Endolysosome involvement in HIV-1 transactivator protein-induced neuronal amyloid beta production

Abstract

The increased life expectancy of people living with HIV-1/AIDS is accompanied by increased prevalence of HIV-1-associated neurocognitive disorder. As well, these individuals are increasingly experiencing Alzheimer's disease (AD)-like neurocognitive problems and neuropathological features such as increased deposition of amyloid beta (Aβ) protein. Findings that Aβ production occurs largely in endolysosomes, that HIV-1 transactivator protein (Tat) disrupts endolysosome function-an early pathological feature of AD-and that HIV-1 Tat can increase Aβ levels prompted us to test the hypothesis that endolysosome dysfunction is associated with HIV-1 Tat-induced increases in neuronal Aβ generation. Using primary cultured rat hippocampal neurons, we found that treatment with HIV-1 Tat caused such morphological changes as enlargement of endolysosomes identified with LysoTracker dye and such functional changes as elevated endolysosome pH measured ratiometrically with LysoSensor dye. The HIV-1 Tat-induced changes in endolysosome function preceded temporally HIV-1 Tat-induced increases in Aβ generation measured using enzyme-linked immunosorbent assay. In addition, we demonstrated that HIV-1 Tat increased endolysosome accumulation of Aβ precursor protein and Aβ identified using immunostaining with 4G8 antibodies. Furthermore, we demonstrated that treatment of neurons with HIV-1 Tat increased endolysosome accumulation of beta amyloid-converting enzyme, the rate-limiting enzymatic step for Aβ production, and enhanced beta amyloid-converting enzyme activity. Together, our findings suggest that HIV-1 Tat increases neuronal Aβ generation and thereby contributes to the development of AD-like pathology in HIV-1-infected individuals by disturbing endolysosome structure and function.

Keywords: Amyloid beta; BACE-1; Endolysosome; HIV-1 Tat; pH.

Figure 1. HIV-1 Tat increased neuronal Aβ generation

(A) HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly levels of Aβ_1-40, when compared with control groups (n=5; ***p<0.001). Neither HIV-1 Tat_1-72 (100 nM) treatment for 1 day nor mutant Tat (Tat_Δ31–61, 100 nM) treatment for 2 days affected significantly levels of Aβ_1-40. (B) HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly levels of Aβ_1-42, when compared with control groups (n=5; ***p<0.001). Neither Tat_1-72 (100 nM) treatment for 1 day nor mutant Tat (Tat_Δ31–61, 100 nM) treatment for 2 days affected significantly levels of Aβ_1-42.

Figure 2. HIV-1 Tat increased endolysosome accumulation of AβPP

(A) Although there is some extent co-localization of AβPP with endosomes (EEA1) in control neurons, HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly the co-localization of AβPP with endosomes (EEA1) (n = 11, ***p<0.001). Bar = 10 μm. (B) There is little co-localization of AβPP with endolysosomes (LAMP1) in control neurons, but HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased markedly the co-colocalization of AβPP with endolysosomes (LAMP1) (n = 9, ***p<0.001). Bar = 10 μm. (C) HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly the protein levels of AβPP (n = 6, *p<0.05).

Figure 3. HIV-1 Tat increased endolysosome accumulation of Aβ

(A) Although there is some extent co-localization of Aβ/AβPP (4G8) with endosomes (EEA1) in control neurons, HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased markedly the co-colocalization of Aβ/AβPP (4G8) with endosomes (EEA1) (n = 10, **p<0.01). Bar = 10 μm. (B) There is little co-localization of Aβ/AβPP (4G8) with endolysosomes (LAMP1) in control neurons, but HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased markedly the co-colocalization of Aβ/AβPP (4G8) with endolysosomes (LAMP1) (n=12, ***p<0.001). Bar = 10 μm.

Figure 4. HIV-1 Tat disturbed the structure of neuronal endolysosomes

(A) HIV-1 Tat_1-72 treatment (100 nM) for 1 day and 2 days increased significantly the size of endolysosomes (lysoTracker) in primary cultured hippocampal neurons (*p<0.05). Mutant Tat_Δ31–61 treatment (100 nM) for 2 days did not affect the size of endolysosomes. Bar = 10 μm. (B) The sizes of endolysosomes from control neurons (n = 40) and neurons treated with mutant Tat_Δ31–61 (n = 37) or HIV-1 Tat_1-72 (n = 67) for 2 days were plotted and analyzed with non-linear regression.

Figure 5. HIV-1 Tat elevated endolysosomes pH

Endolysosome pH was measured ratio-metrically with a LysoSensor dye. HIV-1 Tat_1-72 treatment (100 nM) for 1 day and 2 days increased significantly neuronal endolysosome pH (n=8; *** p<0.001).

Figure 6. HIV-1 Tat increased endolysosome accumulation of BACE-1 and enhanced BACE-1 activity

(A) HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly protein levels of BACE-1 (n=6, *p<0.05). (B) HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased significantly specific activity of BACE-1 (n=8; ***p<0.001). BACE-1 activity was expressed as relative fluorescent units per 10 μg of protein. Specific activities of BACE-1 were expressed as a ratio of BACE-1 activity to protein levels as determined by immunoblotting. (C) There is some extent co-localization of BACE-1 with endosomes (EEA1) in control neurons, and HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased markedly the co-colocalization of BACE-1 with endosomes (EEA1) (n = 12, ***p<0.001). Bar = 10 μm. (D) There is little co-localization of BACE-1 with endolysosomes (LAMP1) in control neurons, but HIV-1 Tat_1-72 treatment (100 nM) for 2 days increased markedly the co-colocalization of BACE-1 with endolysosomes (LAMP1) (n = 10, ***p<0.001). Bar = 10 μm.