David L. Martin

Regulation of γ‐Aminobutyric Acid Synthesis in the Brain

Abstract: γ‐Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD65 and GAD67, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′‐phosphate (pyridoxal‐P), and level of expression among brain regions. GAD65 appears to be localized in nerve terminals to a greater degree than GAD67, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal‐P is a major factor in the short‐term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD65, but GAD67 also contributes to the pool of apoGAD. The apparent localization of GAD65 in nerve terminals and the large reserve of apo‐GAD65 suggest that GAD65 is specialized to respond to short‐term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo‐ to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short‐term regulation by second messengers. The syntheses of GAD65 and GAD67 proteins are regulated separately. GAD67 regulation is complex; it not only is present as apoGAD67, but the expression of GAD67 protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.

Basal expression and induction of glutamate decarboxylase 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.

Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain.

The levels of the two isoforms of glutamate decarboxylase (GAD) were measured in 12 regions of adult rat brain and three regions of mouse brain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting with an antiserum that recognizes the identical C-terminal sequence in both isoforms from both species. In rat brain the amount of smaller isoform, GAD65, was greater than that of the larger isoform, GAD67, in all twelve regions. GAD65 ranged from 77-89% of total GAD in frontal cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, periaqueductal gray matter, substantia nigra, striatum, thalamus and the ventral tegmental area. The proportion of GAD65 was lower in amygdala and cerebellum but still greater than half of the total. There was a strong correlation between total GAD protein and GAD activity. In the three mouse brain regions analysed (cerebellum, cerebral cortex and hippocampus) the proportion of GAD65 (35,47, and 51% of total GAD) was significantly lower than in the corresponding rat-brain regions. The amount of GAD67 was greater than the amount of GAD65 in mouse cerebellum and was approximately equal to the amount of GAD65 in mouse cerebral cortex and hippocampus.

Increased Intracellular γ-Aminobutyric Acid Selectively Lowers the Level of the Larger of Two Glutamate Decarboxylase Proteins in Cultured GABAergic Neurons from Rat Cerebral Cortex

Abstract: The regulation of glutamate decarboxylase (GAD; EC 4.1.1.15) was studied by using cultures of cerebral cortical neurons from rat brain grown in serum‐free medium. About 50% of the neurons in the cultures were γ‐aminobutyric acid (GABA)ergic as determined by two double‐staining procedures. Immunoblotting experiments with four anti‐GAD sera that recognize the two forms to varying degrees, demonstrated that the cultures contained the two forms of GAD that are present in rat brain (apparent molecular masses = 63 and 66 kDa). GAD activity was reduced by 60–70% when intracellular GABA levels were increased by incubating the cultures with the GABA‐transaminase inhibitor γ‐vinyl‐GABA for >5–10 h or with 1 mM GABA itself. Neither baclofen nor muscimol (100 μM) affected GAD activity. Immunoblotting experiments showed that only the larger of the two forms of GAD (66 kDa) was decreased by elevated GABA levels. These results, together with previous results indicating that the smaller form of GAD is more strongly regulated by pyridoxal 5′‐phosphate (the cofactor for GAD), suggest that the two forms of GAD are regulated by different mechanisms.

Structural features and regulatory properties of the brain glutamate decarboxylases.

It is widely recognized that the two major forms of GAD present in adult vertebrate brains are each composed of two major sequence domains that differ in size and degree of similarity. The amino-terminal domain is smaller and shows little sequence identity between the two forms. This domain is thought to mediate the subcellular targeting of the two GADs. Substantial parts of the amino-terminal domain appear to be exposed and flexible, as shown by proteolysis experiments and the locations of posttranslational modifications. The carboxyl-terminal sequence domain contains the catalytic site and shows substantial sequence similarity between the forms. The interaction of GAD with its cofactor, pyridoxal-5 phosphate (pyridoxal-P), plays a key role in the regulation of GAD activity. Although GAD(65) and GAD(67) interact differently with pyridoxal-P, their cofactor-binding sites contain the same set of nine putative cofactor-binding residues and have the same basic structural fold. Thus the cofactor-binding differences cannot be attributed to fundamental structural differences between the GADs but must result from subtle modifications of the basic cofactor-binding fold. The presence of another conserved motif suggests that the carboxyl-terminal domain is composed of two functional domains: the cofactor-binding domain and a small domain that closes when the substrate binds. Finally, GAD is a dimeric enzyme and conserved features of GADs superfamily of pyridoxal-P proteins indicate the dimer-forming interactions are mediated mainly by the carboxyl-terminal domain.

Glutamate-Dependent Active-Site Labeling of Brain Glutamate Decarboxylase

A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5′‐phosphate (pyridoxal‐P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal‐P into rat‐brain GAD was studied by incubating GAD with [32P]pyridoxal‐P, followed by reduction with NaBH4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4‐Deoxypyridoxine 5′‐phosphate (deoxypyridoxine‐P), a close structural analogue of pyridoxal‐P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal‐P (K1= 0.27 μM) and strongly inhibited glutamate‐dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate‐dependent, deoxypyridoxine‐P‐sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl‐Sepharose, labeled with [32P]pyridoxal‐P, purified by immunoaffinity chromatography, and analyzed by SDS‐polyacrylamide gel electrophoresis. Peak I contained only the 59‐kDa labeled protein. Peaks II and III contained the both the 59‐ and 63‐kDa proteins, but in differing proportions. The 59‐kDa protein predominated in peak II and the 63‐kDa protein in peak III. A similar pattern of proteins was observed with each of the immunoaffinity‐purified peaks of enzyme activity on silver‐stained SDS‐polyacrylamide gels. 32P‐Labeled peak‐I GAD and the 59‐ and 63‐kDa bands of peak III were each exhaustively digested with chymotrypsin and chromatographed on sulfopropyl and reverse‐phase HPLC columns. The elution profiles of the digests were very similar, and each contained a single major peak of radioactivity, as would be expected if the same active‐site peptide was labeled. The strong stimulation by glutamate of labeling with [32P]pyridoxal‐P is entirely consistent with the mechanism of GAD inferred from previous studies and provides further evidence for the glutamate‐dependent cyclic interconversion of the apo‐ and holoenzymes.

Direct Casting of Polymer Membranes into Microfluidic Devices

ABSTRACT Fully functional lab-on-a-chip devices for biological analyses require the capability for cell culture, separation, and purification, as well as analyses to be integrated on a single platform. To date, a great deal of research has been focused on analytical methods for the miniaturization of column-based separations. We have created a platform that provides the capability of including membrane separations as an intermediate stage in such devices. Our techniques adapt conventional silicon processing methods to the casting of membranes directly onto the silicon substrate through a wet inversion process. This process allows precise control of membrane thickness and pore size distribution based on the processing conditions. Using this methodology, we were able to fabricate devices that were found to be very robust with molecular weight cutoffs of approximately 350 Da as measured by solute flux in a dialysis mode of operation. These devices were also found to be suitable for cell culture, as evidenced by the high viability of fibroblasts grown within our device. On the basis of these results, a wide range of separations and coculture applications are possible.

GAD and GABA in an enriched population of cultured GABAergic neurons from rat cerebral cortex

To study various aspects of GABAergic metabolism in an easily accessible system, dissociated cells from postnatal rat cerebral cortex were cultured in a serum-based medium and characterized morphologically and biochemically. The majority (70–90%) of the neurons were GABAergic as determined by three double-labeling procedures. The specific activity of glutamine synthetase in the cultures was 4–5% of the levels in rat astrocyte cultures and intact rat brain, indicating that glia were a minor component. The developmental increase of GABA levels preceded the increase of GAD activity in both immunocytochemical and biochemical experiments. GABA turnover rates also increased with culture age and were 20–30% of GAD activity. Four anti-GAD antibodies, which recognize GAD subunits with differing molecular masses to varying degrees, were used to stain cultured neurons and make immunoblots. Immunoblots showed that the neurons contained two major subunits of GAD which differed in mass by 2 kDa. All four antibodies immunostained both neuronal perikarya and neurites but one antibody, which on the immunoblots predominantly labeled the GAD protein with the lower molecular weight, showed a somewhat more pronounced punctate staining, possibly indicating a principal localization to neurites.

Transmitter and Electrical Stimulation of [3H]Taurine Release from Rat Sympathetic Ganglia

Astroglial cells release taurine in response to stimulation with neurotransmitters. This process has been studied most extensively with primary cultures of astrocytes and LRM55 glial cells. These studies have demonstrated that several transmitters can elicit release. The second messenger systems involved in activating release have been characterized (10, 15, 19). An important issue concerning all studies of this type is the applicability of results obtained with glia in culture to glia in vivo. We have chosen the rat superior cervical ganglion as a nervous tissue having the potential for exploring taurine release from glial cells in situ. The major neuronal composition of the ganglion consists of preganglionic nerve terminals providing cholinergic input and principal neurons providing noradrenergic output. The superior cervical ganglion also contains a very small population of dopamine-containing intrinsic neurons known as SIF cells (3). The glial population of the superior cervical ganglion is composed of Schwann cells responsible for myelination and satellite glia, immunoreactive to glial fibrillary acidic protein, that surround the cell bodies of the principal neurons (1, 7). Currently available data suggest that taurine is selectively taken up by the satellite glia. Autoradiographic studies have demonstrated that the ω-amino acids GABA and β-alanine are selectively accumulated by satellite glial cells in the superior cervical ganglion (5, 20), while transport studies have shown that taurine inhibits [3H]GABA uptake and that GABA inhibits [14C]taurine uptake by the superior cervical ganglion (5). The demonstration that potassium-stimulated efflux of [3H]GABA from the superior cervical ganglion is not reduced by preganglionic denervation also supports the glial localization of ω-amino acid transporters in this tissue (4).

Pyridoxal Phosphate, GABA and Seizure Susceptibility

Pyridoxal-P plays a major role in the short term regulation of γ-aminobutyric acid (GABA) synthesis in brain, as the GABA-synthesizing enzyme glutamate decarboxylase (GAD) is regulated in part by a tightly controlled cycle that interconverts the hold- and apoenzyme. This mechanism explains the sensitivity of brain GABA levels to vitamin B6 deficiency and very likely contributes to the enhanced seizure susceptibility that is a prominent symptom of vitamin B6 deficiency in animals and humans.