Neonatal intrahippocampal HIV-1 protein Tat(1-86) injection: neurobehavioral alterations in the absence of increased inflammatory cytokine activation

Abstract

Pediatric AIDS caused by human immunodeficiency virus type 1 (HIV-1) remains one of the leading worldwide causes of childhood morbidity and mortality. HIV-1 proteins, such as Tat and gp120, are believed to play a crucial role in the neurotoxicity of pediatric HIV-1 infection. Detrimental effects on development, behavior, and neuroanatomy follow neonatal exposure to the HIV-1 viral toxins Tat1-72 and gp120. The present study investigated the neurobehavioral effects induced by the HIV-1 neurotoxic protein Tat1-86, which encodes the first and second exons of the Tat protein. In addition, the potential effects of HIV-1 toxic proteins Tat1-86 and gp120 on inflammatory pathways were examined in neonatal brains. Vehicle, 25 μg Tat1-86 or 100 ng gp120 was injected into the hippocampus of male Sprague-Dawley pups on postnatal day 1 (PD1). Tat1-86 induced developmental neurotoxic effects, as witnessed by delays in eye opening, delays in early reflex development and alterations in prepulse inhibition (PPI) and between-session habituation of locomotor activity. Overall, the neurotoxic profile of Tat1-86 appeared more profound in the developing nervous system in vivo relative to that seen with the first exon encoded Tat1-72 (Fitting et al., 2008b), as noted on measures of eye opening, righting reflex, and PPI. Neither the direct PD1 CNS injection of the viral HIV-1 protein variant Tat1-86, nor the HIV-1 envelope protein gp120, at doses sufficient to induce neurotoxicity, necessarily induced significant expression of the inflammatory cytokine IL-1β or inflammatory factors NF-κβ and I-κβ. The findings agree well with clinical observations that indicate delays in developmental milestones of pediatric HIV-1 patients, and suggest that activation of inflammatory pathways is not an obligatory response to viral protein-induced neurotoxicity that is detectable with behavioral assessments. Moreover, the amino acids encoded by the second tat exon may have unique actions on the developing hippocampus.

Keywords: Cytokines; Developmental delay; HIV-1; Neurotoxicity; Tat(1–86); gp120.

Figure 1

Mean (± SEM) body weight across the various test days by treatment. No significant treatment effect or interaction between treatment and day was noted. RR = righting reflex; NG = negative geotaxis; PPI = prepulse inhibition; LA= locomotor activity.

Figure 2

Mean (± SEM) response time with the best fit linear regression across three days for each treatment group. (A) A significant treatment effect was detected for righting reflex across all test days (PD3-5) (p ≤ 0.05); Tat_1-86 -treated animals were much slower in the righting reflex compared to the VEH-treated group. A significant test day effect was found (p ≤ 0.05) with a prominent linear component (p ≤ 0.05), indicating that execution of the righting reflex improved across test days. Planned contrast analyses for each treatment group revealed a significant linear component for the VEH-treated animals (p ≤ 0.05) which was not found in the Tat_1-86-treated group. Thus, the Tat_1-86 treatment appeared to disrupt maturation of the righting reflex across test days. (B) A significant overall treatment effect was detected for negative geotaxis across all test days (PD8-10) (p ≤ 0.05); Tat_1-86 -treated animals had overall slower response latencies compared to VEH-treated animals. A significant day effect was found (p ≤ 0.05) with a prominent linear component (p ≤ 0.01), indicating that execution of the righting reflex improved across test days. No treatment x test day interaction was detected, suggesting that both groups showed a similar rate of improvement across test days, approximating similar terminal levels of acquisition.

Figure 3

Mean (± SEM) peak ASR amplitude by treatment across ISIs (0–4000 ms). No significant treatment effect was found on control trials (0 and 4000 ms ISIs), suggesting that Tat_1-86 did not affect the animals’ baseline startle. Both groups displayed peak inhibition at the 80 ms ISI. The characteristic quadratic fit of the PPI ISI function for the VEH-treated animals was shifted in the Tat_1-86 -treated animals to also include a prominent linear component to the PPI ISI function.

Figure 4

Mean (± SEM) total ambulation for each treatment group across the three test days (PD26-28). Between-session habituation was confirmed by a significant effect of test day with a significant linear decrease across test day (p ≤ 0.005) for the VEH-treated animals; whereas the between-session habituation of the Tat-treated animals was best represented by a quadratic orthogonal component (p ≤ 0.03). The Tat_1-86 -treated group demonstrated significantly greater ambulation on the third session compared to the VEH-treated group (p ≤ 0.05).

Figure 5

Mean (± SEM) optical density ratio values of biomarkers reflecting the potential activation of inflammatory pathways as a function of in vivo neonatal Tat or gp120 treatment. Statistical analysis failed to detect any significant increase in the expression of IL-1β, NFκ-β, or Iκ-β in Tat_1-86 -treated (non-significant % increases of 17.9 %, 4.5 %, and 5.3 %, respectively) or gp120-treated (all values within 2 % of control) animals.