Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus.

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.

Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.

Structure and regulation of expression of the acetylcholinesterase gene.

Acetylcholinesterase, an enzyme essential for the termination of the action of acetylcholine, is encoded by a single gene. Alternative mRNA processing gives rise to the expression of enzyme forms with three distinct carboxyl-termini. These structural differences govern the cellular disposition of the expressed enzyme but do not influence catalytic activity. Alternative polyadenylation signals give rise to distinct 3' non-coding regions which are likely to affect mRNA stability. Alternative splicing also occurs at the 5' end of the gene where two promoter regions can be identified. Hence, regulation of expression of the gene occurs at 3 levels, transcriptional through alternative promoters, translational by affecting mRNA stability and processing of distinct mRNAs and post-translationally by giving rise to distinct peptide chains which are processed differently. Recombinant DNA studies have also been extended to modifying protein structure through site-specific mutagenesis and studying the function of the mutant enzymes.

Promoter elements and transcriptional control of the mouse acetylcholinesterase gene.

The 5'-untranslated region of the mouse acetylcholinesterase gene has been characterized structurally by RNase protection, primer extension, and sequencing. Evidence has been obtained for the use of two alternative promoters in brain. Tissue-specific splicing to alternative acceptor sites in the 5'-untranslated exons occurs in brain, muscle, and erythropoietic cells. cis elements 5' of the cap site that is predominantly used in these tissues and cells have been analyzed by deletion analysis of promoter-reporter gene constructs and by site-specific mutagenesis. The cap site is found 107 base pairs (bp) 5' of the translation start site. This region is devoid of CAAT or TATA sequences; further in the 5' direction 50 and 70 bp are tandem Egr-1 sites. The putative promoter has been coupled to the open reading frame of a luciferase reporter gene. Deletion analysis shows that this region largely accounts for tissue-specific transcription seen upon transfection of neuronal and muscle cells. Mutagenesis of the Egr-1 sites results in a marked loss of reporter gene activity, further substantiating the importance of this region in the control of transcription. cis elements in the promoter differ from those found for the genes encoding the various subunits of the nicotinic acetylcholine receptor, and distinct differences in control of transcription are evident when the respective reporter genes are transfected into C2 muscle cells.

Assignment of the gene for acetylcholinesterase to distal mouse chromosome 5.

Characterization of genomic clones encoding mouse acetylcholinesterase enabled us to identify a restriction fragment length polymorphism that distinguishes between the progenitor strains for the recombinant inbred strain sets AKXD and BXD. The strain distribution pattern for this polymorphism indicates that Ache is located on distal mouse chromosome 5.

Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression.

The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.

Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species.

We have isolated cDNA clones encoding acetylcholinesterase from mouse muscle and brain. The polymerase chain reaction was used to amplify cDNA clones from C2 myotubes encoding the entire open reading frame and large segments of the 5' and 3' untranslated regions. The muscle cDNA clones were used to isolate clones from a brain library encoding the same mRNA species. The mouse clones encode a catalytic subunit containing a C-terminal sequence similar to that of the hydrophilic species of Torpedo. The mouse acetylcholinesterase sequence shares approximately 88% and 61% amino acid identity with bovine and Torpedo acetylcholinesterases, respectively, but only 52% identity with mouse butyrylcholinesterase, the sequence of which we have also deduced by molecular cloning. Northern blot and RNAase protection analyses indicate that the cDNA clones were derived from the acetylcholinesterase transcript that predominates in most expressing tissues. In contrast, erythroid cells are enriched in an mRNA species whose sequence diverges from that of the cDNA in the region encoding the C-terminus of the enzyme.

Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences.

Polymorphic forms of acetylcholinesterase are tethered extracellularly either as dimers membrane-anchored by a glycophospholipid or as catalytic subunits disulfidelinked to a collagen tail that associates with the basal lamina. Genomic clones of acetylcholinesterase from T. californica revealed that individual enzyme forms are encoded within a single gene that yields multiple mRNAs. Each enzyme form is encoded in three exons: the first two exons, bases -22 to 1502 and 1503 to 1669, encode sequence common to both forms, while alternative third exons encode a hydrophobic C-terminal region, to which a glycophospholipid is added upon processing, and a nonprocessed C-terminus, yielding a catalytic subunit that disulfide-links with a collagen-like structural unit. The 3' untranslated region of each alternative exon contains tandem repeat sequences that are inverted with respect to the other exon. This may either dictate alternative exon usage by formation of cis stem-loops or affect the abundance of translatable mRNA by trans-hybridization between the alternative spliced mRNA species.