Forms Of Glutamic Acid Decarboxylase Research Articles

The Evolving Landscape of Autoantigen Discovery and Characterization in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is caused, in part, by T cell–mediated destruction of insulin-producing β-cells. High risk for disease, in those with genetic susceptibility, is predicted by the presence of two or more autoantibodies against insulin, the 65-kDa form of glutamic acid decarboxylase (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8). Despite this knowledge, we still do not know what leads to the breakdown of tolerance to these autoantigens, and we have an incomplete understanding of T1D etiology and pathophysiology. Several new autoantibodies have recently been discovered using innovative technologies, but neither their potential utility in monitoring disease development and treatment nor their role in the pathophysiology and etiology of T1D has been explored. Moreover, neoantigen generation (through posttranslational modification, the formation of hybrid peptides containing two distinct regions of an antigen or antigens, alternative open reading frame usage, and translation of RNA splicing variants) has been reported, and autoreactive T cells that target these neoantigens have been identified. Collectively, these new studies provide a conceptual framework to understand the breakdown of self-tolerance, if such modifications occur in a tissue- or disease-specific context. A recent workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases brought together investigators who are using new methods and technologies to identify autoantigens and characterize immune responses toward these proteins. Researchers with diverse expertise shared ideas and identified resources to accelerate antigen discovery and the detection of autoimmune responses in T1D. The application of this knowledge will direct strategies for the identification of improved biomarkers for disease progression and treatment response monitoring and, ultimately, will form the foundation for novel antigen-specific therapeutics. This Perspective highlights the key issues that were addressed at the workshop and identifies areas for future investigation.

Environmental enrichment as a therapeutic avenue for anxiety in aged Wistar rats: Effect on cat odor exposition and GABAergic interneurons

The use of more ethological animal models to study the neurobiology of anxiety has increased in recent years. We assessed the effect of an environmental enrichment (EE) protocol (24h/day over a period of two months) on anxiety-related behaviors when aged Wistar rats (21months old) were confronted with cat odor stimuli. Owing to the relationship between GABAergic interneurons and the anxiety-related neuronal network, we examined changes in the expression of Parvalbumin (PV) and 67kDa form of glutamic acid decarboxylase (GAD-67) immunoreactive cells in different brain regions involved in stress response. Behavioral results revealed that enriched rats traveled further and made more grooming behaviors during the habituation session. In the cat odor session, they traveled longer distances and they showed more active interaction with the odor stimuli and less time in freezing behavior. Zone analysis revealed that the enriched group spent more time in the intermediate zone according to the proximity of the predator odor. Regarding the neurobiological data, the EE increased the expression of PV-positive cells in some medial prefrontal regions (cingulate (Cg) and prelimbic (PL) cortices), whereas the GAD-67 expression in the basolateral amygdala was reduced in the enriched group. Our results suggest that EE is able to reduce anxiety-like behaviors in aged animals even when ethologically relevant stimuli are used. Moreover, GABAergic interneurons could be involved in mediating this resilient behavior.

Combination of prenatal immune challenge and restraint stress affects prepulse inhibition and dopaminergic/GABAergic markers

Gestational immune challenge with the viral-like antigen poly I:C is a well-established neurodevelopmental model of schizophrenia. However, exposure to inflammation during early life may sensitize the developing brain to secondary insults and enhance the central nervous system vulnerability. To gain a better understanding of the pathophysiology of schizophrenia, we thus developed a two-hit animal model based on prenatal poly I:C immune challenge followed by restraint stress in juvenile mice. C57BL/6 gestational mice were intraperitoneally injected with poly I:C or saline at gestational day 12. Pups were then submitted or not, to restraint stress for 2h, for three consecutive days, from postnatal days 33 to 35. Prepulse inhibition (PPI) of acoustic startle response is commonly used to assess sensorimotor gating, a neural process severely disrupted in patients with schizophrenia. Our results revealed that the combination of prenatal immune challenge with poly I:C followed by a restraint stress period was able to induce a PPI disruption in 36-day-old pups, as opposed to each insult applied separately. PPI deficits were accompanied by dopaminergic and GABAergic abnormalities in the prefrontal cortex and striatum. Indeed, measurements of cortical and striatal dopamine D2 receptor (D2R) mRNA and protein levels revealed that the combination of gestational exposure to poly I:C and postnatal restraint stress induced an increase in D2R protein and mRNA levels. Likewise, the combination of both insults reduced the mRNA and protein expression levels of the 67 kDa form of glutamic acid decarboxylase (GAD67), in those two brain regions. To our knowledge, this two-hit animal model is the first in vivo model reporting PPI deficits at pubertal age. This two-hit animal model may also help in studying innovative therapies dedicated to the treatment of schizophrenia, especially in its early phase.

Effect of honokiol on activity of GAD 65 and GAD 67 in the cortex and hippocampus of mice

Honokiol, an active agent extracted from magnolia bark, has been reported that induces anxiolytic action in a mouse elevated plus-maze test. However, the mechanism of anxiolytic action induced by honokiol remains unclear. This study was to investigate the change in two forms of glutamic acid decarboxylase (GABA synthesized enzymes) GAD 65 and GAD 67 in the cortex and hippocampus areas while the anxiolytic actions induced by chronic administration of honokiol in mice. Mice treated with 7 daily injection of honokiol (1 mg/kg, p.o.) caused anxiolytic action which was similar to that was induced by 7 daily injection of diazepam (2 mg/kg, p.o.) in the elevated plus-maze test. In addition, the activity of hippocampal GAD 65 of honokiol treated mice was significantly increased than that of the vehicle or diazepam treated groups. These data suggest that honokiol causes diazepam-like anxiolytic action, which may be mediated by altering the synthesis of GABA in the brain of mice.

GABA neurotransmitter signaling in the developing mouse lens: Dynamic regulation of components and functionality

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate nervous system, serves as a signaling molecule modulating diverse processes during embryonic development. Earlier we have demonstrated that different forms of glutamic acid decarboxylase (GAD) are differentially regulated during mouse lens development. Here we show that the developing lens expresses also components of GABA signaling downstream of GAD. Multiple GABA(A) and GABA(B) receptor subunits as well as the GABA transporters show expression profiles highly correlated with the expression of different GADs. GABA receptors (GABAR) and the vesicular GABA transporter localize at the apical/basal membranes of the lens epithelia and differentiating fibers and may be involved in conventional GABAR-mediated signaling, while the membrane GABA transporters may also function as Na(+)/Cl(-)/GABA carriers. The functionality of GABAR was verified by calcium imaging in whole lenses. Our data suggest that GABA synthesized locally by GAD, acts through GABA receptors by modulating the intracellular calcium levels.

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for γ-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that μ-calpain is a good candidate for conversion of fGAD67 to tGAD67. This conclusion is based on the following observations: 1. purified recombinant GAD67 is cleaved by μ-calpain at specific sites; 2. in brain synaptosomal preparation, GAD67 is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in μ-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when μ-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. μ-calpain is activated by neuronal stimulation and Ca2+-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.

Post-translational Regulation of l-Glutamic Acid Decarboxylase in the Brain

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA is converted from glutamic acid by the action of glutamic acid decarboxylase (GAD). There are two forms of GAD in the brain, GAD65 and GAD67, referring to a molecular weight of 65 and 67 kDa, respectively. Perturbations in GABAergic neurotransmission have been linked to a number of neurological disorders. Since GAD is the rate-limiting enzyme in controlling GABA synthesis, it is important to understand how GAD is regulated in the brain. It is known that GAD function can be regulated at the transcriptional/translational and post-translational levels. This review focuses briefly on the recent advances in revealing the post-translational regulation of GAD function including protein phosphorylation, palmitoylation and activity-dependent cleavage. The results from these studies have improved our understanding of the regulation of GAD function in the brain.

Learning-induced plasticity of cortical representations does not affect GAD65 mRNA expression and immunolabeling of cortical neuropil

Two forms of glutamic acid decarboxylase (GAD) are present in inhibitory neurons of the mammalian brain, a 65-kDa isoform (GAD65) and a 67-kDa isoform (GAD67). We have previously found that GAD67 is upregulated during learning-dependent plasticity of cortical vibrissal representations of adult mice. After sensory conditioning involving pairing stimulation of vibrissae with a tail shock, the increase in mRNA expression and density of GAD67-immunoreactive neurons was observed in barrels representing vibrissae activated during the training. In the present study, using the same experimental model, we examined GAD65 mRNA and protein levels in the barrel cortex. For this purpose, we used in situ hybridization and immunohistochemistry. No changes in the level of GAD65 mRNA expression were detected after the training. The pattern of GAD65 mRNA expression was complementary to that observed for GAD67. Immunocytochemical analysis found no changes in immunolabeling of neuropil of the barrels representing the vibrissae activated during the training. The results show that, in contrast to GAD67, cortical plasticity induced by sensory learning does not affect the expression of GAD65.

Selective GABAergic Control of Higher-Order Thalamic Relays

GABAergic signaling is central to the function of the thalamus and has been traditionally attributed primarily to the nucleus reticularis thalami (nRT). Here we present a GABAergic pathway, distinct from the nRT, that exerts a powerful inhibitory effect selectively in higher-order thalamic relays of the rat. Axons originating in the anterior pretectal nucleus (APT) innervated the proximal dendrites of relay cells via large GABAergic terminals with multiple release sites. Stimulation of the APT in an in vitro slice preparation revealed a GABA(A) receptor-mediated, monosynaptic IPSC in relay cells. Activation of presumed single APT fibers induced rebound burst firing in relay cells. Different APT neurons recorded in vivo displayed fast bursting, tonic, or rhythmic firing. Our data suggest that selective extrareticular GABAergic control of relay cell activity will result in effective, state-dependent gating of thalamocortical information transfer in higher-order but not in first-order relays.

Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1–95, 96–444, and 445–585), δGAD65 (a truncated GAD65 spanning residues 69–585), and GAD67. δGAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.

Normal incidence of diabetes in NOD mice tolerant to glutamic acid decarboxylase.

Experiments in nonobese diabetic (NOD) mice that lacked expression of glutamic acid decarboxylase (GAD) in β cells have suggested that GAD represents an autoantigen essential for initiating and maintaining the diabetogenic immune response. Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed. Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display. In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

GABA and glutamate in mating-activated cells in the preoptic area and medial amygdala of male gerbils.

The posterodorsal medial amygdala (MeApd), the posterodorsal preoptic nucleus (PdPN), and the medial cell group of the sexually dimorphic preoptic area (mSDA) contain cells that are activated specifically at ejaculation as assessed by Fos expression. The mSDA also expresses Fos early in the mating context. Because little is known about the neurotransmitters of these activated cells, the possibility that they use gamma-aminobutyric acid (GABA) or glutamate was assessed. Putative glutamatergic cells were visualized with immunocytochemistry (ICC) for glutamate and its neuron-specific transporter. Their distributions were compared with those of GABAergic cells visualized with ICC for the 67-kDa form of glutamic acid decarboxylase (GAD(67)) and in situ hybridization for GAD(67) messenger RNA (mRNA). Colocalization of Fos and GAD(67) mRNA in recently mated males indicated that half of the activated cells in the PdPN, mSDA, and lateral MeApd are GABAergic. Colocalization of Fos and glutamate suggested that a quarter of the activated mSDA and lateral MeApd cells are glutamatergic. The PdPN does not appear to have glutamatergic cells. In the lateral MeApd, the percentage of activated cells that are GABAergic (45%) matches the percentage that project to the principal part of the bed nucleus of the stria terminalis (BST; 43%), and the percentage likely to be glutamatergic (27%) matches the percentage projecting to the mSDA (27%). The latter could help to trigger ejaculation. The distribution of GABAergic and putative glutamatergic cells in the caudal preoptic area, caudal BST, and medial amygdala of male gerbils is also described.

Olfactory discrimination learning deficit in heterozygous reeler mice

Adult mice heterozygous for the ‘reeler’ mutation (HR mice) have been found not to have a reeler phenotype but to express a number of abnormal traits including reduced sensorimotor gating, lower density of dendritic spines in frontoparietal cortex and hippocampus, and selectively decreased expression of one form of glutamic acid decarboxylase (GAD67). Since reelin is expressed in olfactory areas of the adult mouse brain, we have tested for olfactory functions in HR mice that express only half of the reelin found in wild-type (WT) mice. HR and WT mice were trained to criterion performance of 90% correct in a block of 20 trials on eight distinct simultaneous-cue, two-odor discriminations. HR mice required significantly more training sessions and made more errors than did WT mice in acquiring the first olfactory discrimination. Subsequent discriminations were learned equally rapidly by both HR and WT mice. Memory retention for the final discrimination was tested 1 week after training and was equally good for both groups. Both HR and WT mice showed equivalent sensitivity in discriminating low concentrations of either ethyl acetate or butanol from non-odorized air. Whether or not the olfactory learning deficit observed in HR mice is related to the low dendritic spine density or GAD67 reduction observed in hippocampus and cortex are currently under investigation.

Regulatory cytokine production stimulated by DNA vaccination against an altered form of glutamic acid decarboxylase 65 in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop a T-cell dependent autoimmune form of diabetes, in which glutamic acid decarboxylase 65 (GAD65) is an important islet target antigen. Intramuscular DNA vaccination with a plasmid encoding native GAD65 (a cytosolic antigen) did not significantly alter the incidence of diabetes, but vaccination against an altered form of GAD65 with a signal peptide (spGAD), which is secreted in vitro, was protective. The preventive effect was further enhanced by repeated injections of the spGAD plasmid. Following DNA injection into muscle GAD65 was expressed for several months, and this was not accompanied by an inflammatory response. Immunization against GAD65 was not associated with substantial alterations in cytokine production by splenic lymphocytes stimulated with immunogenic GAD65 peptides. In contrast, spGAD induced increased secretion of both interleukin 10 and interferon gamma and a striking decrease in the interferon gamma/interleukin 10 ratio in culture supernatants. Similarly, spGAD-immunized mice had higher serum interleukin 10 levels and lower serum interferon gamma levels than other groups, suggesting a systemic effect. In nondiabetic mice there was increased basal production of transforming growth factor beta(1), which was enhanced by antigenic stimulation. These alterations in regulatory cytokine production were apparent both early and late after the treatment was initiated. These findings suggest that DNA vaccination against spGAD protects NOD mice by increasing regulatory cytokine production.

MRNA expression patterns and distribution of white matter neurons in dorsolateral prefrontal cortex of depressed patients differ from those in schizophrenia patients

Background Schizophrenia, bipolar illness, and major depressive disorder have distinct presentations, but share some common symptoms. Hence, some common cellular and molecular abnormalities may be identifiable in these disorders. Methods We examined cell-specific markers in the dorsolateral prefrontal cortex of brains from 18 patients with bipolar or major depressive disorder, and 18 matched controls, using in situ hybridization histochemistry and staining for nicotinamide-dinucleotide phophate-diaphorase (NADPH). The distribution of NADPH-positive interstitial cells of the white matter and the expression of the mRNA for the 67 KD form of glutamic acid decarboxylase (GAD 67) had previously been shown to be altered in prefrontal cortex of schizophrenics. Other markers identifying glutamatergic neuronal populations were α-type II calcium/calmodulin dependent protein kinase (CAMKII-α), brain derived neurotrophic factor, (BDNF) and the putative transcription factor, T-brain–1 (TBR1). Results Expression of GAD 67 and the distribution of NADPH-positive cells in the white matter were not significantly altered in the dorsolateral prefrontal cortex of depressed subjects. Expression of CAMKII-α and TBR1 mRNAs was significantly increased in bipolar patients but not in major depressed patients, and there was a trend toward reduced BDNF expression in both groups. Abnormal patterns of gene expression and neuronal distribution in schizophrenics are markedly different from those in depressed patients. Conclusions The findings that TBR1 and CAMKII-α expression is increased only in bipolar patients suggests abnormalities of specific genes related to a major cortical cell type and its connectivity.

Sequential induction of embryonic and adult forms of glutamic acid decarboxylase during in vitro-induced neurogenesis in cloned neuroectodermal cell-line, NE-7C2.

The expression of different forms of glutamate decarboxylases and GABA was investigated in the course of retinoic acid-induced neuronal differentiation of NE-7C2 cell-line established from brain vesicles of 9-day-old mouse embryos lacking functional p53 gene. Non-induced NE-7C2 cells expressed embryonic GAD mRNAs with a low level of embryonic GAD25 protein and did not contain detectable amounts of GABA. Addition of 10(-6) M retinoic acid induced the expression of N-tubulin and a significant increase in the level of embryonic GAD messages and GAD25 protein in early stage differentiating neurones. The enzymatically active embryonic GAD44 was detected at later stages of induction in neurone-like cells and showed a maximum of expression at the time of neurite elongation and network formation. With the advance of neuronal maturation, the expression of embryonic forms declined while the adult GAD65 and GAD67 transcripts became dominant. GABA-containing neurones were first demonstrated on the sixth day of induction coinciding with the peak of GAD44 expression and the beginning of GAD65 expression. The sequential induction of different GAD forms and the stage-dependent GABA synthesis in NE-7C2 cells is highly reminiscent of the temporal pattern found in vivo and suggests that these processes might be involved in the differentiation of neuronal progenitors.

GABA signalling during development: new data and old questions.

In addition to being the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).

Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation.

Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in gamma-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.

Effects of plasmid DNA injection on cyclophosphamide-accelerated diabetes in NOD mice.

Type 1 diabetes results in most cases from the destruction of insulin-secreting beta cells by the immune system. Several immunization methods based on administration of autoantigenic polypeptides such as insulin and glutamic acid decarboxylase (GAD) have been used to prevent autoimmune diabetes in the non-obese diabetic (NOD) mouse. In the work presented here, a gene-based approach was taken for a similar purpose. A plasmid carrying different cDNAs was used to investigate the effects of injecting naked DNA on cyclophosphamide-accelerated diabetes in female NOD mice. Four-week-old animals received intramuscular injections of plasmid DNA encoding either intracellular GAD, a secreted form of GAD, or a secreted form of a soft coral luciferase. Monitoring of glycosuria and hyperglycemia indicated that injection of plasmid DNA encoding secreted GAD and secreted luciferase could prevent and delay diabetes, respectively. In contrast, injection of DNA encoding intracellular GAD did not suppress the disease significantly. Analysis of anti-GAD IgG(1) antibody titers in animal sera indicated that diabetes prevention after injection of GAD-encoding DNA was possibly associated with increased Th2-type activity. These results suggest that cellular localization of GAD is a factor to consider in the design of GAD-based genetic vaccines for the prevention of autoimmune diabetes.