Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons

Abstract

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.

Figure 1. Glutamate excitotoxicity decreases viability of cultured hippocampal GABAergic neurons.

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 t-test. ***p<0.001.

Figure 2. Glutamate excitotoxicity induces a time-dependent decrease in GAD65 and GAD67 protein levels in cultured hippocampal neurons.

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. **p<0.01; ***p<0.001.

Figure 3. Glutamate excitotoxicity decreases GAD65/67 mRNA.

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 t-test. *p<0.05; **p<0.01.

Figure 4. Proteasome and ubiquitin-activating enzyme (E1) inhibitors prevent glutamate–induced GAD65/67 cleavage.

Cultured hippocampal neurons were pre-incubated or not with 50 µM of UBEI-41 (E1 inhibitor), for 1h, or with the proteasome inhibitors MG132 (1 µM), lactacystin (10 µM) or YU102 (10 µM), for 30 min, before excitotoxic stimulation with glutamate (125 µM), for 20 min. The cells were further incubated in culture conditioned medium (with or without chemical inhibitors) for 5h, and the GAD65/67 immunoreactivity was assessed by western blot (A and B). Incubation with the calpain inhibitor ALLN (10 µM) was performed under the same conditions. The average results in (A) represent the changes in GAD65 or GAD67 immunoreactivity. In panel (B) GAD cleavage was calculated as a percentage of the total enzyme content (GAD65/67). When calpain activity was evaluated through formation of N-terminal spectrin breakdown products (_NSBDPs) the cells were incubated for 30 min after the toxic insult (C). The effect of MG132 and UBEI-41 on calpain activation is expressed as a percentage of the activity measured in the absence of the protease inhibitors. Results are means±SEM of 3–4 different experiments, performed in independent preparations. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni's Multiple Comparison Test (A and B) or the Student's t test (C). ***p<0.001.

Figure 5. GAD 65/67 are captured with an anti-ubiquitin antibody in hippocampal cultures.

(A) Cultured hippocampal neurons were stimulated or not with 125 µM glutamate for 20min, in the presence or in the absence of 10 µM MG132, and the cells were further incubated with culture conditioned medium for 4h before preparation of the extracts. In the top panel, mono- and poly-ubiquitinylated proteins were isolated using the UbiQapture™-Q Kit, and the eluted fraction was subjected to western blot, using an antibody against GAD65/67. GAD65/67 total immunoreactivity in the extracts prepared from control cells and from hippocampal neurons stimulated with glutamate is shown on the right (E.T.). The left lane was loaded with a control provided in the kit, consisting in ubiquitinated protein lysate. The same membranes were probed for mono- and poly-ubiquitin using an antibody included in the kit (A, middle). (B) Human recombinant GAD65 (0.75 µg) were incubated with 20/26S proteasomes (1.5 µg) for 2h at 37°C with or without MG132. The extracts were then probed with GAD65/67 antibody. The activity of the 20S/26S proteasomes used in the experiments was confirmed using the fluorogenic substrate suc-LLVY-MCA. The increase in fluorescence resulting from the cleavage of the substrate was measured in relative fluorescence units (RFU) (C). The results of the capture of ubiquitinated proteins and the assay of the recombinant GAD65 cleavage are representative of two and three independent experiments, respectively.

Figure 6. Excitotoxicity-induced decrease in GAD65/67 activity.

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 t-test. *p<0.05; **p<0.01: ***p<0.001.

Figure 7. Glutamate changes the subcellular distribution of GAD65 along neurites.

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 t-test. *p<0.05; **p<0.01.