Reidun Torp

Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology

Neurofibrillary tangles (NFTs) are composed of abnormal aggregates of the cytoskeletal protein tau. Together with amyloid beta (Abeta) plaques and neuronal and synaptic loss, NFTs constitute the primary pathological hallmarks of Alzheimer disease (AD). Recent evidence also suggests that caspases are activated early in the progression of AD and may play a role in neuronal loss and NFT pathology. Here we demonstrate that tau is cleaved at D421 (DeltaTau) by executioner caspases. Following caspase-cleavage, DeltaTau facilitates nucleation-dependent filament formation and readily adopts a conformational change recognized by the early pathological tau marker MC1. DeltaTau can be phosphorylated by glycogen synthase kinase-3beta and subsequently recognized by the NFT antibody PHF-1. In transgenic mice and AD brains, DeltaTau associates with both early and late markers of NFTs and is correlated with cognitive decline. Additionally, DeltaTau colocalizes with Abeta(1-42) and is induced by Abeta(1-42) in vitro. Collectively, our data imply that Abeta accumulation triggers caspase activation, leading to caspase-cleavage of tau, and that this is an early event that may precede hyperphosphorylation in the evolution of AD tangle pathology. These results suggest that therapeutics aimed at inhibiting tau caspase-cleavage may prove beneficial not only in preventing NFT formation, but also in slowing cognitive decline.

Differential Expression of Two Glial Glutamate Transporters in the Rat Brain: an In Situ Hybridization Study

The distributions of the mRNAs encoding the l‐glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S‐labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.

Reduced postischemic expression of a glial glutamate transporter, GLT1, in the rat hippocampus

Perturbations of the synaptic handling of glutamate have been implicated in the pathogenesis of brain damage after transient ischemia. Notably, the ischemic episode is associated with an increased extracellular level of glutamate and an impaired metabolism of this amino acid in glial cells. Glutamate uptake is reduced during ischemia due to breakdown of the electrochemical ion gradients across neuronal and glial membranes. We have investigated, in the rat hippocampus, whether an ischemic event additionally causes a reduced expression of the glial glutamate transporter GLT1 (Pines et al. 1992) in the postischemic phase. Quantitative immunoblotting, using antibodies recognizing GLT1, revealed a 20% decrease in the hippocampal contents of the transporter protein, 6 h after an ischemic period lasting 20 min induced by four vessel occlusion. In situ hybridization histochemistry with 35S labelled oligonucleotide probes or digoxigenin labelled riboprobes directed to GLT1 mRNA showed a decreased signal in the hippocampus, particularly in CA1. This reduction was more pronounced at 3 h than at 24 h after the ischemic event. We conclude that the levels of GLT1 mRNA and protein show a modest decrease in the postischemic phase. This could contribute to the delayed neuronal death typically seen in the hippocampal formation after transient ischemia.

Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis

Abstract The distributions in rat cerebral cortex and thalamus of the mRNAs encoding the glutamate transporters GLT1 and rEAAC1 (a rat homologue of rabbit EAAC1) were investigated by nonautoradiographic in situ hybridization using digoxigenin-labelled riboprobes. The probe recognizing rEAAC1 mRNA labelled exclusively neurons while GLT1 mRNA was found in glia as well as in select neuronal populations. The neurons containing the GLT1 transcript exhibited a distribution that was different from, and at some sites complementary to, the distribution of neurons containing rEAAC1 mRNA. In the subiculum, neurons positive for GLT1 and rEAAC1 were found in the deep and superficial part of the cell layer, respectively, while in the parietal neocortex GLT1 predominated in layer VI and rEAAC1 in layer V. Very few neuronal populations, most notably cells in hippocampal subfields CA3 and CA4, and in layer II in the entorhinal cortex, appeared to be equipped with both transcripts. In the thalamus the GLT1 labelling predominated in the midline and intralaminar nuclei while rEAAC1 labelling was found throughout this structure. It was concluded that the cerebral cortex and thalamus show cellular, laminar, as well as regional heterogeneities in the expression of the two glutamate transporters.

Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin‐associated tyrosine kinases, and if integrin signaling targets include NMDA‐class glutamate neurotransmitter receptors. The integrin ligand peptide Gly‐Arg‐Gly‐Asp‐Ser‐Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline‐rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to β1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin‐mediated. Both GRGDSP and fibronectin caused rapid Src kinase‐dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and β1 integrin‐dependent increase in NMDA receptor‐mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.

Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: a semiquantitative electron microscopic study in rats.

A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.

Chapter 19: Ultrastructural immunocytochemical observations on the localization, metabolism and transport of glutamate in normal and ischemic brain tissue

Publisher Summary To study how glutamate (Glu) is handled in individual synapses, neuronal perikarya and glial cells, various methods are developed that allow the concentrations of Glu and related amino acids to be examined morphologically at the light and electron microscopic level. This technology is used to study dynamic changes in the localization of the amino acids following experimental manipulations, such as ischemia in vivo and depolarization-induced synaptic release in vitro . This chapter discusses additional examples of such use, and show how the power of the approach is further exploited by estimating the ratios of different amino acids measured simultaneously in individual tissue compartments. The localization of two proteins important for the synaptic handling of Glu— namely, glutamate dehydrogenase (GluDH) and the high-affinity [Na + +K + ]-coupled Glu transporter (GluTp) is discussed. GluDH catalyzes the reversible oxidative deamination of Glu to α-ketoglutarate and NH 4 + , and may therefore take part in the formation as well as in the breakdown of Glu. GluDH deficiency has been implicated as a pathogenetic factor in neurodegenerative disease. GluTp is responsible for clearing the synaptic cleft of Glu conserving it for reuse. However, reversed action of GluTp is probably responsible for the rise in extracellular Glu in energy-deficient state. It may therefore contribute to excitotoxic cell loss in the brain ischemia and related conditions, but may also play a role in neurodegenerative disorders.

Effect of ischaemia and reperfusion on the extra- and intracellular distribution of glutamate, glutamine, aspartate and GABA in the rat hippocampus, with a note on the effect of the sodium channel blocker BW1003C87.

The redistribution of neurotransmitter amino acids resulting from 20 min of ischaemia was studied in the rat hippocampus by quantitative, electron microscopic immunocytochemistry and by in vivo microdialysis. Changes in the distribution of glutamate, glutamine, aspartate and GABA in various cell compartments of CA1 were analysed immediately after ischaemia or after 60 min of reperfusion, by incubating ultrathin sections with antisera raised against protein glutaraldehyde conjugates of the respective amino acids and subsequently with a secondary antibody coupled to colloidal gold particles. Transverse microdialysis probes coupled with HPLC and implanted in the same animals were used to determine the extracellular concentration of amino acids in the left hippocampus and to apply a drug (BW 1003C87) believed to modify the extracellular release of amino acids induced by ischaemia. Forebrain ischaemia was induced by temporary occlusion of the common carotid arteries in rats with permanently occluded vertebral arteries. The extracellular concentrations of glutamate, aspartate and GABA increased markedly during ischaemia, but returned rapidly to normal during reperfusion. BW 1003C87 (250 μM, in the dialysis fluid) did not modify the increase in extracellular concentration of amino acids during ischaemia. Glutamate-like immunoreactivity was reduced in pyramidal cell somata both immediately after ischaemia and after 60 min of reperfusion. This reduction appeared to be somewhat less pronounced for cells in the left hemisphere (perfused with BW 1003C87) than in the contralateral hemisphere. Ischaemia caused no consistent changes in terminals. The ratio between the intracellular levels of glutamate and glutamine was assessed by double-labelling immunocytochemistry, using two different gold particle sizes. The glutamate-glutamine ratio in glial cells was greatly increased after ischaemia, but recovered to a normal level within 1 h of reperfusion. Aspartate-like immunoreactivity was substantially reduced in pyramidal cell somata both immediately and 60 min after ischaemia, while profiles that were immunopositive for GABA in control brains showed increased GABA immunolabelling. These results suggest that postsynaptic neuronal elements as well as glial cells contribute to the extracellular overflow of excitatory amino acids during an ischaemic event: post-synaptic elements by leaking or releasing glutamate and aspartate, and glial cells by losing their ability to convert glutamate to glutamine effectively. The temporal association between the changes in the glial contents of glutamate and glutamine, and the extracellular amino acid fluctuations recorded by microdialysis in the same animals, underline the strategic role of glia in regulating the extracellular level of glutamate.

Genetically augmenting tau levels does not modulate the onset or progression of Aβ pathology in transgenic mice

The two hallmark pathologies of Alzheimer’s disease (AD) are amyloid plaques, composed of the small amyloid‐β (Aβ) peptide, and neurofibrillary tangles, comprised aggregates of the microtubule binding protein, tau. The molecular linkage between these two lesions, however, remains unknown. Based on human and mouse studies, it is clear that the development of Aβ pathology can trigger tau pathology, either directly or indirectly. However, it remains to be established if the interaction between Aβ and tau is bidirectional and whether the modulation of tau will influence Aβ pathology. To address this question, we used the 3xTg‐AD mouse model, which is characterized by the age‐dependent buildup of both plaques and tangles. Here we show that genetically augmenting tau levels and hyperphosphorylation in the 3xTg‐AD mice has no effect on the onset and progression of Aβ pathology. These data suggest that the link between Aβ and tau is predominantly if not exclusively unidirectional, which is consistent with the Aβ cascade hypothesis and may explain why tauopathy‐only disorders are devoid of any Aβ pathology.

A quantitative electron microscopic immunocytochemical study of the distribution and synaptic handling of glutamate in rat hippocampus.

One of the major problems in glutamate immunocytochemistry has been the difficulty involved in separating immunocytochemical labelling due to metabolic glutamate from the labelling caused by transmitter glutamate. Another problem appears to be the accessibility of antigenic sites in conventional light microscopic preparations. In the present report, we have applied the primary glutamate antiserum onto ultrathin tissue sections, followed by the use of a colloidal gold detection system. The use of this postembedding immunogold procedure allows equal access of antibodies to all cellular compartments exposed at the section surface, allows quantitative assessment of the immunoreactivity, and affords a high resolution compatible with studies at the organelle level. When applied to slice preparations the immunogold procedure can be used to identify releasable pools of glutamate. These methodological advances have greatly increased the usefulness of glutamate immunocytochemistry as a tool to study putative glutamatergic terminals in the CNS.