Regulation of γ-Aminobutyric Acid Synthesis in the Brain

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

A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD₆₇ mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD₆₇ in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD⁺, Fos-ir/MCH⁺, and GAD⁺/MCH⁺ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD⁺ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca²⁺ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP₃R) serve to discharge Ca²⁺ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP₃R isoforms and protect cells from apoptosis, reducing ER Ca²⁺ release. However, it has not been elucidated which IP₃R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP₃R I, strongly suppresses IP₃-mediated Ca²⁺ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca²⁺ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca²⁺ release. Moreover, regulating Ca²⁺ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP₃R III, leading to diminished Ca²⁺ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt.     

Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis

Background

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.

Principal Findings

Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K⁺-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca²⁺ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K⁺ (K_ATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.

Conclusions/Significance

We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K_ATP channel responsiveness and the subsequent activity of Ca²⁺ channels through pathways other than CDK5-mediated regulation.     

Long-lasting immune responses 4 years after GAD-alum treatment in children with type 1 diabetes

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD₆₅. Frequencies of naïve, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD₆₅ autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD₆₅, but not with control antigens, compared with placebo subjects. GAD₆₅-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD₆₅ enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD₆₅-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD₆₅ immunity.     

Demand for Zn2+ in acid-secreting gastric mucosa and its requirement for intracellular Ca2+

Background and Aims

Recent work has suggested that Zn²⁺ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn²⁺ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.

Methods and Findings

Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn²⁺ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, ⁷⁰Zn²⁺, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of ⁷⁰Zn²⁺ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn²⁺ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn²⁺ {[Zn²⁺]_i} during exposure to standard extracellular concentrations of Zn²⁺ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn²⁺ increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca²⁺ {[Ca²⁺]_i}. Uptake of Zn²⁺ was abolished following removal of extracellular Ca²⁺ or depletion of intracellular Ca²⁺ stores, suggesting that demand for extracellular Zn²⁺ increases and depends on influx of extracellular Ca²⁺.

Conclusions

This study is the first to characterize the content and distribution of Zn²⁺ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca²⁺ integrates basolateral demand for Zn²⁺ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.     

Seasonal variation in vitamin D₃ levels is paralleled by changes in the peripheral blood human T cell compartment

It is well-recognized that vitamin D₃ has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D₃ status. Here, we investigated if and to what extent seasonality of vitamin D₃ levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D₃ and 1,25(OH)₂D₃ levels in summer were associated with a higher number of peripheral CD4⁺ and CD8⁺ T cells. In addition, an increase in naïve CD4⁺CD45RA⁺ T cells with a reciprocal drop in memory CD4⁺CD45RO⁺ T cells was observed. The increase in CD4⁺CD45RA⁺ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4⁺ and CD8⁺ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D₃ status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings.     

GABA regulates the multidirectional tangential migration of GABAergic interneurons in living neonatal mice

Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT)-Venus transgenic mice from birth (P0) through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr), the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABA_A receptors and of the Na⁺-K⁺-Cl⁻ cotransporters, and chelating intracellular Ca²⁺, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABA_AR by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.     

Down-Regulated GABAergic Expression in the Olfactory Bulb Layers of the Mouse Deficient in Monoamine Oxidase B and Administered With Amphetamine

This study explores primarily the role of the activity of monoamine oxidase B (MAOB) in the regulation of glutamic acid decarboxylase₆₇ (GAD₆₇) expression in distinct layers of main olfactory bulb (OlfB), which links the limbic system. Moreover, the response of GAD₆₇ was investigated to amphetamine perturbation in the absence of MAOB activity. Immunocytochemical analysis was performed on OlfB sections prepared from the adult wild type (WT) and the MAOB gene-knocked-out (KO) mice after receiving repeated intraperitoneal injections (two doses per day, total seven doses) of saline or amphetamine, 5 mg/kg. The levels of the GAD₆₇ immunoreactivity were approximate 25 and 38% lower in respective glomerular (GloL) and mitral cell layers (ML) of saline-treated KO mice than that of WT, whereas similar in the external plexiform or granule cell layers (GraL) of the KO and WT. In the GloL, the level of tyrosine hydroxylase was 39% lower in the KO mice than WT, implicating different dopamine content in the KO from WT. The amphetamine exposure down-regulated the levels of GAD₆₇ in the WT layers by 46 to 52%, and in KO layers 65 to 71%, except ML. The GraL GAD₆₇ level may be regulated by the activation of CREB, as the phosphorylated (p) CREB coexisted with GAD₆₇, and the percentage of GAD₆₇-expressing pCREB neurons was decreased by the amphetamine exposure. The data indicate that the activity of MAOB could modulate the regular and amphetamine-perturbed expression of GAD₆₇ and pCREB. Thus, interactions are suggested among the MAOB activity, GABA content of OlfB, and olfaction.     

High frequency of CD4+ CXCR5+ TFH cells in patients with immune-active chronic hepatitis B

Background

T follicular helper (TFH) cells are a special subpopulation of T helper cells and can regulate humoral immune responses. This study examined whether the frequency of CD4⁺CXCR5⁺ TFH cells could be associated with active immunity in chronic hepatitis B (CHB) patients.

Methodology and Findings

The frequencies of peripheral blood CD4⁺CXCR5⁺ TFH cells, inducible T cell costimulator (ICOS), and/or programmed death 1 (PD-1) positive CD4⁺CXCR5⁺ TFH cells in immune-active (IA), immune-tolerant (IT) CHB, and healthy controls (HC) were characterized by flow cytometry analysis. The effect of adevofir dipivoxil treatment on the frequency of CD4⁺CXCR5⁺ TFH cells, the concentrations of serum IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-21, ALT, AST, HBsAg, HBsAb, HBeAg, HBeAb and HBV loads in IA patients were determined. The potential association of the frequency of CD4⁺CXCR5⁺ TFH cells with clinical measures was analyzed. In addition, the frequency of splenic and liver CD4⁺CXCR5⁺ TFH cells in HBV-transgenic mice was examined. We found that the frequency of CD4⁺CXCR5⁺ TFH cells in IA patients was significantly higher than that of IT patients and HC, and the percentages of CD4⁺CXCR5⁺ TFH in IA patients were positively correlated with AST. Furthermore, the percentages of ICOS⁺, PD-1⁺, and ICOS⁺PD-1⁺ in CD4⁺CXCR5⁺ TFH cells in CHB patients were significantly higher than that of HC. Treatment with adefovir dipivoxil reduced the frequency of CD4⁺CXCR5⁺ TFH, PD-1⁺CD4⁺CXCR5⁺ TFH cells and the concentrations of HBsAg and HBeAg, but increased the concentrations of HBsAb, HBeAb, IL-2 and IFN-γ in IA patients. Moreover, the frequency of splenic and liver CD4⁺CXCR5⁺ TFH cells in HBV-transgenic mice was higher than that of wild-type controls.

Conclusions

These data indicate that CD4⁺CXCR5⁺ TFH cells may participate in the HBV-related immune responses and that high frequency of CD4⁺CXCR5⁺ TFH cells may be a biomarker for the evaluation of active immune stage of CHB patients.     

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells

Background

The present study investigates the effects of high external calcium concentration ([Ca²⁺]_o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.

Methodology/Principal Findings

A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca²⁺]_o (0.37 mM); 2) high [Ca²⁺]_o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca²⁺]_o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca²⁺]_o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca²⁺]_o was not effective in this cell line. In small cells, both higher [Ca²⁺]_o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca²⁺]_o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.

Conclusions/Significance

In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.     

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.     

Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries

Background

As tumor antigen-specific CD4⁺ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4⁺ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients.

Methods and Findings

To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4⁺ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4⁺ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4⁺ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4⁺ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2_60–74 epitope and against the new epitope TRP-2_149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2_149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4⁺ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4⁺ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1_284–298 as a new HLA-DRB1*0301-restricted CD4⁺ T cell epitope.

Conclusions

Our screening approach identified new HLA-DRB1*0301-restricted CD4⁺ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.     

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD₆₅ and GAD₆₇, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD₆₅ and GAD₆₇ exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the K_m of GAD₆₇ and increasing the V_max of both isoforms. Of the 15 cysteines in GAD₆₅, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD₆₇, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD₆₅ and GAD₆₇ are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD₆₅ suggests that alteration of normal redox conditions could affect GAD targeting.     

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K⁺ channels (I_K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action on I_K,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I_K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I_K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K_i of 329 ± 31 nm with cooperative binding of four Ca²⁺ ions per channel. I_K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺ channel blocker Cs⁺. We also found that ryanodine, an antagonist of intracellular Ca²⁺ channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca²⁺]_i control of I_K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.Ca²⁺ underlies many fundamental regulatory processes in plants, including adaptive responses to abiotic environmental stress (Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and programmed cell death evoked by pathogen attack (Low and Merida, 1996; Hammondkosack and Jones, 1997). Coordination of changes in [Ca²⁺]_i and its integration with downstream response elements are central in coupling stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell model, major downstream targets of [Ca²⁺]_i and their roles in stomatal function have been identified. Increasing [Ca²⁺]_i is known to inactivate I_K,in and to activate Cl⁻ channels, events that bias plasma membrane transport for net efflux of osmotically active solute and a loss of turgor, which drives stomatal closure (Blatt and Grabov, 1997). Furthermore, changes in [Ca²⁺]_i are associated with ABA, CO₂, and the growth hormone auxin (Blatt and Grabov, 1997; McAinsh et al., 1997). These [Ca²⁺]_i signals have been observed to oscillate (McAinsh et al., 1995; Webb et al., 1996), characteristics that may constitute “Ca²⁺ signatures” to encode specific downstream responses (Berridge, 1996). Yet, despite the evidence for [Ca²⁺]_i signaling in guard cells, surprisingly little detail is known about the link between [Ca²⁺]_i changes and ion channel activity at the plasma membrane or about the mechanisms mediating such [Ca²⁺]_i changes. To our knowledge, in no instance have the characteristics of ion channel regulation by Ca²⁺ been quantified directly in any higher-plant cell.We recently described the coupling of membrane voltage to [Ca²⁺]_i, demonstrating that hyperpolarization, whether under a voltage clamp or in the presence of low [K⁺]_o, evoked [Ca²⁺]_i increases in guard cells, and that the voltage threshold for [Ca²⁺]_i rise was profoundly altered by ABA (Grabov and Blatt, 1998). Our observations indicated a link to Ca²⁺ influx across the plasma membrane and raised questions about the efficacy of [Ca²⁺]_i in inactivating I_K,in and about the contributions of intracellular Ca²⁺ release to the [Ca²⁺]_i signal. We have used membrane voltage to experimentally manipulate [Ca²⁺]_i and report that I_K,in is strongly dependent on [Ca²⁺]_i, consistent with a cooperative binding of four Ca²⁺ ions to effect inactivation. Additional experiments indicate that voltage-evoked [Ca²⁺]_i increases depend both on Ca²⁺ influx and on release of Ca²⁺ from intracellular stores. These results underscore the role of [Ca²⁺]_i as a high-gain “switch” in the control of I_K,in, and implicate [Ca²⁺]_i in feedback control linking membrane voltage to the activity of the K⁺ channels.     

Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX₃CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX₃CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX₃CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx₃cr1 ^gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX₃CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.     

Presynaptic nicotinic α7 and non-α7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action

Background

Although converging evidence has suggested that nicotinic acetylcholine receptors (nAChR) play a role in the modulation of GABA release in rat hippocampus, the specific involvement of different nAChR subtypes at presynaptic level is still a matter of debate. In the present work we investigated, using selective α7 and α4β2 nAChR agonists, the presence of different nAChR subtypes on hippocampal GABA nerve endings to assess to what extent and through which mechanisms they stimulate endogenous GABA release.

Methodology/Findings

All agonists elicited GABA overflow. Choline (Ch)-evoked GABA overflow was dependent to external Ca²⁺, but unaltered in the presence of Cd²⁺, tetrodotoxin (TTX), dihydro-β-erythroidine (DHβE) and 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride SKF 89976A. The effect of Ch was blocked by methyllycaconitine (MLA), α-bungarotoxin (α-BTX), dantrolene, thapsigargin and xestospongin C, suggesting that GABA release might be triggered by Ca²⁺ entry into synaptosomes through the α7 nAChR channel with the involvement of calcium from intracellular stores. Additionally, 5-Iodo-A-85380 dihydrochloride (5IA85380) elicited GABA overflow, which was Ca²⁺ dependent, blocked by Cd²⁺, and significantly inhibited by TTX and DHβE, but unaffected by MLA, SKF 89976A, thapsigargin and xestospongin C and dantrolene. These findings confirm the involvement of α4β2 nAChR in 5IA85380-induced GABA release that seems to occur following membrane depolarization and opening calcium channels.

Conclusions/Significance

Rat hippocampal synaptosomes possess both α7 and α4β2 nAChR subtypes, which can modulate GABA release via two distinct mechanisms of action. The finding that GABA release evoked by the mixture of sub-maximal concentration of 5IA85380 plus sub-threshold concentrations of Ch was significantly larger than that elicited by the sum of the effects of the two agonists is compatible with the possibility that they coexist on the same nerve terminals. These findings would provide the basis for possible selective pharmacological strategies to treat neuronal disorders that involve the dysfunction of hippocampal cholinergic system.