Loss of hilar mossy cells in Ammon's horn sclerosis.

PURPOSE: Hilar mossy cells represent an important excitatory subpopulation of the hippocampal formation. Several studies have identified this cell type as particularly vulnerable to seizure activity in rat models of limbic epilepsy. Here we have subjected hilar mossy cell loss in the hippocampus of patients with chronic temporal lobe epilepsy (TLE) to a systematic morphological and immunohistochemical analysis. METHODS: Hippocampal specimens from 30 TLE patients were included; 21 patients presented with segmental neuronal cell loss [Ammon's horns clerosis (AHS)] and 8 with focal lesions (tumors, scars, malformations) not involving the hippocampus proper. In one additional TLE patient, no histopathological alteration could be observed. Surgical specimens from tumor patients without epilepsy (n = 2) and nonepileptic autopsy brains (n = 8) were used as controls. Hilar mossy cells in the human hippocampus were visualized using a novel polycloncal antiserum directed against the metabotropic glutamate receptor subtype mGluR7b or by intracellular Lucifer Yellow injection, confocal laser scanning microscopy, and three-dimensional morphological reconstruction. RESULTS: Compared with controls, a significant loss of mGluR7 immunoreactive mossy cells was observed in patients with AHS (p < 0.05). In contrast, TLE patients with focal lesions but structurally intact hippocampus demonstrated only a discrete, nonsignificant reduction of this neuronal subpopulation. This observation was confirmed by analysis of 62 randomly injected hilar neurons from AHS patients, in which we were unable to detect neurons with a morphology like that of hilar mossy cells. CONCLUSION: Our present data indicate significant hilar mossy cell loss in TLE patients with AHS. In contrast, hilar mossy cells appear to be less vulnerable in patients with lesion-associated TLE. Although the significance of mGluR7 immunoreactivity in mossy cells remains to be studied, loss of this cell population is compatible with alterations in hippocampal networks and regional hyperexcitability as pathogenic mechanism of AHS and TLE.

Up-regulation of the metabotropic glutamate receptor mGluR4 in hippocampal neurons with reduced seizure vulnerability.

Selective hippocampal cell loss and altered neurotransmitter receptor expression have been proposed as pathogenic mechanisms in the development of chronic mesial temporal lobe epilepsy (TLE). Studies in animal models point to metabotropic glutamate receptors (mGluRs) as modulators of hippocampal epileptogenesis. In addition, mGluRs may constitute specific targets for the development of novel anticonvulsive drugs. As mGluR4 represents an inhibitory class III mGluR associated with the reduction of intracellular cyclic AMP levels and calcium influx, we have analyzed the regional and cellular expression of mGluR4 in surgical hippocampal specimens obtained from patients with TLE by using immunohistochemistry and in situ hybridization. Although the hippocampi of control specimens (n = 11) were almost devoid of mGluR4 immunolabeling, all TLE specimens (n = 35) showed a striking up-regulation of mGluR4 immunoreactivity, in particular within the dentate gyrus. Immunoelectron microscopy localized the receptor protein to the periphery of presynaptic and postsynaptic membranes. In situ hybridization revealed increased transcript levels of mGluR4 in dentate granule cells and residual CA4 neurons of TLE specimens compared with controls. Our results suggest a potential role of mGluR4 in counteracting excitatory hippocampal activity and in modulating seizure-associated vulnerability of hippocampal neurons. These data may also provide a basis for pharmacological studies of mGluR4 agonists as potential novel drugs in the treatment of TLE.

Hippocampal loss of tenascin boundaries in Ammon's horn sclerosis.

Ammon's horn sclerosis (AHS) is a common finding in patients with temporal lobe epilepsy (TLE). In addition to selective neuronal cell loss and axonal reorganization, AHS is also characterized by a striking astroglial reaction. However, the functional significance of reactive astrogliosis in the pathogenesis of TLE remains to be determined. Reactive astrocytes produce a variety of cell adhesion molecules and other extracellular matrix (ECM) components with potential effects on axonal growth, axonal branching, and neosynaptogenesis in the central nervous system (CNS). In the present study we describe the distribution of the ECM glycoprotein tenascin/cytotactin (TN-C) in 44 human hippocampal specimens from patients with TLE. The distribution of TN-C immunoreactivity was evaluated with the anti-human TN-C monoclonal antibody K8 by densitometrical analysis, and TN-C protein levels were detected by immunoblotting. In the normal human hippocampus, there were distinctive boundaries between areas of high and low TN-C expression. These border zones demarcated areas with major synaptic input, i.e., the dentate gyrus molecular layer (DG-ML) and the gray matter of the Ammon's horn. TN-C and the neurite growth-associated protein GAP-43 exhibited a complementary pattern of distribution. Densitometric and protein biochemical analysis showed a significant, 4.3-fold increase of TN-C in the hippocampus of TLE patients with AHS compared with normal hippocampus obtained at autopsy. This increase in TN-C immunoreactivity was accompanied by a loss of TN-C boundaries and closely correlated with the extent of reactive gliosis, as indicated by immunoreactivity for glial fibrillary acidic protein. Furthermore, a striking colocalization between TN-C and GAP-43 was observed in the DG-ML of patients with AHS. These observations raise the intriguing possibility of pathogenetically relevant glio-neuronal interactions in human TLE.

Immunohistochemical distribution of metabotropic glutamate receptor subtypes mGluR1b, mGluR2/3, mGluR4a and mGluR5 in human hippocampus.

The metabotropic glutamate receptors (mGluRs) can be classified into three families based on amino acid sequence homology, signal transduction mechanisms and pharmacological properties. Generally, class I mGluRs mediate an excitation of neurons while activation of class II and III mGluRs results in a depression of synaptic transmission. In this study we have analyzed the expression pattern of mGluRs in human hippocampus using a panel of polyclonal antibodies specific for mGluR1b, mGluR2/3, mGluR4a, and mGluR5. Immunoreactivity for mGluR1b and mGluR5, i.e., the subtypes representing class I mGluRs, was found in all hippocampal neurons. The mGluR1b antiserum stained perikarya and proximal dendrites, whereas immunoreactivity for mGluR5 was also detectable in the distal dendritic compartments. Immunoreactivity for mGluR2/3, members of class II mGluRs, was present in all principle neurons in the dentate gyrus as well as in the CA4, CA3 and CA2 regions. Pyramidal cells of the CA1 region exhibited only weak labeling for mGluR2/3. Glial cells were also mGluR2/3-immunoreactive. The reaction obtained with an antiserum directed against mGluR4a, a member of class III mGluRs, was confined to the mossy fiber projection field in CA3 stratum lucidum. These data demonstrate differential expression of mGluR variants in the human hippocampus and may provide an important basis for future studies of mGluRs under various neuropathological conditions such as temporal lobe epilepsy, ischemia and neurodegenerative disorders.

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of Fc epsilon R II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the alpha or the beta form of Fc epsilon R II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both alpha and beta forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either alpha or beta, were unaffected after incubation with anti-sense oligonucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of human IgE production via Fc epsilon R-II stimulation results from a decrease in the mRNA for secreted but not membrane epsilon H chains.

We have previously shown, using IgE-anti-IgE immune complexes or mAb directed against CD23, that ongoing production of secreted IgE from the human plasma cell line AF-10 can be inhibited via the low affinity FcR for IgE (CD23). Changes occurring in the various forms of epsilon messenger RNA (epsilon mRNA) during this suppression were investigated. mRNA levels of lambda L chain were also assessed as well as beta-actin controls. Changes in membrane IgE and secreted IgE and lambda protein were simultaneously measured. Using a genomic probe corresponding to the human epsilon C region domains, three sets of epsilon-mRNA bands were identified (2.1, 3.0, and 3.8 kb). Only the largest of these (3.8 kb) contained the full epsilon membrane sequence and coded for true membrane epsilon protein. The 2.1-kb species of epsilon-mRNA contained no membrane sequence and coded for classical secreted epsilon protein. The intermediate epsilon-mRNA species (3.0 kb) was shown to contain membrane sequence but did not contain the full epsilon membrane sequence. The product of this mRNA would, in fact, function as a secreted protein. When IgE production by AF-10 cells was suppressed via Fc epsilon R-II, there was a 50% fall in the steady state levels of both forms of mRNA (2.1 and 3.0 kb) that code for secreted epsilon protein. Similarly, there was a fall in lambda-mRNA corresponding to the observed decrease in free lambda secretion. In marked contrast, levels of both membrane IgE protein and mRNA coding for membrane epsilon were unaltered on the suppressed AF-10 cells. These data suggest that inhibition of IgE production via Fc epsilon R-II is related to a fall in mRNA for secreted proteins (epsilon and lambda) and probably reflects a post-transcriptional mechanism effecting mRNA for secreted vs membrane protein mRNA.