Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats

<div><p>The underlying mechanisms for increased neurodegeneration and neurocognitive deficits in HIV-infected people are unclear. Therefore, this study was aimed to investigate the mechanisms of increased neurodegeneration in 5-month old male HIV-1 Transgenic (Tg) rats compared to the age- and gender-matched wild-type (WT) by evaluating histological changes and biochemical parameters of the key proteins involved in the cell death signaling and apoptosis. Histological and immunohistochemical analyses revealed decreased neuronal cells with elevated astrogliosis in HIV-1 Tg rats compared to WT. Mechanistic studies revealed that increased levels of nitroxidative stress marker proteins such as NADPH-oxidase, cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), the stress-activated mitogen-activated protein kinases such as JNK and p38K, activated cell-cycle dependent CDK5, hypoxia-inducible protein-1α, nitrated proteins, hyperphosphorylated tau, and amyloid plaques in HIV-Tg rats were consistently observed in HIV-1 Tg rats. Confocal microscopy and cell viability analyses showed that treatment with an antioxidant <i>N</i>-acetylcysteine or a specific inhibitor of iNOS 1400W significantly prevented the increased apoptosis of neuro-2A cells by HIV-1 Tat or gp120 protein, demonstrating the causal role of HIV-1 mediated nitroxidative stress and protein nitration in promoting neuronal cell death. Immunoprecipitation and immunoblot analysis confirmed nitration of Hsp90, evaluated as an example of nitrated proteins, suggesting possible involvement of nitrated proteins in neuronal damage. Further, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyperphosphorylation and tauopathy. These changes were accompanied with elevated levels of many apoptosis-related proteins Bax and cleaved (activated) caspase-3 as well as proinflammatory cytokines including TNF-α, IL-6 and MCP-1. Collectively, these results indicate that raised nitroxidative stress accompanied by elevated inflammation, cell death signaling pathway including activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyperphosphorylation are responsible for increased apoptosis of neuronal cells and neurodegeneration in 5-month old HIV-Tg rats.</p></div

Causal role of increased nitroxidative stress in neuronal cell death exposed to recombinant Tat or gp120 protein.

<p>(A-C) Representative images of confocal microscopy (A) of cleaved caspase-3, MTT assay (B) and immunoblot assay with anti-caspase-3 (C) to show the apoptosis rates of neuro-2A cells exposed to the recombinant HIV protein Tat or gp120 in the absence or presence of NAC or 1400W. (B) MTT cell viability analysis to determine the cell death rates of neuro-2A cells exposed to the recombinant Tat or gp120 in the absence or presence of 1 mM NAC or 20 μM 1400W. GADPH was used as a loading control.</p

Hippocampal degeneration in HIV-1 Tg rats.

<p>(A, B) Representative images of the coronal hippocampal CA1 region, CA3 region and dentate gyrus (DG) from WT or HIV-1 Tg rats stained with cresyl violet (A) or H&E (B) to show decreased neuronal cells in HIV-1 Tg rats. Scale bar, 200 μm. (C, D) Densitometric analyses for both stainings (n = 4/group) are presented below. Data are expressed as mean ± SD, n = 4/group. *P < 0.05.</p

Increased tau hyperphosphorylation in the hippocampus of HIV-1 Tg rats.

<p>(A) Immunoblots for detecting tau phosphorylation at indicated amino acids to show different levels of tau hyperphosphorylation in WT and HIV-1 Tg rats. (B) Densitometric results for the immunoblots (n = 4/group) are presented. *<i>P</i> < 0.05.</p

Increased nitroxidative stress marker proteins in the hippocampus of HIV-1 Tg rats.

<p>(A, B) Representative immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the specific antibody to 3-NT, iNOS, HIF1-α, BNIP3, p-IκB, IκB, or GAPDH, as indicated. Densitometric quantitation of each indicated protein relative to GAPDH is shown (n = 4/group). *<i>P</i> < 0.05. (C) Representative immunoblots of immunoprecipitated Hsp90 from WT and HIV-1 Tg rats with the specific antibody to 3-NT or HSP90 are shown to demonstrate nitration of Hsp90 in HIV-1 Tg rats.</p

Increased apoptosis of neuronal cells in HIV-1 Tg rats.

<p>(A) Representative immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the respective antibody to cleaved (activated) caspase-3, Bax, or GAPDH, used as a loading control, as indicated. Densitometric analysis of the immunoblots for caspase-3 or Bax relative to GAPDH is shown below. Data are expressed as mean ± SD, <i>n</i> = 4/group. *<i>P</i> < 0.05. (B) Representative IHC images of cleaved caspase-3 in the cortex and hippocampal CA1 region from WT and HIV-1 Tg rats are shown. Arrows represent positively-stained cells with anti-cleaved caspase-3 antibody.</p

Schematic mechanisms for increased neurodegeneration in HIV-1 Tg rats.

<p>Schematic mechanisms for increased neurodegeneration in HIV-1 Tg rats.</p

Decreased neuronal cells with increased astrocytes in HIV-1 Tg rats.

<p>(A, B) Representative images of the cortex, coronal hippocampal (CA1) region and dentate gyrus from WT or HIV-1 Tg rats stained with NeuN (A) or GFAP (B) to show decreased neuronal cells with increased astrocytes in HIV-1 Tg rats. Scale bar, 100 μm. (C) Immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the respective antibody to NeuN, GFAP, and GAPDH used as a loading control, are shown. Densitometric quantitation of the immunoblots for NeuN or GFAP relative to GAPDH (n = 4/group) is shown. *<i>P</i> < 0.05.</p

Increased proinflammatory cytokines in the hippocampus of HIV-1 Tg rats.

<p>(A) Representative immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the specific antibody to TNF-α, MCP-1, IL6, or GAPDH, as indicated. Densitometric quantitation of each indicated protein relative to GAPDH is shown (n = 4/group), *<i>P</i> < 0.05. (B) ELISA results for TNF-α and MCP-1 in the hippocampal lysates from WT and HIV-1 Tg rats are presented (n = 4/group), *<i>P</i> < 0.05.</p

Increased production of amyloid plaques and C-terminal C99 fragment in the hippocampus of HIV-1 Tg rats.

<p>(A) Representative images of the cerebral cortex region stained with Congo red (A) to show different levels of amyloid plaques in WT and HIV-1 Tg rats. Scale bar, 200 μm. (B) Representative immunoblots for detecting APP and toxic amyloid C-terminal C99 fragment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169945#pone.0169945.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169945#pone.0169945.ref026" target="_blank">26</a>] with the anti-CT19 antibody are shown. The densitometric results for the immunoblots (n = 4/group) are presented below. (C) Representative IHC images for β-amyloid in the cortex region from WT and HIV-1 Tg rat brains are shown. (D) Immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the respective antibody to β-amyloid or GAPDH used as a loading control, are shown. The densitometric results for the immunoblots (n = 4/group) are shown below. *<i>P</i> < 0.05.</p