Fig 1.
Hippocampal degeneration in HIV-1 Tg rats.
(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.
Fig 2.
Increased apoptosis of neuronal cells in HIV-1 Tg rats.
(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, n = 4/group. *P < 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.
Fig 3.
Decreased neuronal cells with increased astrocytes in HIV-1 Tg rats.
(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. *P < 0.05.
Fig 4.
Increased production of amyloid plaques and C-terminal C99 fragment in the hippocampus of HIV-1 Tg rats.
(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 [25, 26] 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. *P < 0.05.
Fig 5.
Increased tau hyperphosphorylation in the hippocampus of HIV-1 Tg rats.
(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. *P < 0.05.
Fig 6.
Activation of different protein kinases and JNK-mediated tau phosphorylation in the hippocampus of HIV-transgenic rats.
(A, B) Representative immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the specific antibody to p-CDK5-Tyr15, CDK5, p-GSK3αβ-Ser9, GSK3αβ, p-JNK, JNK, p-p38K, p38K, or GAPDH, as indicated, are presented. Densitometric results for the immunoblots (n = 4/group) are presented. *P < 0.05. (C) Representative immunoblots of immunoprecipitated tau from WT and HIV-1 Tg rats with the specific antibody to p-JNK, p-tau-Ser396, p-tau-Thr231, or total tau (t-tau) are shown to demonstrate p-JNK binding to tau and hyperphosphorylation of indicated amino acids of tau in HIV-1 Tg rats.
Fig 7.
Increased proinflammatory cytokines in the hippocampus of HIV-1 Tg rats.
(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), *P < 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), *P < 0.05.
Fig 8.
Increased oxidative stress, NADPH oxidase and CYP2E1 in the hippocampus of HIV-1 Tg rats.
(A, B) Plasma ROS levels (A) and hippocampal NADPH oxidase activity in WT and HIV-1 Tg rats (n = 4/group), *P < 0.05. (C) Representative immunoblots of hippocampal lysates from WT and HIV-1 Tg rats with the specific antibody to CYP2E1, CYP4A, or GAPDH, as indicated. Densitometric quantitation of each indicated protein relative to GAPDH is also shown (n = 4/group), *P < 0.05.
Fig 9.
Increased nitroxidative stress marker proteins in the hippocampus of HIV-1 Tg rats.
(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). *P < 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.
Fig 10.
Causal role of increased nitroxidative stress in neuronal cell death exposed to recombinant Tat or gp120 protein.
(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.
Fig 11.
Schematic mechanisms for increased neurodegeneration in HIV-1 Tg rats.