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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-inhibited autophagyAutophagy was estimated by measuring protein levels of LC3, Atg5 and p62. (A) HIV-1 Tat (100 nM) decreased significantly protein levels of LC3. (B) HIV-1 Tat (100 nM) reduced significantly protein levels of Atg5. (C) HIV-1 Tat (100 nM) increased significantly protein levels of p62. Representative Western blots and quantitative data from each of proteins are shown, and actin was used as a loading control (n = 4).
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Figure 6: HIV-1 Tat-inhibited autophagyAutophagy was estimated by measuring protein levels of LC3, Atg5 and p62. (A) HIV-1 Tat (100 nM) decreased significantly protein levels of LC3. (B) HIV-1 Tat (100 nM) reduced significantly protein levels of Atg5. (C) HIV-1 Tat (100 nM) increased significantly protein levels of p62. Representative Western blots and quantitative data from each of proteins are shown, and actin was used as a loading control (n = 4).

Mentions: Endolysosomes also function to control autophagy, a process important for normal physiological functions of neurons. Dysfunctions in autophagy have been implicated in the pathogenesis of a variety of neurodegenerative disorders (Wong and Cuervo, 2010) including HAND (Alirezaei et al., 2008b; Alirezaei et al., 2008a; Spector and Zhou, 2008; Zhou and Spector, 2008; Zhu et al., 2009; Zhou et al., 2011). Based on the findings that HIV-1 Tat disrupts autophagy in immune cells (Van Grol et al., 2010), we determined the extent to which HIV-1 Tat-affected autophagy in primary cultured hippocampal neurons. Three markers were used to evaluate the status of autophagy; MAP1 LC3 which regulates the initiation process of autophagy, Atg5 which regulates the elongation process of autophagy, and p62 which inhibits autophagy. We found that HIV-1 Tat treatment decreased significantly protein levels of LC3 (Figure 6A, n = 4, P<0.05 at 2 day treatment) and Atg5 (Figure 6B, n = 4, P<0.05 at 1 day and P<0.01 at 2 days treatment), but increased significantly protein levels of p62 (Figure 6C, n = 4, P<0.05 at 1 and 2 days treatment).


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

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

HIV-1 Tat-inhibited autophagyAutophagy was estimated by measuring protein levels of LC3, Atg5 and p62. (A) HIV-1 Tat (100 nM) decreased significantly protein levels of LC3. (B) HIV-1 Tat (100 nM) reduced significantly protein levels of Atg5. (C) HIV-1 Tat (100 nM) increased significantly protein levels of p62. Representative Western blots and quantitative data from each of proteins are shown, and actin was used as a loading control (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: HIV-1 Tat-inhibited autophagyAutophagy was estimated by measuring protein levels of LC3, Atg5 and p62. (A) HIV-1 Tat (100 nM) decreased significantly protein levels of LC3. (B) HIV-1 Tat (100 nM) reduced significantly protein levels of Atg5. (C) HIV-1 Tat (100 nM) increased significantly protein levels of p62. Representative Western blots and quantitative data from each of proteins are shown, and actin was used as a loading control (n = 4).
Mentions: Endolysosomes also function to control autophagy, a process important for normal physiological functions of neurons. Dysfunctions in autophagy have been implicated in the pathogenesis of a variety of neurodegenerative disorders (Wong and Cuervo, 2010) including HAND (Alirezaei et al., 2008b; Alirezaei et al., 2008a; Spector and Zhou, 2008; Zhou and Spector, 2008; Zhu et al., 2009; Zhou et al., 2011). Based on the findings that HIV-1 Tat disrupts autophagy in immune cells (Van Grol et al., 2010), we determined the extent to which HIV-1 Tat-affected autophagy in primary cultured hippocampal neurons. Three markers were used to evaluate the status of autophagy; MAP1 LC3 which regulates the initiation process of autophagy, Atg5 which regulates the elongation process of autophagy, and p62 which inhibits autophagy. We found that HIV-1 Tat treatment decreased significantly protein levels of LC3 (Figure 6A, n = 4, P<0.05 at 2 day treatment) and Atg5 (Figure 6B, n = 4, P<0.05 at 1 day and P<0.01 at 2 days treatment), but increased significantly protein levels of p62 (Figure 6C, n = 4, P<0.05 at 1 and 2 days treatment).

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