Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory

Abstract

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment.

Keywords: Astrocyte; CCL2; CD163; Cognition; HAND.

Figure 1

Nef is expressed for seven days after transfection of primary rat astrocytes in vitro and in vivo from infused cells. (A) Primary culture composition was assessed for astrocytes (GFAP) and microglia (Iba1/2). Western blot of three, independent primary cell extractions (lanes 1–3) and SVGA astrocytes (lane 4) show positive for GFAP and negative for Iba1/2. Positive control for microglia in whole brain (WB) tissue shows positive reaction with Iba1/2. Molecular weight markers are labeled (STD). In the third panel, immunofluorescence for GFAP (green) and Iba1/2 (red) show cultures are astrocytes. DAPI was used for nuclear counterstaining. (B) Cells and culture supernatants were collected each day for seven days after transfection with Nef plasmid. Western blots, lower part, measured Nef protein expression. Densitometry of western blots was used to quantify (upper part) Nef from cell lysates and supernatants from the same cell cultures. Concentrations of Nef were estimated using a Nef protein standard (refer to methods). Actin served as a loading control for cell lysates. Immunofluorescence indicates Nef (red) expression from GFAP positive (green) astrocytes. (C) Immunostaining of hippocampal tissue proximal to infusion site: left to right, DAPI, GFAP (green), and Nef (red) in split channel, and merged image at seven days post-surgery. Scale bars = 25μM.

Figure 2

Nef expression unilaterally in the hippocampus causes learning impairment in spatial and non-spatial memory. (A) A schematic chart of the novel location recognition (NLR) and novel object recognition (NOR) tasks. Average exploration time of rats from the three groups (Naïve, n=8; GFP, n=12; or Nef, n=14): (B) for each object during acclimation; (C) for the test object prior to (open bars) and after moving the location (closed bars); and (D) for the test object prior to (open bars) and after replacing with a novel object (closed bars). All error bars represent standard error. *p<0.05; **p<0.005.

Figure 3

Neither Nef exposure nor surgery caused widespread behavioral changes. Locomotor and anxiety behavior for each group during the acclimation phase are presented (mean plus standard error) as representative data. (A) Total distance traveled and (B) velocity were assessed for locomotor function. (C) Time spent in the center served as a measure of anxiety.

Figure 4

Rats in the Nef group had fewer hippocampal neurons in CA3. (A) Neurons were detected by Nissl staining. Representative right hippocampal sections at 100x (left) and 400x (right) magnification from rats implanted with astrocytes expressing GFP or Nef. CA3 panels indicate region counted for lower graph. (B) Neurons were counted in CA3 and mean (plus standard error) counts for Nef and GFP groups were calculated in both hemispheres. Graph shows average number of neurons per animal per CA3 region. *p<0.05, **p<0.01. Scale bars = 100μM.

Figure 5

The Nef group shows CCL2 induction and enhanced infiltration of CD163-positive macrophages proximal to injection site compared to GFP controls. Real time, quantitative RT-PCR was used to measure CCL2 expression in: (A) cultures of transfected primary astrocytes expressing GFP (open bars) or Nef (closed bars) or (B) hippocampal tissue implanted with GFP-expressing (open bars) or Nef-expressing (closed bars) astrocytes. Data are presented for both non-infused (left) and infused (right) hemispheres. (C) Representative tissue sections immunostained for macrophage infiltration using the CD163 marker, GFP group (top) and Nef group (bottom) at 100x (left) and 400x (right) magnification. (D) Densitometric analysis was used to quantify CD163 staining. Scatter plot shows mean (+/− standard error) for the GFP (open circles) and Nef closed circles) groups. Scale bar = 100μM. *p<0.05; **p<0.005.

Figure 6

Proposed components of learning impairment caused by astrocytic Nef expression. Nef protein is produced in astrocytes (1) resulting in induction of CCL2 (2). The secreted CCL2 serves as a powerful attractant to peripheral monocytes that migrate to the brain (2a) and differentiate to macrophages expressing CD163 (2b). These macrophages contribute to the local inflammatory environment resulting in the loss of neurons (3). Nef released from astrocytes (4) may compromise the blood brain barrier (4a) and cause direct neurotoxicity (4b). Finally, Nef production in astrocytes can promote release of proinflammatory molecules (5) further contributing to the neurotoxic environment. The loss of neuron numbers and function results in learning impairment. Solid lines indicate findings from this study; dashed lines are based on other published data.