Insulin Treatment Prevents Neuroinflammation and Neuronal Injury with Restored Neurobehavioral Function in Models of HIV/AIDS Neurodegeneration

Abstract

HIV-1 infection of the brain causes the neurodegenerative syndrome HIV-associated neurocognitive disorders (HAND), for which there is no specific treatment. Herein, we investigated the actions of insulin using ex vivo and in vivo models of HAND. Increased neuroinflammatory gene expression was observed in brains from patients with HIV/AIDS. The insulin receptor was detected on both neurons and glia, but its expression was unaffected by HIV-1 infection. Insulin treatment of HIV-infected primary human microglia suppressed supernatant HIV-1 p24 levels, reduced CXCL10 and IL-6 transcript levels, and induced peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. Insulin treatment of primary human neurons prevented HIV-1 Vpr-mediated cell process retraction and death. In feline immunodeficiency virus (FIV) infected cats, daily intranasal insulin treatment (20.0 IU/200 μl for 6 weeks) reduced CXCL10, IL-6, and FIV RNA detection in brain, although PPAR-γ in glia was increased compared with PBS-treated FIV+ control animals. These molecular changes were accompanied by diminished glial activation in cerebral cortex and white matter of insulin-treated FIV+ animals, with associated preservation of cortical neurons. Neuronal counts in parietal cortex, striatum, and hippocampus were higher in the FIV+/insulin-treated group compared with the FIV+/PBS-treated group. Moreover, intranasal insulin treatment improved neurobehavioral performance, including both memory and motor functions, in FIV+ animals. Therefore, insulin exerted ex vivo and in vivo antiviral, anti-inflammatory, and neuroprotective effects in models of HAND, representing a new therapeutic option for patients with inflammatory or infectious neurodegenerative disorders including HAND.

Significance statement: HIV-associated neurocognitive disorders (HAND) represent a spectrum disorder of neurocognitive dysfunctions resulting from HIV-1 infection. Although the exact mechanisms causing HAND are unknown, productive HIV-1 infection in the brain with associated neuroinflammation is a potential pathogenic mechanism resulting in neuronal damage and death. We report that, in HIV-infected microglia cultures, insulin treatment led to reduced viral replication and inflammatory gene expression. In addition, intranasal insulin treatment of experimentally feline immunodeficiency virus-infected animals resulted in improved motor and memory performances. We show that insulin restored expression of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which is suppressed by HIV-1 replication. Our findings indicate a unique function for insulin in improving neurological outcomes in lentiviral infections, implicating insulin as a therapeutic intervention for HAND.

Keywords: FIV; HIV-1; insulin; microglia; neuropathology; neurotoxicity.

Copyright © 2016 the authors 0270-6474/16/3610683-13$15.00/0.

Figures

Figure 1.

Expression of proinflammatory genes in HIV-infected human brain white matter. Analyses of transcript levels in frontal white matter disclosed that HLA-DQA1 (A), IL-6 (B), and CXCL10 (C) were increased in the HIV+ group, whereas PPAR-γ (D), IR (E), and IGF-1R (F) were similar in HIV-1-infected samples (HIV+, n = 6) compared with uninfected control samples (HIV−, n = 6), as measured by real-time PCR normalized to GAPDH and expressed as relative fold change (RFC). Immunohistochemical detection in HIV+ and HIV− frontal lobe brain sections showed that MHC Class II on macrophages/microglia (G) and IL-6 on glia (H) were increased in HIV+ brain sections, but PPAR-γ (I), detected in glial cytoplasm and nuclei, was reduced in HIV+ sections, whereas IR-β immunoreactivity (J), detected on neurons and glia, was similar in HIV− and HIV+ brain sections. Data represent mean ± SEM (Student's t test, *p < 0.05). Original magnification, 200×; scale bar, 20 μm.

Figure 2.

Insulin inhibits HIV-1 replication in primary human cells. HIV-1 p24 levels were measured by ELISA in supernatants collected from HIV-1-infected PBMCs (day 4 after infection; A) exposed to different concentrations of insulin (IU/ml). AZT (zidovudine) was used as a positive treatment control. Both insulin and AZT exerted antiviral effects. B, p24 levels in supernatants from HIV-infected primary human microglia were reduced with insulin treatment (day 5 after infection). C, Supernatant p24 levels from VSV-pseudotyped HIV-1 of primary human microglia were reduced by insulin treatment (1.0 IU/ml, 1 h before transfection). D, Reverse transcriptase activity was measured in FIV-infected lymphoid cells treated with different concentrations of insulin (IU/ml) (day 4 after infection). Data represent mean ± SEM with a minimum of three replicates per group (one-way ANOVA, post hoc Tukey's test, *p < 0.05).

Figure 3.

Expression of proinflammatory and viral genes in HIV-infected human microglia treated with insulin. Primary human microglia were infected with HIV-1YU2, followed by insulin treatment (1.0 IU/ml). Analyses of expression revealed that insulin treatment suppressed HIV pol (A), HIV vpr (B), IL-6 (C), and CXCL10 (D), but not IL-1β (E), whereas insulin treatment (F) increased PPAR-γ expression in HIV-infected microglia measured in real-time PCR (day 5 after infection). Data represent mean ± SEM with a minimum of three replicates per group (one-way ANOVA, post hoc Tukey's test, *p < 0.05).

Figure 4.

Insulin protects primary human neurons against Vpr-induced neurotoxicity. Primary human neurons were exposed to HIV-1 Vpr (100 nm), followed by treatment with insulin. A, MAP-2 immunolabeling (green) merged with DAPI nuclear staining (blue) of healthy neurons (A1) showed a loss of neuronal processes and nuclei with Vpr exposure (A2) that were rescued by insulin treatment at 1.0 IU/ml (A3) and 3.0 IU/ml (A4). B, Class III β-tubulin (βIII-tubulin) expression measured by in-cell Western assay was diminished in Vpr-exposed neurons, but preserved by insulin treatment in a concentration-dependent manner. C, Similarly, neuronal nuclear DAPI staining measured by florescent quantitative analysis at 48 h after treatment also showed a loss of nuclei with Vpr exposure that was prevent by insulin treatment. Values were normalized to background signals and are expressed as the percentage increase relative to control cells. Data represent mean ± SEM with a minimum of six replicates per group (one-way ANOVA, post hoc Tukey's test, ***p < 0.01). Original magnification, 200×.

Figure 5.

Expression of proinflammatory genes in feline cortex (CTX) and striatum (ST). FIV+ animals were treated with intranasal insulin or PBS for 6 weeks and compared with uninfected FIV− animals. Examination of transcript levels showed that IL-6 (A) and CXCL10 (B) were suppressed in cortex by intranasal insulin treatment in FIV+ animals, whereas MX1 (C) and IL-1β (D) were induced in both CTX and ST but were unaffected by intranasal insulin treatment. E, PPAR-γ expression was induced, but FIV pol (F) was suppressed in cortex of FIV+ animals afater intranasal insulin treatment. Data represent mean ± SEM (one-way ANOVA, post hoc Tukey's test, *p < 0.05).

Figure 6.

Neuropathological analyses of FIV-infected and uninfected animals. Immunohistochemical studies indicated that, in representative brain sections Iba-1 in cortical microglia (A, arrowheads) and IL-6 in white matter glia (B, arrowheads) displayed reduced immunoreactivity with insulin treatment of FIV+ animals. FIV p24 immunoreactvity was detected in cells resembling microglia (A2, inset), whereas IL-6 immunodetection was evident in cells resembling astrocytes (B2, inset). In contrast, PPAR-γ immunoreactivity (C) was increased in cortical glia (arrowheads) in both cytoplasm and nuclei (C1, inset) from FIV+ animals treated with intranasal insulin. D, MAP-2 immunoreactivity was apparent in cortical neuronal cell bodies and processes, but it expression was increased by intranasal insulin treatment of FIV+ animals compared with PBS-treated animals. Original magnification: A–C, 20×; D, 40×. Scale bar, 20 μm.

Figure 7.

Intranasal insulin protects neurons in vivo. A, cresyl violet (Nissl) staining in FIV−, FIV+/PBS-treated, and FIV+/insulin-treated animals revealed that fewer nucleolated neurons (arrows) in the middle suprasylvian gyrus were detected in FIV+/PBS-treated animals. Neurons with nucleoli were counted in the cortex (suprasylvian gyrus; B), striatum (caudate; C), and CA1 region (D) of the hippocampus. Mean neuronal counts (B, C, cell number/1.0 mm2 low-power field, lpf, and D, cell number/0.25 mm2 high-power field, hpf) were lower in each anatomic site among PBS-treated FIV+ animals compared with FIV− animals, but intranasal insulin treatment prevented neuronal loss in all brain regions. Data represent mean ± SEM (one-way ANOVA, post hoc Tukey's test, *p < 0.05).

Figure 8.

Insulin improves neurobehavioral outcomes in FIV-infected animals. Analyses of neurobehavioral functions in FIV− (n = 5), FIV+/PBS-treated (n = 6), and FIV+/insulin-treated (n = 6) animals showing improved neurobehavioral performance with insulin treatment, as assessed by maze completion time (A) and gait variance (B). Insulin treatment improved decision making and motor memory in FIV+ animals, as assessed by maze errors (C) and object memory task (D). Data represent mean ± SEM (one-way ANOVA, post hoc Tukey's test, *p < 0.05).