Glutamic Acid Decarboxylase GAD65 Research Articles

Autoantibodies to N-terminally Truncated GAD65(96-585): HLA Associations and Predictive Value for Type 1 Diabetes.

ObjectiveTo evaluate the role of autoantibodies to N-terminally truncated glutamic acid decarboxylase GAD65(96-585) (t-GADA) as a marker for type 1 diabetes (T1D) and to assess the potential human leukocyte antigen (HLA) associations with such autoantibodies.DesignIn this cross-sectional study combining data from the Finnish Pediatric Diabetes Register, the Type 1 Diabetes Prediction and Prevention study, the DIABIMMUNE study, and the Early Dietary Intervention and Later Signs of Beta-Cell Autoimmunity study, venous blood samples from 760 individuals (53.7% males) were analyzed for t-GADA, autoantibodies to full-length GAD65 (f-GADA), and islet cell antibodies. Epitope-specific GAD autoantibodies were analyzed from 189 study participants.ResultsT1D had been diagnosed in 174 (23%) participants. Altogether 631 (83%) individuals tested positive for f-GADA and 451 (59%) for t-GADA at a median age of 9.0 (range 0.2-61.5) years. t-GADA demonstrated higher specificity (46%) and positive predictive value (30%) for T1D than positivity for f-GADA alone (15% and 21%, respectively). Among participants positive for f-GADA, those who tested positive for t-GADA carried more frequently HLA genotypes conferring increased risk for T1D than those who tested negative for t-GADA (77% vs 53%; P < 0.001).ConclusionsAutoantibodies to N-terminally truncated GAD improve the screening for T1D compared to f-GADA and may facilitate the selection of participants for clinical trials. HLA class II-mediated antigen presentation of GAD(96–585)-derived or structurally similar peptides might comprise an important pathomechanism in T1D.

BDNF eksikliği ve endoplazmik retikulum stresinin GABAerjik sistem üzerindeki etkileri

Purpose: Protective pathways against endoplasmic reticulum stress in neurons are activated via brain-derived neurotrophic factor. However, it is not known how the inhibitory intermediate neuron types expressing the different Ca2+-binding proteins of GABAergic system will be affected with changes in Ca2+ homeostasis, in conditions of chronic reduction of brain-derived neurotrophic factor and endoplasmic reticulum stress. The study was planned to reveal the interaction of these factors. Materials and methods: 6-8 months old (30-40 g), wild-type (WT) and brain-derived neurotrophic factor heterozygous (BDNF(+/-)) male mice were used and 4 groups were formed. Groups 3 and 4 were treated with a single dose of tunicamycin to induce endoplasmic reticulum stress. On the 3rd day of tunicamycin injection, animals were sacrificed and blood and brain tissues were taken. In serum samples BDNF, in tissue homogenates GRP78, CHOP, Caspase-12, parvalbumin, calretinin, calbindin, GAD65 and GAD67 levels were investigated by ELISA method. One-way ANOVA and Tukey post-hoc tests were used for statistical evaluation. Results: Serum BDNF levels were significantly lower in BDNF(+/-) and tunicamycin-treated BDNF(+/-) groups. Caspase-12 and CHOP levels significantly increased with tunicamycin injection. Calbindin level decreased significantly with endoplasmic reticulum stress. GAD65 and GAD67 levels were similar in WT and BDNF(+/-) groups. However, GAD65 level was significantly decreased during endoplasmic reticulum stress in WT and BDNF(+/-) groups. Conclusion: Endoplasmic reticulum stress caused a significant decrease in glutamic acid decarboxylase GAD65 isoform and caldindin levels. This result indicates that the sensitivity of varied intermediate neurons in GABAergic system to endoplasmic reticulum stress may be different.

Semicarbazide-induced thyroid disruption in Japanese flounder (Paralichthys olivaceus) and its potential mechanisms

Semicarbazide (SMC) is a carcinogenic and genotoxic substance that has been found in aquatic systems. SMC may also cause thyroid follicular epithelial cell injury in rats, but the thyroid-disrupting properties of SMC and its potential mechanisms remain unclear. In this study, we exposed fertilized eggs of Japanese flounder (Paralichthys olivaceus) to 1, 10, 100, and 1000μg/L SMC for 55 d to assess the impact of SMC exposure on the thyroid system. The number of larvae in each metamorphic stage was counted, the concentrations of whole-body thyroid hormones (THs) 3,5,3′-triiodothyronine (T3) and thyroxine (T4) were measured, and the transcription levels of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis and gamma-aminobutyric acid (GABA) synthesis were quantified. The results showed that 10μg/L SMC significantly increased whole-body T3 levels, and 100 and 1000μg/L SMC markedly enhanced whole-body T4 and T3 levels. Furthermore, 100μg/L SMC exposure shortened the time it took for flounder larvae to complete metamorphosis by 2 d as compared to the control group. Thus, this study demonstrated that SMC exerted thyroid-disrupting effects on Japanese flounder. SMC-mediated stimulation of TH levels was primarily related to transcriptional alterations of pituitary-derived thyroid stimulating hormone β-subunit (tshβ) and hepatic deiodinase (id). In the 10 and 100μg/L SMC exposure groups, the increased TH levels may have resulted from inhibition of TH metabolism caused by down-regulation of id3 mRNA expression, while at 1000μg/L SMC-exposed group, up-regulation of tshβ and id1 transcripts was expected to enhance the synthesis of T4 and the conversion of T4 to T3 and, consequently, result in higher T4 and T3 levels. In addition, 1000μg/L SMC-induced down-regulation in glutamic acid decarboxylase gad65 and gad67 transcription may have also contributed to the increased TH levels. The thyroid-disrupting effects of 10 and 100μg/L SMC indicated that environmentally relevant concentrations of SMC posed potential environmental risks to aquatic organisms. Overall, our results demonstrated for the first time that SMC exhibited thyroid-disrupting properties by affecting the HPT axis and GABA synthesis, providing theoretical support for environmental risk assessment.

GAD‐treatment of children and adolescents with recent‐onset type 1 diabetes preserves residual insulin secretion after 30 months

This study aimed to analyse data from two different studies (phase II and phase III) regarding the safety and efficacy of treatment with alum formulated glutamic acid decarboxylase GAD65 (GAD-alum) at 30 months after administration to children and adolescents with type 1 diabetes. The phase II trial was a double-blind, randomised placebo-controlled study, including 70 children and adolescents who were followed for 30 months. Participants received a subcutaneous injection of either 20 µg of GAD-alum or placebo at baseline and 1 month later. During a subsequent larger European phase III trial including three treatment arms, participants received two or four subcutaneous injections of either 20 µg of GAD-alum and/or placebo at baseline, 1, 3 and 9 months. The phase III trial was prematurely interrupted at 15 months, but of the 148 Swedish patients, a majority completed the 21 months follow-up, and 45 patients completed the trial at 30 months. Both studies included GAD65 auto-antibodies-positive patients with fasting C-peptide ≥ 0.10 nmol/l. We have now combined the results of these two trials. There were no treatment related adverse events. In patients treated with 2 GAD-alum doses, stimulated C-peptide area under the curve had decreased significantly less (9 m: p < 0.037; 15 m: p < 0.032; 21 m: p < 0.003 and 30 m: p < 0.004), and a larger proportion of these patients were also able to achieve a peak stimulated C-peptide > 0.2 nmol/L (p < 0.05), as compared with placebo. Treatment with two doses of GAD-alum in children and adolescents with recent-onset type 1 diabetes shows no adverse events and preserves residual insulin secretion.

Altered glutamate/GABA equilibrium in aged mice cortex influences cortical plasticity

Age-related molecular changes in the synapse can cause plasticity decline. We found an impairment of experience-dependent cortical plasticity is induced by short lasting sensory conditioning in aged mice. However, extending the training procedure from 3 to 7 days triggered plasticity in the aged cortex of the same range as in young mice. Additionally, GABAergic markers (GABA, GAD67, VGAT) in young and aged groups that showed the plastic changes were upregulated. This effect was absent in the aged group with impaired plasticity, while the expression of Vglut1 increased in all trained groups. This may reflect the inefficiency of inhibitory mechanisms in the aging brain used to control increased excitation after training and to shape proper signal to noise ratio, which is essential for appropriate stimuli processing. HPLC analysis showed that the glutamate/GABA ratio was significantly reduced in aged animals due to a significant decrease in glutamate level. We also observed a decreased expression of several presynaptic markers involved in excitatory (vesicular glutamate transporter-vglut2) and inhibitory (glutamic acid decarboxylase-GAD67, vesicular GABA transporter VGAT) transmission in the aged barrel cortex. These changes may weaken the plasticity potential of neurons and impede the experience-dependent reorganization of cortical connections. We suggest that the imbalance toward inhibition resulting from a decrease of glutamate content in the aging cerebral cortex, together with GABAergic system ineffectiveness in upregulating GABA level after sensory training, contributes to the impairment of learning-dependent cortical plasticity.

The GABA-synthetic enzyme GAD65 controls circadian activation of conditioned fear pathways

Circadian fluctuations of fear and anxiety symptoms are observable in persons with post-traumatic stress disorder, generalized anxiety, and panic disorder; however, the underlying neurobiological mechanisms are not sufficiently understood. In the present study, we investigated the putative role of inhibitory neurotransmission in the circadian fluctuation of fear symptoms, using mice with genetic ablation of the γ-amino butyric acid (GABA) synthesizing isoenzyme, glutamic acid decarboxylase GAD65. We observed in these mutant mice an altered expression of conditioned fear with a profound reduction of freezing, and an increase of hyperactivity bouts occurring only when both fear conditioning training and retrieval testing were done at the beginning of their active phase. Mutants further showed an increased arousal response at this time of the day, although, circadian rhythm of home cage activity was unaltered. Hyperactivity and reduced freezing during fear memory retrieval were accompanied by an increased induction of the immediate early gene cFos suggesting hyperactivation of the hippocampus, amygdala, and medial hypothalamus. Our data suggest a role of GAD65-mediated GABA synthesis in the encoding of circadian information to fear memory. GAD65 deficits in a state-dependent manner result in increased neural activation in fear circuits and elicit panic-like flight responses during fear memory retrieval.

IS 9. Modulation of cortical inhibition by rTMS-a rat model

Introduction Human cortical excitability can be modified by repetitive transcranial magnetic stimulation (rTMS) but the factors that determine the direction of change remain still elusive. Classical forms of synaptic plasticity and metaplastic processes like homeostatic synaptic scaling and priming-dependent synaptic plasticity are possible mechanistic scenarios. Although the activity of excitatory neurons finally represents the degree of cortical activation, a multitude of inhibitory cortical systems governs about magnitude and pattern of cortical activity and the degree of plasticity allowed without loosing essential excitation-inhibition balance. It is thus likely that rTMS-induced changes in cortical excitability are associated with changes in cortical inhibition and that classes of interneurons differently contribute. Objectives We established a rat model of rTMS enabling to study cellular effects of rTMS in more detail and with possible relation to behavioural changes. To focus on changes in inhibitory cortical activity, we studied the expression of certain activity markers which are specific to inhibitory neurons in general, like the two isoforms of the glutamic acid decarboxylase GAD65 and GAD67, or relate to a certain class of interneurons, like the expression of the calcium-binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR). Our recent studies focus on the temporal dynamics of these activity markers which is of particular interest in case of combining rTMS with other artificial or natural interventions or repeated rTMS. Materials and methods Adult male Sprague–Dawley rats ( n = 42.15 weeks old) were trained to tolerate the procedure of manual restrain and rTMS without signs of stress before receiving a single block (600 pulses) of either verum or sham intermittent theta-burst stimulation protocol (iTBS) at an intensity just subthreshold for evoking motor responses (MagStim rapid, 70 mm figure-of-eight coil). Then, rat were deeply anesthetized and perfused at different intervals post-rTMS (10, 20, 40, 80, 160 min) for immunohistochemical analysis of neuronal activity marker expression (GAD65/67, PV, CB for inhibitory neurons; c-Fos and zif268 mainly for excitatory neurons). Results Three phases of changes in neuronal activity marker expression could be distinguished: an early phase (10–20 min) of strongly increased c-Fos and GAD65 expression, a following phase (40–80 min) of reduced GAD67, PV and CB expression and a late phase (>160 min) of reduced c-Fos and GAD65 expression. Conclusion Early increased levels of c-Fos and GAD65 indicate a strong co-activation of excitatory and inhibitory neurons due to iTBS. The delayed decrease in GAD67, PV and CB expression indicates a secondary depression of activity of inhibitory neurons without signs of increased excitatory activity (c-Fos, zif268). The late decrease in c-Fos and GAD65 finally indicates a matched reduction in the activity of both excitatory and inhibitory activities. Obviously, excitation-inhibition balance is never violated but the phases of changed synaptic (GAD65) and somatic (GAD67, PV, CB) inhibitory neuron activity to not co-vary in time. This study has been supported by the Deutsche Forschungsgemeinschaft, DFG (FU 256/3-2, SFB 874, TP A4).

Novel object recognition ability in female mice following exposure to nanoparticle-rich diesel exhaust

Recently, our laboratory reported that exposure to nanoparticle-rich diesel exhaust (NRDE) for 3months impaired hippocampus-dependent spatial learning ability and up-regulated the expressions of memory function-related genes in the hippocampus of female mice. However, whether NRDE affects the hippocampus-dependent non-spatial learning ability and the mechanism of NRDE-induced neurotoxicity was unknown. Female BALB/c mice were exposed to clean air, middle-dose NRDE (M-NRDE, 47μg/m3), high-dose NRDE (H-NRDE, 129μg/m3), or filtered H-NRDE (F-DE) for 3months. We then investigated the effect of NRDE exposure on non-spatial learning ability and the expression of genes related to glutamate neurotransmission using a novel object recognition test and a real-time RT-PCR analysis, respectively. We also examined microglia marker Iba1 immunoreactivity in the hippocampus using immunohistochemical analyses. Mice exposed to H-NRDE or F-DE could not discriminate between familiar and novel objects. The control and M-NRDE-exposed groups showed a significantly increased discrimination index, compared to the H-NRDE-exposed group. Although no significant changes in the expression levels of the NMDA receptor subunits were observed, the expression of glutamate transporter EAAT4 was decreased and that of glutamic acid decarboxylase GAD65 was increased in the hippocampus of H-NRDE-exposed mice, compared with the expression levels in control mice. We also found that microglia activation was prominent in the hippocampal area of the H-NRDE-exposed mice, compared with the other groups. These results indicated that exposure to NRDE for 3months impaired the novel object recognition ability. The present study suggests that genes related to glutamate metabolism may be involved in the NRDE-induced neurotoxicity observed in the present mouse model.

Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial

The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4years of follow-up. Seventy children and adolescents aged 10-18years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20μg of GAD-alum or placebo at baseline and 1month later. The study was blinded to participants and investigators until month30. The study was unblinded at 15months to the sponsor and statistician in order to evaluate the data. At follow-up after 30months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with <6months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 ± 0.032nmol/l at day1 and 0.215 ± 0.031nmol/l at month15. The corresponding figures for the placebo group were 0.354 ± 0.039 and 0.184 ± 0.033nmol/l, respectively. The decline in fasting C-peptide levels between day1 and month1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4years. Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. ClinicalTrials.gov NCT00435981 The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB.

Blocking L-type Voltage-gated Ca2+ Channels with Dihydropyridines Reduces γ-Aminobutyric Acid Type A Receptor Expression and Synaptic Inhibition

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the major sites of fast inhibitory neurotransmission in the brain, and the numbers of these receptors at the cell surface can determine the strength of GABAergic neurotransmission. Chronic changes in neuronal activity lead to an adaptive modulation in the efficacy of GABAergic synaptic inhibition, brought about in part by changes in the number of synaptic GABA(A)Rs, a mechanism known as homeostatic synaptic plasticity. Reduction in the number of GABA(A)Rs in response to prolonged neuronal activity blockade is dependent on the ubiquitin-proteasome system. The underlying biochemical pathways linking chronic activity blockade to proteasome-dependent degradation of GABA(A)Rs are unknown. Here, we show that chronic blockade of L-type voltage-gated calcium channels (VGCCs) with nifedipine decreases the number of GABA(A)Rs at synaptic sites but not the overall number of inhibitory synapses. In parallel, blockade of L-type VGCCs decreases the amplitude but not the frequency of miniature inhibitory postsynaptic currents or expression of the glutamic acid decarboxylase GAD65. We further reveal that the activation of L-type VGCCs regulates the turnover of newly translated GABA(A)R subunits in a mechanism dependent upon the activity of the proteasome and thus regulates GABA(A)R insertion into the plasma membrane. Together, these observations suggest that activation of L-type VGCCs can regulate the abundance of synaptic GABA(A)Rs and the efficacy of synaptic inhibition, revealing a potential mechanism underlying the homeostatic adaptation of fast GABAergic inhibition to prolonged changes in activity.

GABAC receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex

GABA(C) receptors are enriched in the upper grey layers of the mammalian superior colliculus and contribute to synaptic processing. Electrophysiological data suggested that the GABA(C) receptor ρ subunits are expressed by GABAergic interneurons which represent about half of the neurons in the stratum griseum superficiale (SGS). Combining in situ hybridization for ρ2 receptor mRNA and the glutamic acid decarboxylase GAD-65 mRNA confirmed this assumption. A majority of ρ-labeled neurons in SGS and pretectum are GABAergic. Combining in situ hybridization with immunohistochemistry for the two projection neuron markers calbindin and parvalbumin revealed that a few ρ2 mRNA expressing cells coexpressed calbindin, but not parvalbumin. In visual cortex, ρ2 mRNA was present in pyramidal neurons and parvalbumin-containing interneurons. The results show that in the SGS primarily GABAergic neurons express GABA(C) receptors whereas the majority of tectothalamic calbindin neurons and intrinsically projecting parvalbumin neurons do not.

Determination of Glutamic Acid Decarboxylase (GAD65) in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.