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Role of endolysosomes in HIV-1 Tat-induced neurotoxicity.

Hui L, Chen X, Haughey NJ, Geiger JD - ASN Neuro (2012)

Bottom Line: Combined anti-retroviral therapeutic drugs effectively increase the lifespan of HIV-1-infected individuals who then have a higher prevalence of HAND (HIV-1 associated neurocognitive disorder).As early as 24 h after HIV-1 Tat was applied to neurons, HIV-1 Tat accumulated in endolysosomes, endolysosome morphology was affected and their size increased, endolysosome membrane integrity was disrupted, endolysosome pH increased, specific activities of endolysosome enzymes decreased and autophagy was inhibited, as indicated by the significant changes in three markers for autophagy.In contrast, statistically significant levels of HIV-1 Tat-induced neuronal cell death were observed only after 48 h of HIV-1 Tat treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203, USA.

ABSTRACT
Combined anti-retroviral therapeutic drugs effectively increase the lifespan of HIV-1-infected individuals who then have a higher prevalence of HAND (HIV-1 associated neurocognitive disorder). Soluble factors including HIV-1 proteins released from HIV-1-infected cells have been implicated in the pathogenesis of HAND, and particular attention has been paid to the HIV-1 Tat (transactivator of transcription) protein because of its ability to directly excite neurons and cause neuronal cell death. Since HIV-1 Tat enters cells by receptor-mediated endocytosis and since endolysosomes play an important role in neuronal cell life and death, we tested here the hypothesis that HIV-1 Tat neurotoxicity is associated with changes in the endolysosome structure and function and also autophagy. Following the treatment of primary cultured rat hippocampal neurons with HIV-1 Tat or as controls mutant-Tat or PBS, neuronal viability was determined using a triple staining method. Preceding observations of HIV-1 Tat-induced neuronal cell death, we observed statistically significant changes in the structure and membrane integrity of endolysosomes, endolysosome pH and autophagy. As early as 24 h after HIV-1 Tat was applied to neurons, HIV-1 Tat accumulated in endolysosomes, endolysosome morphology was affected and their size increased, endolysosome membrane integrity was disrupted, endolysosome pH increased, specific activities of endolysosome enzymes decreased and autophagy was inhibited, as indicated by the significant changes in three markers for autophagy. In contrast, statistically significant levels of HIV-1 Tat-induced neuronal cell death were observed only after 48 h of HIV-1 Tat treatment. Our findings suggest that endolysosomes are involved in HIV-1 Tat-induced neurotoxicity and may represent a target for therapeutic intervention against HAND.

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HIV-1 Tat-altered the structure of neuronal endolysosomes(A) HIV-1 Tat is accumulated in endolysosomes of primary cultured neurons. FITC-labelled Tat47-57 peptide (100 nM, green) was co-localized with endolysosomes (red, LysoTracker) and DAPI (4′,6-diamidino-2-phenylindole; blue) was used for staining nuclei. Bar = 50 μm. (B) Live cell imaging showed that HIV-1 Tat (100 nM) treatment increased the sizes of neuronal endolysosomes. LysoTracker (red) was used to identify endolysosomes, and calcein AM (green) was used to stain living cells (top panel), and the sizes of endolysosomes were quantified with Image J software. HIV-1 Tat-enlarged endosomes as identified with EEA1 staining (middle panel). HIV-1 Tat-enlarged lysosomes as identified with LAMP1 staining (bottom panel). (C) Quantification of the top panel of (B) showed that HIV-1 Tat (100 nM) treatment for 1 and 2 days increased significantly the size of neuronal endolysosomes (n = 15).
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Figure 2: HIV-1 Tat-altered the structure of neuronal endolysosomes(A) HIV-1 Tat is accumulated in endolysosomes of primary cultured neurons. FITC-labelled Tat47-57 peptide (100 nM, green) was co-localized with endolysosomes (red, LysoTracker) and DAPI (4′,6-diamidino-2-phenylindole; blue) was used for staining nuclei. Bar = 50 μm. (B) Live cell imaging showed that HIV-1 Tat (100 nM) treatment increased the sizes of neuronal endolysosomes. LysoTracker (red) was used to identify endolysosomes, and calcein AM (green) was used to stain living cells (top panel), and the sizes of endolysosomes were quantified with Image J software. HIV-1 Tat-enlarged endosomes as identified with EEA1 staining (middle panel). HIV-1 Tat-enlarged lysosomes as identified with LAMP1 staining (bottom panel). (C) Quantification of the top panel of (B) showed that HIV-1 Tat (100 nM) treatment for 1 and 2 days increased significantly the size of neuronal endolysosomes (n = 15).

Mentions: In neurons and other cells, HIV-1 Tat uses receptor-mediated endocytotic mechanisms (Liu et al., 2000; Vendeville et al., 2004; King et al., 2006) to enter cells where HIV-1 Tat accumulates first in endolysosomes. Since the basic region of amino acids 49–57 of HIV-1 Tat is required for binding to membrane receptor proteins (Sabatier et al., 1991) and exerting its neurotoxic effects (Weeks et al., 1995), we used a FITC-labelled Tat47–57 to determine first the extent to which HIV-1 Tat accumulated in endolysosomes in neurons. After incubating neurons with the FITC-labelled HIV-1 Tat47–57 (FITC-Tat), we observed significant intracellular accumulation of FITC-Tat, which was mainly compartmentalized in endolysosomes as identified with LysoTracker dye (red, Figure 2A).


Role of endolysosomes in HIV-1 Tat-induced neurotoxicity.

Hui L, Chen X, Haughey NJ, Geiger JD - ASN Neuro (2012)

HIV-1 Tat-altered the structure of neuronal endolysosomes(A) HIV-1 Tat is accumulated in endolysosomes of primary cultured neurons. FITC-labelled Tat47-57 peptide (100 nM, green) was co-localized with endolysosomes (red, LysoTracker) and DAPI (4′,6-diamidino-2-phenylindole; blue) was used for staining nuclei. Bar = 50 μm. (B) Live cell imaging showed that HIV-1 Tat (100 nM) treatment increased the sizes of neuronal endolysosomes. LysoTracker (red) was used to identify endolysosomes, and calcein AM (green) was used to stain living cells (top panel), and the sizes of endolysosomes were quantified with Image J software. HIV-1 Tat-enlarged endosomes as identified with EEA1 staining (middle panel). HIV-1 Tat-enlarged lysosomes as identified with LAMP1 staining (bottom panel). (C) Quantification of the top panel of (B) showed that HIV-1 Tat (100 nM) treatment for 1 and 2 days increased significantly the size of neuronal endolysosomes (n = 15).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3379000&req=5

Figure 2: HIV-1 Tat-altered the structure of neuronal endolysosomes(A) HIV-1 Tat is accumulated in endolysosomes of primary cultured neurons. FITC-labelled Tat47-57 peptide (100 nM, green) was co-localized with endolysosomes (red, LysoTracker) and DAPI (4′,6-diamidino-2-phenylindole; blue) was used for staining nuclei. Bar = 50 μm. (B) Live cell imaging showed that HIV-1 Tat (100 nM) treatment increased the sizes of neuronal endolysosomes. LysoTracker (red) was used to identify endolysosomes, and calcein AM (green) was used to stain living cells (top panel), and the sizes of endolysosomes were quantified with Image J software. HIV-1 Tat-enlarged endosomes as identified with EEA1 staining (middle panel). HIV-1 Tat-enlarged lysosomes as identified with LAMP1 staining (bottom panel). (C) Quantification of the top panel of (B) showed that HIV-1 Tat (100 nM) treatment for 1 and 2 days increased significantly the size of neuronal endolysosomes (n = 15).
Mentions: In neurons and other cells, HIV-1 Tat uses receptor-mediated endocytotic mechanisms (Liu et al., 2000; Vendeville et al., 2004; King et al., 2006) to enter cells where HIV-1 Tat accumulates first in endolysosomes. Since the basic region of amino acids 49–57 of HIV-1 Tat is required for binding to membrane receptor proteins (Sabatier et al., 1991) and exerting its neurotoxic effects (Weeks et al., 1995), we used a FITC-labelled Tat47–57 to determine first the extent to which HIV-1 Tat accumulated in endolysosomes in neurons. After incubating neurons with the FITC-labelled HIV-1 Tat47–57 (FITC-Tat), we observed significant intracellular accumulation of FITC-Tat, which was mainly compartmentalized in endolysosomes as identified with LysoTracker dye (red, Figure 2A).

Bottom Line: Combined anti-retroviral therapeutic drugs effectively increase the lifespan of HIV-1-infected individuals who then have a higher prevalence of HAND (HIV-1 associated neurocognitive disorder).As early as 24 h after HIV-1 Tat was applied to neurons, HIV-1 Tat accumulated in endolysosomes, endolysosome morphology was affected and their size increased, endolysosome membrane integrity was disrupted, endolysosome pH increased, specific activities of endolysosome enzymes decreased and autophagy was inhibited, as indicated by the significant changes in three markers for autophagy.In contrast, statistically significant levels of HIV-1 Tat-induced neuronal cell death were observed only after 48 h of HIV-1 Tat treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203, USA.

ABSTRACT
Combined anti-retroviral therapeutic drugs effectively increase the lifespan of HIV-1-infected individuals who then have a higher prevalence of HAND (HIV-1 associated neurocognitive disorder). Soluble factors including HIV-1 proteins released from HIV-1-infected cells have been implicated in the pathogenesis of HAND, and particular attention has been paid to the HIV-1 Tat (transactivator of transcription) protein because of its ability to directly excite neurons and cause neuronal cell death. Since HIV-1 Tat enters cells by receptor-mediated endocytosis and since endolysosomes play an important role in neuronal cell life and death, we tested here the hypothesis that HIV-1 Tat neurotoxicity is associated with changes in the endolysosome structure and function and also autophagy. Following the treatment of primary cultured rat hippocampal neurons with HIV-1 Tat or as controls mutant-Tat or PBS, neuronal viability was determined using a triple staining method. Preceding observations of HIV-1 Tat-induced neuronal cell death, we observed statistically significant changes in the structure and membrane integrity of endolysosomes, endolysosome pH and autophagy. As early as 24 h after HIV-1 Tat was applied to neurons, HIV-1 Tat accumulated in endolysosomes, endolysosome morphology was affected and their size increased, endolysosome membrane integrity was disrupted, endolysosome pH increased, specific activities of endolysosome enzymes decreased and autophagy was inhibited, as indicated by the significant changes in three markers for autophagy. In contrast, statistically significant levels of HIV-1 Tat-induced neuronal cell death were observed only after 48 h of HIV-1 Tat treatment. Our findings suggest that endolysosomes are involved in HIV-1 Tat-induced neurotoxicity and may represent a target for therapeutic intervention against HAND.

Show MeSH
Related in: MedlinePlus