Cytochrome C dysregulation induced by HIV infection of astrocytes results in bystander apoptosis of uninfected astrocytes by an IP3 and calcium-dependent mechanism

Abstract

HIV entry into the CNS is an early event after peripheral infection, resulting in neurologic dysfunction in a significant number of individuals despite successful anti-retroviral therapy. The mechanisms by which HIV mediates CNS dysfunction are not well understood. Our group recently demonstrated that HIV infection of astrocytes results in survival of HIV infected cells and apoptosis of surrounding uninfected astrocytes by the transmission of toxic intracellular signals through gap junctions. In the current report, we characterize the intracellular signaling responsible for this bystander apoptosis. Here, we demonstrate that HIV infection of astrocytes results in release of cytochrome C from the mitochondria into the cytoplasm, and dysregulation of inositol trisphosphate/intracellular calcium that leads to toxicity to neighboring uninfected astrocytes. Blocking these dysregulated pathways results in protection from bystander apoptosis. These secondary messengers that are toxic in uninfected cells are not toxic in HIV infected cells, suggesting that HIV protects these cells from apoptosis. Thus, our data provide novel mechanisms of HIV mediated toxicity and generation of HIV reservoirs. Our findings provide new potential therapeutic targets to reduce the CNS damage resulting from HIV infection and to eradicate the generation of viral reservoirs. We demonstrated that HIV infection of astrocytes protects infected cells from apoptosis but results in cell death of surrounding uninfected astrocytes by a mechanism that is dependent on gap junction channels, dysregulation of mitochondrial cytochrome C (CytC), and cell to cell diffusion of inositol trisphosphate (IP3 ) and calcium. Our data provide essential information about generation of brain reservoirs and the mechanism of toxicity mediated by the virus.

Keywords: AIDS; HIV; connexin; gap junctions; reservoirs.

Fig. 1

HIV infection of astrocytes results in apoptosis of uninfected astrocytes, but not in infected cells. Using immunofluorescence staining of uninfected and HIV_ADA infected cultures of astrocytes for HIV-p24 (to demonstrate HIV infection), TUNEL (to determine apoptosis) and DAPI (to quantify total numbers of cells); cell death was quantified for up to 120 days post-infection. HIV infection of primary cultures of astrocytes resulted in apoptosis, mostly of uninfected cells (lines with squares). Apoptosis was minimally detected in uninfected cultures of primary astrocytes (control cells, lines with circles). Quantification of apoptosis in just HIV-p24 positive astrocytes indicated that most of infected cells survive infection (lines with triangles). n = 5, *p < 0.005, represents significance as compared to control uninfected cultures (control cells).

Fig. 2

Cytochrome c (CytC), normally concentrated inside of the mitochondria, is released into the cytoplasm of HIV infected astrocytes. We stained CytC (FITC staining, green staining), mitochondria with Mito-tracker (Mtrack, to observe mitochondria, red staining), HIV-p24 (Cy5 staining, to identify HIV infected astrocytes, white), and DAPI (to identify nucleus, blue staining) in uninfected and HIV infected primary cultures of astrocytes. Colocalization of mitochondria and CytC was quantified by using an imaging software, NIS Elements. Mitochondria in uninfected astrocytes (control) were localized throughout the cytoplasm and 100% of CytC was localized inside these structures (black bars and pictures labeled uninfected cells, perfect colocalization in the merge picture). Staining of HIV infected primary cultures (HIV) indicated that cells are positive for HIV-p24, and CytC did not totally colocalized with mito-tracker (see graph, white bars). The loss of colocalization was because of the release of CytC into the cytoplasm of infected astrocytes. Pictures in the right side correspond to a high magnification to observe the colocalization between the different markers. Despite the dysregulation of CytC, no apoptosis was detected in HIV infected cells as described in Fig. 1. n = 6, *p < 0.0042, represents significance as compared to control uninfected cultures. Bar: 50 µm to uninfected cells and 15 to HIV-infected cells.

Fig. 3

Blocking cytochrome c (CytC), IP₃ and the increase in intracellular calcium induced by the virus reduces bystander toxicity from HIV infected to uninfected astrocytes. Control cultures have minimal apoptosis (Control). HIV infection of astrocytes cultures with HIV_ADA, resulted as we described previously, in bystader apoptosis of uninfected astrocytes after 7–14 days post-infection (Eugenin and Berman 2007; Eugenin et al. 2011) (HIV_ADA). Blocking gap junction channels using 18-α-glycerritenic acid (AGA, 35 µM) to reduce the spread of toxic signals generated from HIV infected to uninfected astrocytes completely abrogates bystander apoptosis (HIV_ADA+AGA). Blocking increases in intracellular calcium with BAPTA-AM (HIV_ADA+BAPTA-AM) or CytC induced activation of IP₃ and calcium release with a specific peptide reduced bystander apoptosis induced by the virus to control levels (HIV_ADA+IP₃/CytC pep). Scramble peptides did not alter bystander apoptosis (data not shown). Calcium ionophore (A23187, 5 µM), cell permeable cAMP (8Br-cAMP, 1 mM) and cGMP (8Brc-GMP, 1 mM) did not alter bystander apoptosis (HIV_ADA+ cAMP or cGMP). All of these factors can cross gap junction channels. (n = 5, *p < 0.007, represent significance from control conditions, and #p < 0.001, represent significance from HIV_ADA infected cultures).

Fig. 4

HIV infection protects infected astrocytes from apoptosis induced by cytoplasmic cytochrome c (CytC). (a) Microinjection by single cell electroporation of CytC into uninfected U87CD4CCR5 astrocytes resulted in apoptosis as determined by TUNEL staining (control+CytC) as compared to cells microinjected with phosphate-buffered saline (PBS) only (control). Microinjection of CytC into the cytoplasm of HIV-infected U87CD4CCR5 astrocytes (95% of the cells are infected) did not result in apoptosis, suggesting that HIV_ADA infection protects these cells from the toxicity of cytoplasmic CytC (HIV+CytC). Microinjection of PBS into cells did not trigger apoptosis (HIV). (b) Primary cultures of human astrocytes were microinjected with PBS and this did not result in apoptosis (control). However, microinjection of CytC into uninfected astrocytes resulted in 100% apoptosis, supporting the high toxicity of free cytoplasmic CytC. Microinjection of HIV infected primary cultures, where 5.9 ± 3.2% of the cells are infected with HIV, with PBS resulted in minimal apoptosis (HIV). However, microinjection of CytC into the cytoplasm of HIV infected cultures resulted in a decrease in apoptosis of 6.2 ± 2.9% that corresponds to the fraction of cells infected with HIV (HIV+CytC). n = 6, *p < 0.0005 as compared to control conditions and #p < 0.05 as compared to control+CytC.

Fig. 5

Schematic representation of the mechanism of generation of HIV reservoirs and amplification of toxicity to uninfected astrocytes by a bystander mechanism. Our proposed model is that, cytochrome c (CytC), inositol trisphosphate (IP₃) and calcium mediate apoptosis of uninfected astrocytes. HIV infection of astrocytes dysregulates mitochondrial function and CytC metabolism resulting in release of CytC into the cytoplasm that acts as a toxic factor in neighboring uninfected cells. IP₃ and calcium can diffuse through gap junctions resulting in bystander apoptosis of uninfected astrocytes that do not have the protection provided by HIV. These mechanisms assure the perpetuation of HIV CNS reservoirs and result in amplification of toxicity in uninfected cells. The crosses in the cartoon denote the potential target pathways for the virus to maintain the survival of the infected astrocytes.