Chronic low-level expression of HIV-1 Tat promotes a neurodegenerative phenotype with aging

Abstract

The widespread use of combinational antiretroviral therapies (cART) in developed countries has changed the course of Human Immunodeficiency Virus (HIV) infection from an almost universally fatal disease to a chronic infection for the majority of individuals. Although cART has reduced the severity of neurological damage in HIV-infected individuals, the likelihood of cognitive impairment increases with age, and duration of infection. As cART does not suppress the expression of HIV non-structural proteins, it has been proposed that a constitutive production of HIV regulatory proteins in infected brain cells may contribute to neurological damage. However, this assumption has never been experimentally tested. Here we take advantage of the leaky tetracycline promoter system in the Tat-transgenic mouse to show that a chronic very low-level expression of Tat is associated with astrocyte activation, inflammatory cytokine expression, ceramide accumulation, reductions in brain volume, synaptic, and axonal damage that occurs over a time frame of 1 year. These data suggest that a chronic low-level production of Tat may contribute to progressive neurological damage in virally suppressed HIV-infected individuals.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Brain volume loss is observed in doxycycline naïve rtTA-Tat mice at 12 months of age. qRT-PCR analysis showing expression of tat mRNA in cortex of (a) 3-month old control rtTA mice, and doxycycline naïve rtTA-Tat mice, and (b) 11–12 month old doxycycline treated control rtTA mice, doxycycline naïve rtTA-Tat mice, and doxycycline treated rtTA-Tat mice. (c) Tat protein expression in cortex of 11–12 month old doxycycline treated control rtTA mice, doxycycline naïve rtTA-Tat mice, and doxycycline treated rtTA-Tat mice. (d) Representative T2 weighted images from 3–5 and 11–12 month old mice of the indicated genotype and treatment conditions showing enlargement of ventricles in 11–12 month old doxycycline naïve and doxycycline treated rtTA-Tat mice compare with doxycycline treated rtTA control mice. (e,f) Bar graphs show volumetric quantification of the ventricle, hippocampus, dentate gyrus, striatum, and motor cortex of 3–5 month animals and (g,h) of 11–12 month animals. (i) Representative fiber tracking maps of the corpus callosum calculated from the diffusion weighted MRI images. Quantification of the fractional anisotropy (j) and parallel eigenvalue (k) the diffusion weighted images in 11–12 month old animals. Data are mean ± SEM of n = 6–7 animals/group. ANOVA with Tukey post hoc comparisons, *p < 0.05 compared to control, ***p < 0.001 compared to rtTA control.

Figure 2

Evidence for axonal and synaptic damage in doxycycline naïve rtTA-Tat mice. Immunoblots and density quantitation of (a) the axonal marker βIII-tubulin, (b) the presynaptic marker Synaptophysin, and (c) the postsynaptic marker PSD95 in cortex of 11–12 month old mice with the indicated genotype and treatment condition. (d) qRT-PCR analysis showing expression of the indicated cytokines in cortex of 11–12 month old mice with the indicated genotype and treatment condition. Data are expressed as mean ± SEM. N = 3 mice per condition for western blots, and n = 6–7 per group for qRT-PCR. ***p < 0.001, **p < 0.01, *p < 0.05, and #p < 0.05 compared to rtTA control group.

Figure 3

Accumulation of ceramide in cortex of rtTA-Tat mice. Concentrations of (a) dihydroceramides, (b) ceramides, (c) monohexosylceramides, and (d) dihexosylceramides, were determined in cortex of mice with the indicated genotypes. Hierarchal clustering analysis is shown to the left of each class of lipid. Blue colors indicates increase and red colors indicates decrease compared to rtTA control mice. Data are expressed as mean ± SEM, n = 6–7 per group. Bold text in table indicates p < 0.05 compared to rtTA control group. ANOVA with Tukey post hoc comparisons.

Figure 4

Accumulation of sphingomyelin in cortex of rtTA-Tat mice. Concentrations of sphingomyelins were determined in cortex of mice with the indicated genotypes. Hierarchal clustering analysis is shown to the left of each class of lipid. Blue colors indicates increase and red colors indicates decrease compared to rtTA control mice. Data are expressed as mean ± SEM, n = 6–7 per group. Bold text in table indicates p < 0.05 and italicized entries indicates p < 0.001 compared to rtTA control group. ANOVA with Tukey post hoc comparisons.

Figure 5

The increase in dentate gyrus volume is linked to an increase in reactive astrocytes in this brain region. (a) Representative images and (b) quantitative analysis of GFAP fluorescence from the dentate gyrus of 11–12 month old mice with the indicated genotypes. (c) qRT-PCR analysis showing expression of the indicated cytokines in hippocampus of 11–12 month old mice with the indicated genotype and treatment condition. Data are expressed as mean ± SEM for n = 6–7 per group. * p < 0.05, **p < 0.01. ANOVA with Tukey post hoc comparisons.