Role of the Proteasome in Excitotoxicity-Induced Cleavage of Glutamic Acid Decarboxylase in Cultured Hippocampal Neurons

Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms—GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): study protocol for a randomized controlled trial

BACKGROUND: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). METHODS/DESIGN: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH2O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure 6430 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. DISCUSSION: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration metho

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Brain-derived neurotrophic factor regulates the expression and synaptic delivery of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity in the hippocampus, but the mechanisms involved are not fully understood. The neurotrophin couples synaptic activation to changes in gene expression underlying long term potentiation and short term plasticity. Here we show that BDNF acutely up-regulates GluR1, GluR2, and GluR3 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits in 7-day in vitro cultured hippocampal neurons. The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a tropomyosin-related [corrected] kinase (Trk) inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors [corrected] The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a Trk inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors. Accordingly, BDNF increased the mRNA levels for GluR1 and GluR2 subunits. Biotinylation studies showed that stimulation with BDNF for 30 min selectively increased the amount of GluR1 associated with the plasma membrane, and this effect was abrogated by emetine. Under the same conditions, BDNF induced GluR1 phosphorylation on Ser-831 through activation of protein kinase C and Ca(2+)-calmodulin-dependent protein kinase II. Chelation of endogenous extracellular BDNF with TrkB-IgG selectively decreased GluR1 protein levels in 14-day in vitro cultures of hippocampal neurons. Moreover, BDNF promoted synaptic delivery of homomeric GluR1 AMPA receptors in cultured organotypic slices, by a mechanism independent of NMDA receptor activation. Taken together, the results indicate that BDNF up-regulates the protein levels of AMPA receptor subunits in hippocampal neurons and induces the delivery of AMPA receptors to the synaps

Glutamate excitotoxicity induces a time-dependent decrease in GAD65 and GAD67 protein levels in cultured hippocampal neurons.

<p>Neurons were stimulated with 125 µM glutamate, for 20 min, and further incubated in culture conditioned medium for the indicated period of time. Full length GAD 65/67 protein levels were determined by Western Blot with an antibody that recognizes both isoforms. Control protein levels of GAD65/67 were set to 100%. Actin was used as loading control (A). Panel A shows a representative experiment and mean±SEM of 9 independent experiments. The cleavage of GAD67 was also analysed with an antibody directed against amino acids 70–130 of this isoform (B). In this case the results obtained under control conditions were compared with the immunoreactivity in extracts prepared 14 h after the toxic insult. The amino acid sequence of GAD65 (lower sequence) and 67 (top sequence) are aligned in panel C, which also show the binding sites for the antibodies used in this study. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni's multiple comparison test. **<i>p</i><0.01; ***<i>p</i><0.001.</p

Excitotoxicity-induced decrease in GAD65/67 activity.

<p>Heads of adult Wistar rats were decapitated and processed immediately or kept for 24 h at room temperature. The extracts were used for both GAD activity measurements and Western Blot analysis. GAD activity was determined using a trapping technique for radiolabelled [¹⁴C]CO₂ brought by GAD65/67 activity, and was expressed as nmol CO₂/hr/mg of protein (A). Full-length GAD65/67 protein levels from the same extracts were determined by Western Blot using an anti-GAD65/67 antibody, and control protein levels of GAD65/67 were set to 100% (B). Data are presented as mean±SEM of 3 to 4 independent experiments. Statistical analysis was performed using Student's <i>t</i>-test. *<i>p</i><0.05; **<i>p</i><0.01: ***<i>p</i><0.001.</p

Glutamate excitotoxicity decreases viability of cultured hippocampal GABAergic neurons.

<p>GABAergic and glutamatergic neurons in the cultures (DIV7) were identified by immunocytochemistry, using antibodies against GABA (A, E) and VGLUT1+2 (A, D). The total number of cells present in the analysed fields was calculated based on the number of nuclei, stained with the fluorescent dye Hoechst 33342. Data are presented as mean±SEM of 5 independent preparations (A). Excitotoxic stimulation of hippocampal neurons was performed by incubation with 125 µM glutamate, for 20 min, in fresh Neurobasal medium containing B27 supplement, and the cells were further incubated in the original medium for 14 h. Cell death was assessed with the recombinant Luciferase chemoluminescence assay with CellTiterGlo (B), or by fluorescence microscopy using the fluorescence dye Hoechst 33342 (C). In the latter condition GABAergic cells were identified by immunocytochemistry, using an antibody against GABA. Data are presented as mean±SEM of 5 independent experiments. Statistical analysis was performed using Student's <i>t</i>-test. ***<i>p</i><0.001.</p

Glutamate excitotoxicity decreases GAD65/67 mRNA.

<p>Gene expression was analysed in cultured hippocampal neurons (7 DIV) exposed or not to 125 µM Glutamate, for 20 min, and then returned to the original culture medium for 4 h. For the reverse transcription reaction 1 µg of total RNA was used. The results were normalized with two internal control genes, GAPDH and Tubulin. Data are presented as mean±SEM of four to five independent transcription reactions, performed in independent preparations. Statistical analysis was performed using Student's <i>t</i>-test. *<i>p</i><0.05; **<i>p</i><0.01.</p

Glutamate changes the subcellular distribution of GAD65 along neurites.

<p>10 DIV hippocampal neurons were incubated with or without glutamate (125 µM) for 20 min, and then returned to the original culture medium for 4 h.(A) Cells were fixed, permeabilized and probed with specific antibodies to GAD65 and GABA_A receptor subunits β2/3. (B) Arrows indicate GAD65 clustering (red), a pattern that is changed in glutamate treated cells. Images are representative of three different experiments performed in independent preparations. Images in (B) show colocalization of the immunoreactivity for GAD65 (red) and GABA_A receptor subunits β2/3 (green) under control conditions, and the redistribution of GAD65 in the axons of hippocampal neurons subjected to excitotoxic conditions. The scale bar corresponds to 10 µm. (C) Quantification of the images for the colocalization of GAD65 and GABA_A subunits β2/3, expressed as percentage of total GAD65 puncta (left), and the average number of GAD65 puncta per axon unit length (right). Data are presented as mean±SEM of 3 independent experiments performed in different preparations. Statistical analysis was performed using Student's <i>t</i>-test. *<i>p</i><0.05; **<i>p</i><0.01.</p