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

Differential patterns of post-migration HIV-1 infection acquisition among Portuguese immigrants of different geographical origin

OBJECTIVE: To investigate the dynamics of phylogenetic transmission clusters involving immigrants of Portuguese Speaking Countries living in Portugal. DESIGN/METHODS: We included genomic sequences, sociodemographic and clinical data from 772 HIV migrants followed in Portugal between 2001 and 2017. To reconstruct HIV-1 transmission clusters, we applied phylogenetic inference from 16 454 patients: 772 migrants, 2973 Portuguese and 12 709 global controls linked to demographic and clinical data. Transmission clusters were defined using: clusters with SH greater than 90% (phylogenetic support), genetic distance less than 3.5% and clusters that included greater than 66% of patients from one specific geographic origin compared with the total of sequences within the cluster. Logistic regression was performed to assess factors associated with clustering. RESULTS: Three hundred and six (39.6%) of migrants were included in transmission clusters. This proportion differed substantially by region of origin [Brazil 54% vs. Portuguese Speaking African Countries (PALOPs) 36%, P < 0.0001] and HIV-1 infecting subtype (B 52%, 43% subtype G and 32% CRF02_AG, P < 0.001). Belonging to a transmission cluster was independently associated with treatment-naive patients, CD4+ greater than 500, with recent calendar years of sampling, origin from PALOPs and with seroconversion. Among Brazilian migrants - mainly infected with subtype B - 40.6% were infected by Portuguese. Among migrants from PALOPs - mainly infected with subtypes G and CFR02_AG - the transmission occurred predominantly within the migrants' community (53 and 80%, respectively). CONCLUSION: The acquisition of infection among immigrants living in Portugal differs according to the country of origin. These results can contribute to monitor the HIV epidemic and prevent new HIV infections among migrants.publishersversionpublishe

Molecular Epidemiology of HIV-1 Infected Migrants Followed Up in Portugal: Trends between 2001-2017

Migration is associated with HIV-1 vulnerability. OBJECTIVES: To identify long-term trends in HIV-1 molecular epidemiology and antiretroviral drug resistance (ARV) among migrants followed up in Portugal Methods: 5177 patients were included between 2001 and 2017. Rega, Scuel, Comet, and jPHMM algorithms were used for subtyping. Transmitted drug resistance (TDR) and Acquired drug resistance (ADR) were defined as the presence of surveillance drug resistance mutations (SDRMs) and as mutations of the IAS-USA 2015 algorithm, respectively. Statistical analyses were performed. RESULTS: HIV-1 subtypes infecting migrants were consistent with the ones prevailing in their countries of origin. Over time, overall TDR significantly increased and specifically for Non-nucleoside reverse transcriptase inhibitor (NNRTIs) and Nucleoside reverse transcriptase inhibitor (NRTIs). TDR was higher in patients from Mozambique. Country of origin Mozambique and subtype B were independently associated with TDR. Overall, ADR significantly decreased over time and specifically for NRTIs and Protease Inhibitors (PIs). Age, subtype B, and viral load were independently associated with ADR. CONCLUSIONS: HIV-1 molecular epidemiology in migrants suggests high levels of connectivity with their country of origin. The increasing levels of TDR in migrants could indicate an increase also in their countries of origin, where more efficient surveillance should occur.status: publishe

Molecular epidemiology of hiv-1 infected migrants followed up in Portugal: Trends between 2001-2017

Migration is associated with HIV-1 vulnerability. Objectives: To identify long-term trends in HIV-1 molecular epidemiology and antiretroviral drug resistance (ARV) among migrants followed up in Portugal Methods: 5177 patients were included between 2001 and 2017. Rega, Scuel, Comet, and jPHMM algorithms were used for subtyping. Transmitted drug resistance (TDR) and Acquired drug resistance (ADR) were defined as the presence of surveillance drug resistance mutations (SDRMs) and as mutations of the IAS-USA 2015 algorithm, respectively. Statistical analyses were performed. Results: HIV-1 subtypes infecting migrants were consistent with the ones prevailing in their countries of origin. Over time, overall TDR significantly increased and specifically for Non-nucleoside reverse transcriptase inhibitor (NNRTIs) andNucleoside reverse transcriptase inhibitor (NRTIs). TDR was higher in patients from Mozambique. Country of origin Mozambique and subtype B were independently associated with TDR. Overall, ADR significantly decreased over time and specifically for NRTIs and Protease Inhibitors (PIs). Age, subtype B, and viral load were independently associated with ADR. Conclusions: HIV-1 molecular epidemiology in migrants suggests high levels of connectivity with their country of origin. The increasing levels of TDR in migrants could indicate an increase also in their countries of origin, where more efficient surveillance should occur. © 2020 by the authors

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