Profile of miR-23 Expression and Possible Role in Regulation of Glutamic Acid Decarboxylase during Postnatal Retinal Development.

As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development.

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription.

Interaction of cannabinoid receptor type 1 (CB1) and GABAergic neuronal activity is involved in drug abuse-related behavior. However, its role in drug-dependent Pavlovian conditioning is not well understood. In this study, we aimed to evaluate the effects of a CB1 agonist, JWH-210, on the development of conditioned place preference (CPP)-induced by methamphetamine (METH). Pretreatment with a synthetic cannabinoid, JWH-210 (CB1 agonist), increased METH-induced CPP score and METH-induced dopamine release in acute striatal slices. Interestingly, CB1 was expressed in glutamate decarboxylase 67 (GAD67) positive cells, and overexpression of CB1 increased GAD67 expression, while CB1 knockdown reduced GAD67 expression in vivo and in vitro. GAD67 is known as an enzyme involved in the synthesis of GABA. CB1 knockdown in the mice striatum increased METH-induced CPP. When GAD67 decreased in the mice striatum, mRNA level of CB1 did not change, suggesting that CB1 can regulate GAD67 expression. GAD67 knockdown in the mouse striatum augmented apomorphine (dopamine receptor D2 agonist)-induced climbing behavior and METH-induced CPP score. Moreover, in the human brain, mRNA level of GAD67 was found to be decreased in drug users. Therefore, we suggest that CB1 potentiates METH-induced CPP through inhibitory GABAergic regulation of dopaminergic neuronal activity.

Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.

Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

BACKGROUND: Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. RESULTS: When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. CONCLUSIONS: This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

Molecular engineering of L-aspartate-α-decarboxylase for improved activity and catalytic stability.

ß-Alanine is an important precursor for the production of food additives, pharmaceuticals, and nitrogen-containing chemicals. Compared with the conventional chemical routes for ß-alanine production, the biocatalytic routes using L-aspartate-α-decarboxylase (ADC) are more attractive when energy and environment are concerned. However, ADC's poorly understood properties and its inherent mechanism-based inactivation significantly limited the application of this enzyme. In this study, three genes encoding the ADC enzymes from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis were overexpressed in E. coli. Their properties including specific activity, thermostability, and mechanism-based inactivation were characterized. The ADC enzyme from B. subtilis, which had higher specific activity and thermostability than the others, was selected for further study. In order to improve its activity and relieve its mechanism-based inactivation by molecular engineering so as to improve its catalytic stability, a high-throughput fluorometric assay of ß-alanine was developed. From a library of 4000 mutated enzymes, two variants with 18-22% higher specific activity and 29-64% higher catalytic stability were obtained. The best variant showed 50% higher ß-alanine production than the wild type after 8 h of conversion of L-aspartate, showing great potential for industrial biocatalytic production of ß-alanine.

HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a.

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.

Epigenetic Suppression of GADs Expression is Involved in Temporal Lobe Epilepsy and Pilocarpine-Induced Mice Epilepsy.

Recent studies have shown that histone acetylation is involved with the regulation of enzyme glutamate decarboxylases (GADs), including GAD67 and GAD65. Here, we investigated the histone acetylation modifications of GADs in the pathogenesis of epilepsy and explored the therapeutic effect of a novel second-generation histone deacetylase inhibitor (HDACi) JNJ-26481585 in epilepsy animals. We revealed the suppression of GADs protein and mRNA level, and histone hypoacetylation in patients with temporal lobe epilepsy and pilocarpine-induced epilepsy mice model. Double-immunofluorescence also indicated that the hypoacetyl-H3 was located in hippocampal GAD67/GAD65 positive neurons in epilepsy mice. JNJ-26481585 significantly reversed the decrease of the GAD67/GAD65 both protein and mRNA levels, and the histone hypoacetylation of GABAergic neurons in epilepsy mice. Meanwhile, single-cell real-time PCR performed in GFP-GAD67/GAD65 transgenic mice demonstrated that JNJ-26481585 induced increase of GAD67/GAD65 mRNA level in GABAergic neurons. Furthermore, JNJ-26481585 significantly alleviated the epileptic seizures in mice model. Together, our findings demonstrate inhibition of GADs gene via histone acetylation plays an important role in the pathgenesis of epilepsy, and suggest JNJ-26481585 as a promising therapeutic strategy for epilepsy.

Garcinol Upregulates GABAA and GAD65 Expression, Modulates BDNF-TrkB Pathway to Reduce Seizures in Pentylenetetrazole (PTZ)-Induced Epilepsy.

BACKGROUND Epilepsy is the most predominant neurological disorder characterized by recurrent seizures. Despite treatment with antiepileptic drugs, epilepsy still is a challenge to treat, due to the associated adverse effects of the drugs. Previous investigations have shown critical roles of BDNF-TrkB signalling and expression of glutamic acid decarboxylase 65 (GAD65) and GABAA in the brain during epilepsy. Thus, drugs that could modulate BDNF-TrkB signal and expression of GAD65 and GABAA could aid in therapy. Recent experimental data have focussed on plant-derived compounds in treatments. Garcinol (camboginol), is a polyisoprenylated benzophenone derived from the fruit of Garcinia indica. We investigated the effects of garcinol in pentylenetetrazole (PTZ)-induced epileptic models. MATERIAL AND METHODS Seizure scores were measured in epilepsy kindled mice. Neuronal degeneration and apoptosis were assessed by Nissl staining, TUNEL assay, and Fluoro-Jade B staining. Immunohistochemistry was performed to evaluate cleaved caspase-3 expressions. Expression of BDNF, TrkB, GABAA, GAD65, Bad, Bcl-2, Bcl-xL, and Bax were determined by western blots. RESULTS Significantly reduced seizure scores and mortality rates were observed with pretreatment with garcinol. Elevated expression of apoptotic proteins and caspase-3 in kindled mice were effectively downregulated by garcinol. Epileptogenic mice presented increased BDNF and TrkB with considerably decreased GABAA and GAD65 expression. Garcinol significantly enhanced GABAA and GAD65 while it suppressed BDNF and TrkB. Garcinol enhanced the performance of mice in Morris water maze tests. CONCLUSIONS Garcinol exerts neuroprotective effects via supressing apoptosis and modulating BDNF-TrkB signalling and GAD65/GABAA expressions and also enhanced cognition and memory of the mice.

Up-Regulation of GABAergic Signal Events in Bone Marrow Lymphocytes in Childhood Acute Lymphoblastic Leukemia.

Gamma-aminobutyric acid (GABA) is involved in the proliferation, differentiation, and migration of several cell types including cancer cells. Whether GABA may be involved with acute lymphoblastic leukemia (ALL) is unclear. Therefore, the goal of this report was to examine if GABAergic signaling expression is altered in bone marrow lymphocytes of ALL children. RT-PCR and western blot analysis were used to examine the expression of the GABA synthetizing enzyme glutamic acid decarboxylase (GAD) isoforms (GAD65 and GAD67), and type-A GABA receptor (GABAAR) subunits [α(1-6), ß(1-3), γ(1-3), δ, ε, θ, π, and ρ(1-3)] in bone marrow lymphocytes of 19 ALL children before chemotherapy. The data obtained were compared with those in 13 age-matched non-ALL children. Immunofluorescent staining was used to examine the cellular localization of GAD. We found that GAD and GABAAR subunits were expressed in bone marrow lymphocytes of ALL children. Moreover, RT-PCR and western blot showed that GAD and several GABAAR subunits were significantly increased in ALL children as compared with the data of non-ALL children. Our present study reveals up-regulation of GABAergic signaling events in bone marrow lymphocytes in ALL children. However, the role of this signaling system in lymphocyte proliferation and invasion as related to the progression of ALL requires further investigation.

Injection of Toll-like receptor 4 siRNA into the ventrolateral periaqueductal gray attenuates withdrawal syndrome in morphine-dependent rats.

We assessed the role of the Toll-like receptor 4 (TLR4) gene in the ventrolateral periaqueductal gray (vlPAG) region of morphine-dependent rats on attenuating withdrawal syndrome, and regulating glutamic acid decarboxylase (GAD67), glutamic acid (Glu), and gamma-aminobutyric acid (GABA). After siRNA-mediated downregulation of TLR4, changes were observed in withdrawal behavior and downstream signaling molecules. Rats were injected into the vlPAG with TLR4 siRNA, followed by intraperitoneal injection of morphine for 5 consecutive days, and then naloxone, and the behavioral indices of morphine withdrawal were observed. 'Wet-dog' shakes, teeth chattering, and the total scores of withdrawal reactions were reduced. TLR4 expression and Glu levels were reduced, whereas GAD67 and GABA levels were increased. Overall, these findings indicate that modifying TLR4 gene expression in the vlPAG stimulates expression of the downstream signaling molecule, GAD67, which decreases Glu levels and increases GABA levels. This mechanism may explain the inhibition of withdrawal syndrome in morphine-dependent rats.

Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

BACKGROUND: Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. METHODS: The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPß was tested using immunohistochemistry. RESULTS: In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the control vectors (P = 0.0005). Intrathecal GABA-A/B agonists elevated mechanical threshold in the pain model. The HSV vectors expressing GAD67 reversed the lowered GABA immunoreactivity in the spinal dorsal horn in the neuropathic rats. HSV vectors expressing GAD67 in the neuropathic rats reversed the increased signals of mitochondrial superoxide in the spinal dorsal horn. The vectors expressing GAD67 reversed the upregulated immunoreactivity expression of pCREB and pC/EBPß in the spinal dorsal horn in rats exhibiting NP. CONCLUSIONS: Based on our results, we suggest that GAD67 mediated by HSV vectors acting through the suppression of mitochondrial reactive oxygen species and transcriptional factors in the spinal cord decreases pain in the HIV-related neuropathic pain model, providing preclinical evidence for gene therapy applications in patients with HIV-related pain states.

Impaired selenoprotein expression in brain triggers striatal neuronal loss leading to co-ordination defects in mice.

Secisbp2 [SECIS (selenocysteine insertion sequence)-binding protein 2] binds to SECIS elements located in the 3'-UTR region of eukaryotic selenoprotein mRNAs. It facilitates the incorporation of the rare amino acid selenocysteine in response to UGA codons. Inactivation of Secisbp2 in hepatocytes greatly reduced selenoprotein levels. Neuron-specific inactivation of Secisbp2 (CamK-Cre; Secisbp2fl/fl) reduced cerebral expression of selenoproteins to a lesser extent than inactivation of tRNA[Ser]Sec. This allowed us to study the development of cortical PV (parvalbumin)+ interneurons, which are completely lost in tRNA[Ser]Sec mutants. PV+ interneuron density was reduced in the somatosensory cortex, hippocampus and striatum. In situ hybridization for Gad67 (glutamic acid decarboxylase 67) confirmed the reduction of GABAergic (where GABA is γ-aminobutyric acid) interneurons. Because of the obvious movement phenotype involving a broad dystonic gait, we suspected basal ganglia dysfunction. Tyrosine hydroxylase expression was normal in substantia nigra neurons and their striatal terminals. However the densities of striatal PV+ and Gad67+ neurons were decreased by 65% and 49% respectively. Likewise, the density of striatal cholinergic neurons was reduced by 68%. Our observations demonstrate that several classes of striatal interneurons depend on selenoprotein expression. These findings may offer an explanation for the movement phenotype of selenoprotein P-deficient mice and the movement disorder and mental retardation described in a patient carrying SECISBP2 mutations.

Prenatal ethanol exposure has sex-specific effects on hippocampal long-term potentiation.

Alcohol consumption during pregnancy is deleterious to the developing brain of the fetus and leads to persistent deficits in adulthood. Long-term potentiation (LTP) is a biological model for learning and memory processes and previous evidence has shown that prenatal ethanol exposure (PNEE) affects LTP in a sex specific manner during adolescence. The objective of this study was to determine if there are sex specific differences in adult animals and to elucidate the underlying molecular mechanisms that contribute to these differences. Pregnant Sprague-Dawley dams were assigned to either; liquid ethanol, pair-fed or standard chow diet. In vivo electrophysiology was performed in the hippocampal dentate gyrus (DG) of adult offspring. LTP was induced by administering 400 Hz stimuli. Western blot analysis for glutamine synthetase (GS) and glutamate decarboxylase from tissue of the DG indicated that GS expression was increased following PNEE. Surprisingly, adult females did not show any deficit in N-methyl-D-aspartate (NMDA)-dependent LTP after PNEE. In contrast, males showed a 40% reduction in LTP. It was indicated that glutamine synthetase expression was increased in PNEE females, suggesting that altered excitatory neurotransmitter replenishment may serve as a compensatory mechanism. Ovariectomizing females did not influence LTP in control or PNEE animals, suggesting that circulating estradiol levels do not play a major role in maintaining LTP levels in PNEE females. These results demonstrate the sexually dimorphic effects of PNEE on the ability for the adult brain to elicit LTP in the DG. The mechanisms for these effects are not fully understood, but an increase in glutamine synthetase in females may underlie this phenomenon.

Comparative analysis of different biofactories for the production of a major diabetes autoantigen.

The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 µg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 µg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.

Disruption of hippocampal neuregulin 1-ErbB4 signaling contributes to the hippocampus-dependent cognitive impairment induced by isoflurane in aged mice.

BACKGROUND: A prolonged isoflurane exposure may lead to cognitive decline in rodents. Neuregulin 1 (NRG1)-ErbB4 signaling plays a key role in the modulation of hippocampal synaptic plasticity through regulating the neurotransmission. The authors hypothesized that hippocampal NRG1-ErbB4 signaling is involved in isoflurane-induced cognitive impairments in aged mice. METHODS: Fourteen-month-old C57BL/6 mice were randomized to receive 100% O2 exposure, vehicle injection after 100% O2 exposure, vehicle injection after exposure to isoflurane carried by 100% O2, NRG1-ß1 injection after exposure to isoflurane carried by 100% O2, and NRG1-ß1 and an ErbB4 inhibitor AG1478 injection after exposure to isoflurane carried by 100% O2. Fear conditioning test was used to assess the cognitive function of mice 48-h postexposure. The brain tissues were harvested 48-h postexposure to determine the levels of NRG1, ErbB4, p-ErbB4, parvalbumin, and glutamic acid decarboxylase 67 in the hippocampus using Western blotting, enzyme-linked immunosorbent assay, and immunofluorescence. RESULTS: The percentage of freezing time to context was decreased from 50.28 ± 11.53% to 30.82 ± 10.00%, and the hippocampal levels of NRG1, p-ErbB4/ErbB4, parvalbumin, and glutamic acid decarboxylase 67 were decreased from 172.79 ± 20.85 ng/g, 69.15 ± 12.20%, 101.68 ± 11.21%, and 104.71 ± 6.85% to 112.92 ± 16.65 ng/g, 42.26 ± 9.71%, 75.89 ± 10.26%, and 73.87 ± 16.89%, respectively, after isoflurane exposure. NRG1-ß1 attenuated the isoflurane-induced hippocampus-dependent cognitive impairment and the declines in the hippocampal NRG1, p-ErbB4/ErbB4, parvalbumin, and glutamic acid decarboxylase 67. AG1478 inhibited the rescuing effects of NRG1-ß1. CONCLUSION: Disruption of NRG1-ErbB4 signaling in the parvalbumin-positive interneurons might, at least partially, contribute to the isoflurane-induced hippocampus-dependent cognitive impairment after exposure to isoflurane carried by 100% O2 in aged mice.

Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

Differential regulation of glutamic acid decarboxylase gene expression after extinction of a recent memory vs. intermediate memory.

Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65 isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD). However, a detailed analysis of changes in gene expression of GAD in the subregions comprising the extinction network has not been undertaken. Here, we report changes in gene expression of the GAD65 and GAD67 isoforms of GAD, as measured by relative quantitative real-time RT-PCR, in subregions of the amygdala, hippocampus, and prefrontal cortex 24-26 h after extinction of a recent (1-d) or intermediate (14-d) fear memory. Our results show that extinction of a recent memory induces a down-regulation of Gad65 gene expression in the hippocampus (CA1, dentate gyrus) and an up-regulation of Gad67 gene expression in the infralimbic cortex. Extinguishing an intermediate memory increased Gad65 gene expression in the central amygdala. These results indicate a differential regulation of Gad gene expression after extinction of a recent memory vs. intermediate memory.

Number and regional distribution of GAD65 mRNA-expressing interneurons in the rat hippocampal formation.

In rodent models for neuropsychiatric disorders reduced number of hippocampal interneurons have been reported, but the total number of GABAergic neurons in the normal rat hippocampus is yet unknown. We used in situ hybridization method to label the 65 isoform of glutamic acid decarboxylase (GAD65) and counted the number of GAD65 mRNA-expressing neurons along the entire septo-temporal axis of the hippocampus. We found that 2/3 of the interneurons were in Ammon's horn (61,590) and 1/3 in the dentate gyrus (28,000). We observed the following numbers in Ammon's horn: CA3 area 33,400, CA2 area 4,190, CA1 area 24,000 and in the dentate gyrus: 6,000 in the molecular and 9,000 in the granule cell layers and 13,000 in the hilus. GAD65 mRNA-expressing neurons were significantly more numerous in dorsal than in ventral hippocampus. The ratio between interneurons and principal cells was lowest in the granule cell layer (0.9%) and highest in hilus (21%). In Ammon's horn this ratio was constant being 13% in CA3 and 8% in CA1-2 areas. In the entire hippocampal formation, the interneuron/principal cell ratio was 6%, with a significant difference between Ammon's horn (9.5%) and the dentate gyrus (2.8%) including the hilus. Such low ratios could suggest that even a limited loss of GABAergic neurons in the hippocampus may have a considerable functional impact.

Participation of calbindin-D28K in nociception: results from calbindin-D28K knockout mice.

Since calbindin-D(28K) (CB-D(28K))-positive neurons have been related to nociceptive sensory processing, we have hypothesized that altered CB-D(28K) expression could alter nociceptive transmission. We have used +/+ and -/- knockout (KO) mice for CB-D(28k) in different behavioral models of pain and sensory responses at the caudalis subdivision of the trigeminal spinal nucleus in order to understand how this protein may participate in nociception. Behavioral responses to formalin injection in the hind paw or at the whisker pad or in the hind paw glutamate or i.p. acetic acid tests showed an increase of the pain threshold in CB-D(28k) -/- mice. KO mice showed a diminution of the inhibitory activity at Sp5C nucleus and a marked reduction of GABA content. Sp5C neurons from CB-D(28k) -/- mice did not change their spontaneous activity or tactile response after formalin injection in the whisker pad. In contrast, Sp5C neurons increased their spontaneous firing rate and tactile response after formalin injection in their receptive field in CB-D(28k) +/+ mice. The results of this study demonstrate the active role played by CB-D(28k) in nociceptive sensory transmission. The lack of this calcium binding protein, associated to deficient GABAergic neurotransmission, translates into dysfunction of sensory processing of nociceptive stimuli.

Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection neurons.

The GABA-synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium-sized projection neurons with high-level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5'-noncoding, 3'-noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia-thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium-sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.