Metformin promotes PEN2 expression to attenuate microglia-mediated neurotoxicity induced by HIV-1 Tat

Abstract

Metformin, a first-line drug used to treat type 2 diabetes mellitus (T2DM), also reduces neuroinflammation and improves motor and cognitive outcomes. Metformin binds to presenilin enhancer 2 (PEN2) and further enhances its therapeutic benefits. The mechanisms of HIV-associated neurocognitive disorders (HANDs) remain unclear. HIV-1 trans-activator of transcription (Tat) contributes to neurotoxicity in HAND. We revealed that PEN2 expression decreased markedly in HAND patients and Tat-infected microglia. Metformin (200 µM) treatment significantly reduced Tat-induced decreases in cell viability, oxidative stress, the proinflammatory response and excessive glutamate and iNOS release and had neuroprotective effects. Tat subsequently increased NF-κB activity, which was prominently suppressed during treatment. In addition, PEN2 knockdown in microglia dramatically reversed the neuroprotective effect of metformin against Tat. Our findings indicate that metformin binds PEN2 and modulates microglia-mediated HIV-1 Tat neurotoxicity in HAND.

Keywords: HAND Tat; Metformin; Neurotoxicity; PEN2.

Fig. 1

BV-2 microglial cells were transduced with LV-Vector or LV-Tat for 48 h. A Based on the gene expression dataset GSE28160, PEN2 expression decreased in HAND patients. B Agarose gel Electrophoresis analyzed Tat and PEN2 expression at mRNA levels. C Representative Western blot analysis of Tat and PEN2 expression at protein level. n = 3 for each group, * P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 2

BV-2 microglial cells were transduced with LV-Tat for 48 h. A Representative Western blot analysis of Tat and IBA1 expression at protein levels and quantitative analysis of gray levels under different concentrations of metformin. B Immunofluorescence staining for IBA1 (red) and the histogram showed quantitative immunofluorescence analysis of IBA1 when metformin at concentrations of 200 µm. Scale bars, 50 μm. n = 3 for each group, *P < 0.05, **P < 0.01, two-tailed t-test

Fig. 3

BV-2 microglial cells were transduced with LV-Vector or LV-flag-Tat for 48 h, treated with saline or metformin for 24 h, as well as LV-shScramble or LV-shPEN2. A Representative Western blot analysis of Tat and PEN2 expression at protein levels and quantitative analysis of gray levels, respectively. B DCFH-DA analysis of ROS. C Measurement of the Levels of MDA. D Determination of the activity of SOD. The results revealed that metformin reduces Tat-induced oxidative stress in BV-2 microglia cells by regulating PEN2 expression. n = 3 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 4

BV-2 microglial cells were transduced with LV-Vector or LV-flag-Tat for 48 h, treated with saline or metformin for 24 h, as well as LV-shScramble or LV-shPEN2. A Representative Western blot analysis of Tat, PEN2, P-NF-κB p65, and NF-κB p65 expression at protein levels and quantitative analysis of gray levels, respectively. B ELISA analysis of IL-6, TNF-α, CCL2 and CXCL10 in cell culture supernatants. The studies showed that metformin promotes PEN2 expression to suppress Tat-induced NF‑κB signalling activation and the inflammatory response in BV-2 microglia. n = 3 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 5

BV-2 microglial cells were transduced with LV-Vector or LV-flag-Tat for 48 h, treated with saline or metformin for 24 h, as well as LV-shScramble or LV-shPEN2. A Representative Western blot analysis of Tat, PEN2 and EAAT2 expression at protein levels and quantitative analysis of gray levels, respectively. B Measurement of extracellular glutamate levels. We detected that metformin inhibits glutamate neurotoxicity in Tat-transduced BV-2 microglia by targeting PEN2. n = 3 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 6

BV-2 microglial cells were transduced with LV-Vector or LV-flag-Tat for 48 h, treated with saline or metformin for 24 h, as well as LV-shScramble or LV-shPEN2. A. Representative Western blot analysis of Tat, PEN2 and iNOS expression at protein levels and quantitative analysis of gray levels, respectively. B. Measurement of nitric oxide levels. The metformin reverses Tat-induced iNOS expression and NO production in BV-2 microglia. n = 3 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 7

BV-2 microglial cells were transduced with LV-Vector or LV-flag-Tat for 48 h, treated with saline or metformin for 24 h, as well as LV-shScramble or LV-shPEN2. A Cell culture supernatants were collected as the MCM and added into HT-22 neuronal cells to incubate for another 24 h. CCK-8 analysis of cell viability. B Measurement of extracellular LDH levels. n = 3 for each group, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test

Fig. 8

Schematic diagram of metformin promotes PEN2 expression to attenuate microglia-mediated HIV-1 Tat neurotoxicity. The metformin treatment attenuated neuroinflammatory, inhibited NF-κB activity, suppressed oxidative stress and promoted cell activation in Tat infected microglia cells via targeting to PEN2