Niranjala J.K. Tillakaratne

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.

Retraining the injured spinal cord.

The present review presents a series of concepts that may be useful in developing rehabilitative strategies to enhance recovery of posture and locomotion following spinal cord injury. First, the loss of supraspinal input results in a marked change in the functional efficacy of the remaining synapses and neurons of intraspinal and peripheral afferent (dorsal root ganglion) origin. Second, following a complete transection the lumbrosacral spinal cord can recover greater levels of motor performance if it has been exposed to the afferent and intraspinal activation patterns that are associated with standing and stepping. Third, the spinal cord can more readily reacquire the ability to stand and step following spinal cord transection with repetitive exposure to standing and stepping. Fourth, robotic assistive devices can be used to guide the kinematics of the limbs and thus expose the spinal cord to the new normal activity patterns associated with a particular motor task following spinal cord injury. In addition, such robotic assistive devices can provide immediate quantification of the limb kinematics. Fifth, the behavioural and physiological effects of spinal cord transection are reflected in adaptations in most, if not all, neurotransmitter systems in the lumbosacral spinal cord. Evidence is presented that both the GABAergic and glycinergic inhibitory systems are up‐regulated following complete spinal cord transection and that step training results in some aspects of these transmitter systems being down‐regulated towards control levels. These concepts and observations demonstrate that (a) the spinal cord can interpret complex afferent information and generate the appropriate motor task; and (b) motor ability can be defined to a large degree by training.

Chronic Intermittent Ethanol Treatment in Rats Increases GABAA Receptor α4-Subunit Expression: Possible Relevance to Alcohol Dependence

Abstract: Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long‐lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABAA antagonist, pentylenetetrazol. We have shown previously that muscimol‐evoked 36Cl− efflux and paired‐pulse inhibition (involving GABAA‐mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)‐treated rats. We now report elevated levels of mRNA in forebrain for the α4 subunit of the GABAA receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin‐labeled RNA probes, we show that at 2 days withdrawal, 60‐dose CIE leads to a significant 30% increase in α4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the α5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit‐selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.

GAMMA-AMINOBUTYRIC ACID (GABA) METABOLISM IN MAMMALIAN NEURAL AND NONNEURAL TISSUES

4-Aminobutyric acid (GABA), a major inhibitory neurotransmitter of mammalian central nervous system, is found in a wide range of organisms, from prokaryotes to vertebrates. GABA is widely distributed in nonneural tissue including peripheral nervous and endocrine systems. GABA acts on GABAA and GABAB receptors. GABAA receptors are ligand-gated chloride channels modulated by a variety of drugs. GABAB receptors are essentially presynaptic, usually coupled to potassium or calcium channels, and they function via a GTP binding protein. In neural and nonneural tissues, GABA is metabolized by three enzymes--glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle. Alternate sources of GABA include putrescine, spermine, spermidine and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination. GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to subunit relative molecular weight (Mr) in kilodaltons). These different forms of GAD are the product of different genes, differing in nucleotide sequence, immunoreactivity and subcellular localization. The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract and circulating erythrocytes. In some tissues, one form (GAD65 or GAD67) predominates. GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified, although the presence of this enzyme has not been in mammalian pancreas, ova, oviduct, testis or sympathetic ganglia.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

Independent cellular and ontogenetic expression of mRNAS encoding three α polypeptides of the rat GABAA receptor

Previous studies have shown that several distinct but related polypeptides can serve as alpha subunits of functional GABAA receptors. Furthermore, the diversity of these polypeptides at least partially accounts for the functional heterogeneity of GABAA receptors. In this paper, we report the results of in situ hybridization studies using probes derived from our recently reported cDNAs for alpha 1, alpha 2, and alpha 4 GABAA receptor polypeptides. We show that the mRNAs that encode these isoforms have distinct regional and cellular distributions and are present at widely varying levels within the rat brain. In addition, our Northern blot analyses indicate that each of these three alpha mRNAs has a distinct pattern of ontogenetic regulation. Differential regulation of alpha polypeptide isoforms may lead to changes in GABAA receptor function during ontogeny as well as to distinct cellular responses to GABA and GABA-related drugs.

Increased expression of glutamate decarboxylase (GAD67) in feline lumbar spinal cord after complete thoracic spinal cord transection

To determine changes in γ‐aminobutyric acid (GABA) in the spinal cord in response to a complete transection, we examined the cellular and tissue changes of the two forms of GABA synthetic enzyme glutamate decarboxylase (GAD65 and GAD67). In situ hybridization, immunohistochemistry, and Western blot analyses show that spinal cord transection between thoracic segments 12 and 13 results in an increase of GAD67, but not GAD65, protein and mRNA in the lumbar spinal cord. This increase occurs mainly in the dorsal horn and persists for at least 12 months. In addition, there was relatively high GAD67‐immunoreactivity around the central canal, with dorsolateral GAD67‐immunoreactive fibers extending toward the ependyma and into the central canal in the transected animals. We suggest that an increase in GAD67 leads to increased GABA production in spinal neurons below the injury site, resulting in altered inhibition and trophic support during posttrauma recovery and adaptation. Increased GABA synthesis around the central canal, in the vicinity of ependymal cells, may represent part of a regenerative process in the mammalian spinal cord, reminiscent of that observed in lower vertebrates. J. Neurosci. Res. 60:219–230, 2000

Glutamate decarboxylases in nonneural cells of rat testis and oviduct : differential expression of GAD65 and GAD67

Abstract: γ‐Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5′‐phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

Postnatal expression of glutamate decarboxylases in developing rat cerebellum.

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD67, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD67 mRNA predominates in the cerebellum. The mRNA for GAD65, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD65 is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD67 mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.

Cyclic AMP Decreases the Expression of a Neuronal Marker (GAD67) and Increases the Expression of an Astroglial Marker (GFAP) in C6 Cells

Abstract: C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2‐kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP‐mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.