiation (LTP) induction. Because of the higher NR2B contribution in the young hippocampus, LTP of extracellular field potentials could be enhanced by repeated tetanic stimulation in young but not in adult mice. Similarly, NR2B-specific antagonists reduced LTP in young but only marginally in adult wild-type mice, further demonstrating that in mature CA3-to-CA1 connections LTP induction results primarily from NR2A-type signaling. This finding is also supported by gene-targeted mutant mice expressing C-terminally truncated NR2A subunits, which participate in synaptic NMDAR channel formation and Ca2+ signaling, as indicated by immunopurified synaptic receptors, postembedding immunogold labeling, and spinous Ca2+ transients in the presence of NR2B blockers. These blockers abolished LTP in the mutant at all ages, revealing that, without the intracellular C-terminal domain, NR2A-type receptors are deficient in LTP signaling. Without NR2B blockade, CA3-to-CA1 LTP was more strongly reduced in adult than young mutant mice but could be restored to wild-type levels by repeated tetanic stimulation. Thus, besides NMDA receptor-mediated Ca2+ influx, subtype-specific signaling is critical for LTP induction, with the intracellular C-terminal domain of the NR2 subunits directing signaling pathways with an age-dependent preference.

Animals↗
PubMed2003

Delayed K+ clearance associated with aquaporin-4 mislocalization: phenotypic defects in brains of alpha-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 alpha-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 alpha-syntrophin-null mice (alpha-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the alpha-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 alpha-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the alpha-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.

Animals↗
PubMed2003

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb.

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.

Animals↗
PubMed2003

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.

Animals↗
PubMed2002

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells.

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis. However, confocal visualization of antibody-stained A549 cells in the z-plane revealed the majority of the peri-nuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z-plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z-section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near-complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z-plane. The antibody also provided initial visualization of the compartmentalization of protein-GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti-GSH antibody-stained Jurkat T lymphocytes, we also demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide. In addition, we showed cell-cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

Animals↗