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A J Tobin

Publications and source records attributed to A J Tobin.

At least 37 records · Page 2Linked to original sources
PubMed1997

Multiple elements regulate GAD65 transcription.

GAD65 and GAD67, the two forms of GABA-synthesizing enzyme, are usually coexpressed, but their levels are regulated independently. The GAD67 promoter has been described. We have now characterized transcriptional regulatory elements in the 5' flanking region of the GAD65 gene, extending 2.4 kb from the ATG translation initiation site. Primer extension assays revealed that transcription begins at -228 in both adult rat brain and in P19 embryonal carcinoma cells, with additional start sites at -280 in brain and at -360 in P19 cells. These sites are in a GC-rich (72%) region lacking a TATA box. Transient transfection assays revealed that the basal promoter is between -740 and -60, and elements conferring cell-type specificity are further 5'. DNA sequences between -1652 and -1420 can 'silence' transcription from a heterologous promoter. GAD65 and GAD67 promoters share little sequence identity, consistent with differences in their transcriptional regulation.

Animals↗
PubMed1997

Pentobarbital decreases the gamma-aminobutyric acidA receptor subunit gamma-2 long/short mRNA ratio by a mechanism distinct from receptor occupation.

Treatment with pentobarbital of primary cultured cerebellar granule cells decreased the gamma-aminobutyric acid, (GABA)A receptor subunit gamma-2 long/short (gamma-2L/S) mRNA ratio. A high dose of pentobarbital (500 microM) decreased the gamma-2L/S ratio by 64%; the decrease was dose and time dependent and reversible. (-)-Hexobarbital (500 microM), the less potent stereoisomer for GABA(A) receptor activation, decreased the ratio slightly (30%) but significantly more than (+)-hexobarbital (20%). Other GABA(A) receptor activators had no (100 mM ethanol) or little (2 microM 5alpha-pregnane-3alpha-ol-20-one) effect on the gamma-2L/S ratio. Furthermore, picrotoxin (10 microM), which blocks the GABA- and pentobarbital-activated GABA(A) receptor channel, neither changed the gamma-2L/S ratio nor blocked the pentobarbital-induced changes. These data suggest that barbiturates alter the gamma-2L/S mRNA ratio by a mechanism that does not require GABA(A) receptor activation. The gamma-2L/S subunit mRNA includes an exon encoding an octapeptide that contains a protein kinase C phosphorylation consensus site. This exon-encoded peptide, occurring in the putative intracellular loop, can be phosphorylated, and in vitro, this phosphorylation has been shown to have functional consequences. This is the first report of a drug-induced alteration in receptor mRNA splicing. Furthermore, the changes in the gamma-2L/S ratio produced by pentobarbital exposure may have significant effects on the function of an important brain protein, the GABA(A) receptor.

Animals↗
PubMed1996

Presence of calbindin D28K and GAD67 mRNAs in both orthotopic and ectopic Purkinje cells of staggerer mice suggests that staggerer acts after the onset of cytodifferentiation.

We used in situ hybridization to study the expression of GAD67 and calbindin D28K mRNAs in developing mouse cerebellar Purkinje cells. Both genes are expressed prenatally; calbindin D28K mRNAs can be detected in Purkinje cells of embryonic day (E) 15 mice, whereas GAD67 mRNAs first appear slightly later, in E16 mice. The stunted Purkinje cells of staggerer (sg/sg) mutant mice maintain calbindin D28K and GAD67 expression. Our data suggest that the sg/sg mutation does not interfere with the transcriptional activation of these two genes, and might therefore act after the induction of specific gene expression in developing Purkinje cells.

Animals↗
PubMed1996

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Adoptive Transfer↗
PubMed1996

Beta-adrenergic receptor activation promotes process outgrowth in an embryonic rat basal forebrain cell line and in primary neurons.

A clonal cell line, AS583-8.E4.22, from the embryonic day 15 rat basal forebrain was established using retrovirus-mediated transduction of a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen. The cell line expresses cytoskeletal and neurotransmitter features indicative of neuronal commitment. In response to agents that increase intracellular cAMP, including forskolin and catecholamines, the cell line exhibits rapid process outgrowth and growth cone formation that does not require new gene expression or protein synthesis. The neurite outgrowth induced by catecholamines is mediated by beta 2-adrenergic receptors and is characterized by a rapid, reversible redistribution of filamentous actin. Neurons from primary cultures of embryonic day 15 basal forebrain were also found to respond to beta-adrenergic receptor agonists by enhancing growth cone formation. These results suggest that catecholamines provide cues that induce cytoskeletal rearrangements leading to neuronal process outgrowth and growth cone formation in the developing basal forebrain and possibly other neuronal progenitor cell populations. The neuronal basal forebrain cell line provides an ideal model to study the signalling mechanisms underlying the catecholamine-induced process outgrowth.

Adrenergic beta-Agonists↗
PubMed1995

The gamma-aminobutyric acid receptor gamma 3 subunit gene (GABRG3) is tightly linked to the alpha 5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation.

GABAA receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter gamma-aminobutyric acid. The GABAA receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies (alpha, beta, gamma, delta, rho) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABAA receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABAA receptor beta 3 (GABRB3) and alpha 5 (GABRA5) subunit genes, is located in chromosome 15q11-q13. It is shown here that the GABAA receptor gamma 3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5' end of GABRG3 were linked to an existing phage contig spanning the 3' end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation.

Animals↗
PubMed1994

Differential localization of two glutamic acid decarboxylases (GAD65 and GAD67) in adult monkey visual cortex.

Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.

Animals↗
PubMed1994

The exon-intron organization of the genes (GAD1 and GAD2) encoding two human glutamate decarboxylases (GAD67 and GAD65) suggests that they derive from a common ancestral GAD.

We have cloned and characterized human genes (GAD1 and GAD2) encoding the two human glutamate decarboxylases, GAD67 and GAD65. The coding region of the GAD65 gene consists of 16 exons, spanning more than 79 kb of genomic DNA. Exon 1 contains the 5' untranslated region of GAD65 mRNA, and exon 16 specifies the protein's carboxy terminal and at least part of the mRNA's 3' untranslated sequence. Similarly, the coding region of the GAD67 gene consists of 16 exons, spread over more than 45 kb of genomic DNA. The GAD67 gene contains an additional exon (exon 0) that, together with part of exon 1, specifies the 5' untranslated region of GAD67 mRNA. Exon 16 specifies the entire 3' untranslated region of GAD67 mRNA. Exons 1-3 encode the most divergent region of GAD65 and GAD67. The remaining exon-intron boundaries occur at identical positions in the two cDNAs, suggesting that they derive from a common ancestral GAD gene.

Amino Acid Sequence↗
PubMed1994

Cellular distribution of calbindin D28K mRNAs in the adult mouse brain.

We have determined the cellular distribution of calbindin D28K mRNAs throughout the mouse brain by in situ hybridization. While these studies identified neuronal populations similar to those previously identified in rat brain by immunohistochemistry, some discrepancies exist. These may derive from species differences or from the immunological cross-reactivity of calbindin D28K antiserum with other proteins. We note an intriguing association between the distribution of neurons containing calbindin D28K mRNA and those reported by others to contain the inositol 1,4,5-triphosphate (InsP3) receptor.

Animals↗
PubMed1994

A rat brain cDNA encodes enzymatically active GABA transaminase and provides a molecular probe for GABA-catabolizing cells.

cDNAs encoding gamma-aminobutyric acid aminotransferase (GABA-T) were isolated from a lambda ZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD65 and GAD67.

4-Aminobutyrate Transaminase↗
PubMed1994

Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells.

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.

Actins↗
PubMed1994

Distinctive patterns of GABAA receptor subunit mRNAs in 13 cell lines.

We have investigated the GABAA receptor mRNA composition in 13 cell lines, using 13 subunit-specific oligo-primers (alpha 1-6, beta 1-3, gamma 1-3, and delta) and reverse transcriptase PCR amplification. Cell lines (B35, B65, B103, B104, RINm5F, Rat1, PC12, C6, C17, C27, beta TC3, NB41A3, AtT-20), derived from diverse tissue origins, were investigated in order to identify homogeneous cellular sources with distinctive GABAA receptor subunits. Fifteen GABAA receptor subunits have been cloned from mammalian tissue (those listed above plus the retinal subunits rho 1 and rho 2). This multiplicity of GABAA receptor subunits underlies the diverse pharmacology of the GABAA receptor. Attempts to understand the regulation and pharmacology of individual subunits and of the heterooligomeric receptor combinations have been impeded by a lack of pure populations of cells expressing GABAA receptor subunits. Permanent cell lines provide such a resource. Each GABAA receptor subunit mRNA, alpha 1-5, beta 1-3, gamma 1-3, and delta, was detected in at least one cell line. All cell lines examined contained detectable levels of at least one GABAA receptor subunit mRNA. Each cell line contained distinctive combinations of subunit mRNAs. None of the cell lines examined contained detectable amounts of alpha 6 mRNA. These cell lines, which transcribe GABAA receptor subunit mRNAs, provide useful cellular sources for transcriptional and pharmacological studies. Our data also suggest that endogenous GABAA receptor subunit mRNAs may be present in cells that are routinely used for transfection studies, and that this expression might confound interpretation of the studies. In the following companion article, we have looked for functional GABAA receptor Cl- ion channels in these cell lines, using the patch-clamp technique (Hales and Tyndale, 1994).

Animals↗
PubMed1994

Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms.

Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. In most brain regions, both GAD isoforms were present in neuronal cell bodies as well as axon terminals. A few populations of neurons, such as those in the reticular nucleus of the thalamus, exhibited similar cell body labeling for both GADs. However, in many brain regions, the cell bodies that were immunoreactive for GAD67 were often more numerous than those that were immunoreactive for GAD65. In contrast, the density (quantity) of GAD65-immunoreactive axon terminals was higher than that of GAD67-immunoreactive terminals. Strong parallels were observed between the intensity of immunohistochemical labeling of cell bodies and the levels of mRNA labeling for both GAD isoforms. Many groups of GAD-containing cell bodies were distinctly labeled for GAD67, and these same groups of neurons were heavily labeled for GAD67 mRNA. Such neurons included Purkinje cells of the cerebellar cortex, nonpyramidal cells in the cerebral cortex, and neurons of the reticular nucleus of the thalamus. Similar parallels in labeling were observed for GAD65 and its mRNA. Distinct cell body labeling for the protein and associated high levels of GAD65 mRNA were found in neurons of the reticular nucleus of the thalamus and periglomerular cells in the olfactory bulb. However, many cell bodies were not readily labeled for GAD65 with immunohistochemical methods. Such absence or weakness of cell body labeling for the protein was associated with low or moderate levels of GAD65 mRNA. Even though light cell body staining was frequently observed for GAD65 and its mRNA, strong axon terminal labeling for GAD65 was present. Thus, in the deep cerebellar nuclei to which the Purkinje cells of the cerebellar cortex project, strong terminal labeling was observed for both GAD isoforms even though only light cell body labeling of the Purkinje cells was obtained for GAD65 and its mRNA. The findings suggest that the two isoforms of GAD are present in most classes of GABA neurons but that they are not similarly distributed within the neurons. GAD67 is present in readily detectable amounts in many GAD-containing cell bodies whereas GAD65 is particularly prominent in many axon terminals. In addition, neurons that express either form of GAD mRNA also express the corresponding protein. Levels of labeling for the GAD mRNAs suggest that, under normal conditions, the synthesis of GAD65 is frequently lower than that of GAD67.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice results from the T-lymphocyte-mediated destruction of the insulin-producing pancreatic beta-cells and serves as a model for human IDDM. Whereas a number of autoantibodies are associated with IDDM, it is unclear when and to what beta-cell antigens pathogenic T cells become activated during the disease process. We report here that a T-helper-1 (Th1) response to glutamate decarboxylase develops in NOD mice at the same time as the onset of insulitis. This response is initially limited to a confined region of glutamate decarboxylase, but later spreads intramolecularly to additional determinants. Subsequently, T-cell reactivity arises to other beta-cell antigens, consistent with intermolecular diversification of the response. Prevention of the spontaneous anti-glutamate decarboxylase response, by tolerization of glutamate decarboxylase-reactive T cells, blocks the development of T-cell autoimmunity to other beta-cell antigens, as well as insulitis and diabetes. Our data suggest that (1) glutamate decarboxylase is a key target antigen in the induction of murine IDDM; (2) autoimmunity to glutamate decarboxylase triggers T-cell responses to other beta-cell antigens, and (3) spontaneous autoimmune disease can be prevented by tolerization to the initiating target antigen.

Aging↗
PubMed1993

In situ hybridization of GAD mRNA in monkey and human brain: quantification at both regional and cellular levels.

GAD mRNA was detected in human and monkey brain postmortem by in situ hybridization with an [35S]-labelled copy RNA corresponding to a 2.7-kb fragment of the coding region of human GAD mRNA. A characteristic and reproducible pattern of hybridization was obtained with the anti-sense, but not the sense probe in both monkey and human brain. Microscopic examination of tissue sections showed that only neuronal perikarya, not glial cells, were labelled. The data confirm the heterogeneity of GAD mRNA distribution reported in rodent brain and non-human primate brain.

Aged↗
PubMed1993

Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods.

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD) mRNAs in rat brain. These mRNAs encode two forms of GAD that both synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs was virtually identical, as in the reticular nucleus of the thalamus. In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences in the functional properties of these neurons and their means of regulating GABA synthesis.

Animals↗
PubMed1993

Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarboxylases play distinctive functional roles.

Two genes encode two forms of glutamate decarboxylase, GAD65 and GAD67. Because the two GADs differ in subcellular distribution and interactions with the cofactor pyridoxal phosphate, the two enzymes may play different roles in gamma-aminobutyric acid (GABA) production. In this study we have used in situ hybridization to compare the regional and cellular distributions of the two GAD mRNAs in rat brain. Both GAD mRNAs are abundant in olfactory bulb, olfactory tubercle, zona incerta, reticular nucleus of the thalamus, oculomotor nuclei, and pontine tegmental area. GAD65 mRNA is more abundant in several structures of the visual system, including the lateral geniculate nuclei, superior colliculi, and olivary pretectal nucleus, as well as in several hypothalamic and pontine nuclei. In contrast, GAD67 mRNA is more abundant in neocortex, the granular layer of olfactory bulb, lateral and medial septum, globus pallidus, inferior colliculi, and cerebellar cortex. Both GAD mRNAs are present in interneurons as well as in projection neurons, and both are present in neurons with different types of synapses, including dendrodendiritic, axosomatic, and axodendritic synapses. GAD65 mRNA predominates in the visual and the neuroendocrine systems, which are more subject to phasic changes, while GAD67 is present at relatively higher concentrations in many tonically active neurons. GAD65 and GAD67 together may provide more flexibility in the regulation of GABA synthesis than either could alone.

Animals↗