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K P Lehre

Publications and source records attributed to K P Lehre.

15 recordsLinked to original sources
PubMed2006

Specificity controls for immunocytochemistry.

Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.

Antibodies, Monoclonal↗
PubMed1999

Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease.

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N-terminal and central parts), GLAST (C-terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)-triphosphate (IP3)-receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na+-dependent "binding" of D-[3H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N-terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT.

ATP-Binding Cassette Transporters↗
PubMed1998

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain.

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.

ATP-Binding Cassette Transporters↗
PubMed1998

The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia.

Antibodies to an excitatory amino acid transporter (EAAT4) label a glycoprotein of approximately 65 kDa strongly in the cerebellum and weakly in the forebrain. Cross-linking of cerebellar proteins with bis(sulfosuccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters GLAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cerebellar molecular layer, being strongly expressed in parasagittal zones. It is located in cerebellar Purkinje cells, and the highest concentrations are seen in ones expressing high levels of zebrin II (aldolase C). The labeling of Purkinje cell spines and thin dendrites is stronger than that of large diameter dendrites and cell bodies. EAAT4 is present at low concentrations in the synaptic membrane, but is highly enriched in the parts of the dendritic and spine membranes facing astrocytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and multivesicular organelles may reflect turnover of transporter in the plasma membrane. The total Purkinje cell spine surface and the EAAT4 concentration were found to be 1.1 m2/cm3 and 0.2 mg/cm3, respectively, in the molecular layer, corresponding to 1800 molecules/microm2. The juxtasynaptic location of EAAT4 may explain electrophysiological observations predicting the presence of a neuronal glutamate transporter near the release site at a Purkinje cell spine synapse. EAAT4 may function as a combined transporter and inhibitory glutamate receptor.

ATP-Binding Cassette Transporters↗
PubMed1997

Localization of the glutamate transporter protein GLAST in rat retina.

Glutamate is a neurotransmitter in retina. Glutamate transporter proteins keep the resting extracellular glutamate concentration low. This is required for normal neurotransmission and prevents the extracellular concentration of glutamate from reaching toxic levels. Here we describe the light and electron microscopic localization of the glutamate transporter protein GLAST in rat retina using an antibody raised and affinity purified against a peptide corresponding to amino acid residues 522-541. The strongest immunocytochemical labelling was observed in the outer plexiform layer, ganglion cell layer, and optic disc. GLAST was found in Müller cell processes in all retinal layers, notably ensheathing the photoreceptor terminals in the outer plexiform layer, and in astrocytes close to vessels in the inner retina and optic disc. No labelling was observed in neurons. The electrophoretic mobility of GLAST in retina was similar to that in cerebellum. In conclusion, the findings are in agreement with those reported by Derouiche and Rauen [7], except that we did not detect any GLAST in the retinal pigment epithelium.

Amino Acid Sequence↗
PubMed1997

Immunocytochemical localization of a high-affinity glutamate-aspartate transporter, GLAST, in the rat and guinea-pig cochlea.

Glutamate transporters play an important role in the reuptake of glutamate after its release from glutamatergic synapses. Four such transporters have so far been cloned from the rat brain. One, the glutamate-aspartate transporter GLAST, has been detected in the mammalian cochlea, in which the principal afferent synapse of the auditory nerve, between the inner hair cells and neurites of type I spiral ganglion neurons, has been suggested to be glutamatergic. The distribution of GLAST was therefore investigated to provide clues to the handling of glutamate in the cochlea. This was studied using light and electron microscopic immunocytochemistry in rats and guinea pigs with antibodies raised against synthetic peptides based on the sequence for GLAST. Significant immunoreactivity was found in the myelin sheath formed by satellite cells surrounding the type I spiral ganglion neurons, and along the plasma membranes of supporting cells around the inner hair cells; other cells in both locations were only weakly labelled, if at all. The absence of substantial numbers of synapses in the spiral ganglion suggests that GLAST is unlikely to be associated with the uptake of synaptic glutamate after release in this region. Immunoreactivity associated with the inner hair cells is consistent with the utilization of glutamate at the afferent synapse.

ATP-Binding Cassette Transporters↗
PubMed1997

Differential developmental expression of the two rat brain glutamate transporter proteins GLAST and GLT.

The extracellular concentration of the excitatory neurotransmitter glutamate is kept low by the action of glutamate transporters in the plasma membranes of both neurons and glial cells. These transporters may play important roles, not only in the adult brain, but also in the developing brain, as glutamate is thought to modulate the formation and elimination of synapses as well as neuronal migration, proliferation and apoptosis. Here we demonstrate the developmental changes in the expression of two glutamate transporters, GLAST and GLT, by quantitative immunoblotting and by light and electron microscopic immunocytochemistry. At birth, GLT is not detectable, but GLAST is present at significant concentrations both in the forebrain and in the cerebellum. GLT is first detected in the forebrain and cerebellum in the second and third week, respectively. Both transporters reach adult levels by postnatal week 5. The development of the total glutamate uptake activity in the forebrain, as determined by solubilization and reconstitution of the transporters in liposomes, parallels that of GLT, in agreement with the observation that GLT is the predominant transporter in the adult brain. The regional distributions of both GLAST and GLT in the tissue are similar in young and adult rats. Only GLAST is detectable in the external germinal layer of the cerebellar cortex. Electron microscopical investigation demonstrated GLAST and GLT exclusively in glial cells in young as well as in adult animals.

ATP-Binding Cassette Transporters↗
PubMed1996

Brain glutamate transporter proteins form homomultimers.

Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

ATP-Binding Cassette Transporters↗
PubMed1995

Down-regulation of glial glutamate transporters after glutamatergic denervation in the rat brain.

Membrane-localized transporter proteins, expressed in both neurons and glial cells, are responsible for removal of extracellular glutamate in the mammalian CNS. The amounts and activities of these transporters may be under regulatory control. We demonstrate here that cortical lesions, which decrease striatal glutamate uptake in synaptosome-containing homogenates by approximately 50%, also decrease the striatal concentrations of the astrocytic glutamate transporter proteins, GLT-1 and GLAST by approximately 20-30%. Since GABA uptake activity was not decreased and glial fibrillary acidic protein was increased in the same samples, the lesion-induced losses of GLT-1 and GLAST were not caused by a general impairment of neuronal or glial function. The observed reduction in the two astrocytic glutamate transporters after corticostriatal nerve terminal degeneration indicates that their levels of expression are dependent on glutamatergic innervation.

ATP-Binding Cassette Transporters↗
PubMed1995

Arachidonic acid inhibits a purified and reconstituted glutamate transporter directly from the water phase and not via the phospholipid membrane.

Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous system. Keeping the extracellular concentration of glutamate low, the glutamate transporters are required for normal brain function. Arachidonic acid (AA) inhibits glutamate uptake in relatively intact preparations (cells, tissue slices, and synaptosomes (Rhoads, D.E., Ockner, R. K., Peterson, N. A., and Raghupathy, E. (1983) Biochemistry 22, 1965-1970 and Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A., Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59, 600-606). The present study demonstrates that the effect of AA occurs also in a reconstituted system, consisting of a purified glutamate transporter protein incorporated into artificial cell membranes (liposomes). The characteristics of the AA effect in this system and in intact cells are similar with regard to specificity, sensitivity, time course, changes in Vmax, and affinity. AA-ethyl ester is inactive, suggesting that the free carboxylic group is required for inhibitory activity. When incubated with proteoliposomes, AA (300 microM, 15 min) mostly partitions to the lipid phase (lipid/water about 95:5). However, uptake inhibition is abolished by rapid dilution (6.5-fold) of the incubation medium (water phase), a procedure that does not modify the amount of AA associated with lipids. On the contrary, inhibition remains sustained if the same dilution volume contains as little as 5 microM AA, a concentration inactive before saturation of liposome lipids with 300 microM AA. The same degree of inhibition (60%) is obtained by 5 microM AA following preincubation with the inactive AA-ethyl ester (300 microM) instead of AA. The lipids apparently inactivate AA by extracting it from the water phase. The results suggest that AA acts on the transporter from the water phase rather than via the membrane. This could be true for other proteins as well since gamma-aminobutyric acid uptake is similarly affected by AA.

ATP-Binding Cassette Transporters↗
PubMed1995

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.

ATP-Binding Cassette Transporters↗
PubMed1995

Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations.

Glutamate, the major excitatory neurotransmitter in brain, is almost exclusively intracellular due to the action of the glutamate transporters in the plasma membranes. To study the localization and properties of these proteins, we have raised antibodies specifically recognizing parts of the sequences of two cloned rat glutamate transporters, GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992). On immunoblots the antibodies against GLT-1 label a broad heterogeneous band with maximum density at around 73 kDa, while the antibody against GLAST labels a similarly broad band at around 66 kDa in the cerebellum and a few kilodaltons lower in other brain regions. GLT-1 is expressed at the highest concentrations in the hippocampus, lateral septum, cerebral cortex, and striatum, while GLAST is preferentially expressed in the molecular layer of the cerebellum. However, both transporters are present throughout the brain, and have roughly parallel distributions in the cerebral hemispheres and brainstem. Preembedding light and electron microscopical immunocytochemistry shows that both GLT-1 and GLAST are restricted to astrocytes, which appear to express both proteins concomitantly, but in different proportions in different parts of the brain. Nerve terminal labeling was not observed. Both the amino and carboxyl terminals of GLT-1 and GLAST are located intracellularly, indicating an even number of transmembrane segments. Antibodies against a synthetic peptide corresponding to amino acid residues 2-11 of the proposed sequence of GLT-1 recognize the native rat brain GLT-1 protein, confirming that the translation initiation site is at the first ATG.

ATP-Binding Cassette Transporters↗
PubMed1993

A monoclonal antibody raised against an [Na(+)+K+]coupled L-glutamate transporter purified from rat brain confirms glial cell localization.

A monoclonal antibody (9C4) shows that an [Na(+)+K+]coupled glutamate transporter protein purified from rat brain runs electrophoretically as a wide band and is localized in neuroglial cell bodies and processes, but not in neurons. This confirms the findings with polyclonal antibodies [Neuroscience 51 (1992) 295-310], and shows that the apparent heterogeneity in relative molecular mass is accounted for by a single antigenic epitope. By testing several synthetic peptides derived from the deduced amino acid sequences of two cloned rat brain glutamate transporters, the antigenic epitope was identified as residing within the peptide TQSVYDDTKNHRESNSNQC (residues 518-536) of one of these [Nature 360 (1992) 464-467].

Amino Acid Sequence↗