Increased vulnerability of ApoE4 neurons to HIV proteins and opiates: Protection by diosgenin and l-deprenyl

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Abstract

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations and causes a dementing illness in a subset of individuals. Factors contributing to the development of dementia in this population remain unknown. We found that HIV-infected individuals with the E4 allele of Apolipoprotein E (ApoE) or history of intravenous drug abuse had increased oxidative stress in the CNS. In vitro studies showed that HIV proteins, gp120 and Tat, Tat + morphine but not tumor necrosis factor-α (TNF-α), caused increased neurotoxicity in human neuronal cultures with ApoE4 allele. Microarray analysis showed a differential alteration of transcripts involved in energy metabolism in cultures of ApoE3 and 4 neurons upon treatment with Tat + morphine. This was confirmed using assays of mitochondrial function and exposure of the neurons to Tat + morphine. Using this in vitro model, we screened a number of novel antioxidants and found that only l-deprenyl and diosgenin protected against the neurotoxicity of Tat + morphine. Furthermore, Tat-induced oxidative stress impaired morphine metabolism which could also be prevented by diosgenin. In conclusion, opiate abusers with HIV infection and the ApoE4 allele may be at increased risk of developing dementia. l-deprenyl and a plant estrogen, diosgenin, may have therapeutic potential in this population.

Introduction

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations (Davies et al., 1997), and some drug abusers with HIV infection have a severe encephalitis (Bell et al., 1998). Both drugs of abuse and HIV target the basal ganglia and cortex, areas rich in opioidergic receptors. Yet, only 20% of patients with HIV infection develop dementia and in some patients encephalitis may occur without dementia (Sacktor, 2002). These observations suggest that genetic factors may predispose to neuronal toxicity following HIV infection. The E4 allele of Apolipoprotein E (ApoE) has been associated with Alzheimer disease and with poor recovery following brain injury (Koponen et al., 2004) but not with Parkinson disease (Eerola et al., 2002) or Creutzfeldt–Jakob disease (Chapman et al., 1998). While ApoE4 was not associated with higher risk of developing multiple sclerosis, it was associated with disease activity and accumulation of disability (Fazekas et al., 2001). The role of ApoE in pathogenesis of HIV dementia (HIVD) remains unclear. In a previous cross-sectional study, no association between ApoE4 and HIVD was found (Dunlop et al., 1997); however, a subsequent longitudinal study found that the presence of ApoE4 allele resulted in a two-fold increase in the risk of developing HIVD (Corder et al., 1998). Recent studies from our laboratory suggest that HIVD patients with an ApoE4 genotype show a dysregulated lipid and sterol metabolism (Cutler et al., 2004). We have also shown that patients with HIVD have massive increases in oxidative stress as measured by immunostaining for lipid peroxidation product 4-hydroxynonenol (HNE) in brain tissue and by semiquantitative analysis of protein carbonyls in cerebrospinal fluid (CSF) (Turchan et al., 2003a). No information about drug-abusing populations was available from these studies, and they also did not determine if ApoE4 influenced the severity of dementia or oxidative stress in the central nervous system (CNS).

ApoE normally plays a role in the distribution of cholesterol for repairing nerve cells during development and after injury. There are three common isoforms of ApoE, namely, E2, E3, and E4, as a result of nucleic acid substitutions at codons 112 and 158. ApoE is a major serum lipoprotein involved in cholesterol metabolism. Lipoproteins containing ApoE4 are cleared more efficiently from blood than those containing ApoE3 and ApoE2. ApoE does not cross the blood–brain barrier but is synthesized in the brain by astrocytes and neurons. In the brain, astrocytes are the major source of ApoE which maintains lipid homeostasis in the central nervous system (Huang et al., 2004). In the brain, ApoE is thought to be involved in the mobilization and redistribution of cholesterol and phospholipid during membrane remodeling associated with plasticity and synaptogenesis. Neurite extension and branching are more extensive in ApoE3-treated cells compared with ApoE4-treated cells (Qiu et al., 2004).

To further investigate the role of ApoE in the pathogenesis of HIVD, we initially measured HNE levels by mass spectroscopy in autopsy brain tissue and CSF of patients characterized for ApoE alleles and history of intravenous drug abuse (IVDA) respectively. To confirm these observations and to determine the underlying mechanisms involved in the differential susceptibility of neurons of different ApoE alleles to the combined effects of HIV and drugs of abuse, we used an in vitro human brain culture model. We chose a human system because only humans and not rodents have all the ApoE alleles. Mixed human neuronal cultures derived from the different ApoE alleles were exposed to HIV proteins Tat and gp120 that have been previously implicated in HIV neuropathogenesis in combination with morphine which is the major metabolite of heroin. The cultures were compared by gene arrays and confirmed by neurotoxicity and other functional assays. Our observations suggest that the presence of ApoE4 allele predisposes neurons to toxicity via viral proteins and opiate drugs and suggest that the neurotoxicity is mediated by increased oxidative stress.

Section snippets

Patient populations

All human samples were obtained following approval of the Johns Hopkins University institutional review board for research on human subjects. Human brain tissues from the pre-highly active antiretroviral therapy (HAART) era (pre-1996) were obtained from the Johns Hopkins AIDS brain bank. All brain tissues used had histopathological evidence of encephalitis as indicated by the infiltration of macrophages or presence of multinucleated giant cells and were free of opportunistic infections as

Influence of ApoE allele on lipid peroxidation in brain of patients with HIV encephalitis

To determine if ApoE allele could influence the amount of oxidative stress in the brain of HIV-infected patients, we chose brain samples from patients with HIVD from the pre-HAART era where the course of dementia was subacute in all patients. We measured 4-HNE levels bound to lysine or histidine in extracts of autopsy brain tissue which is formed as a result of lipid peroxidation. Patients with ApoE4 allele had increased levels of HNE in the middle frontal gyrus, parietal lobe, and the

Discussion

Even though the HIV epidemic is being driven by drug abusers in North America and several other regions in the world, clinical studies to determine the combined effects of HIV and drugs of abuse have been few and hard to interpret. These patients are often polydrug abusers and have other comorbidities such as hepatitis C infection. Despite these caveats, a longitudinal study in HIV-infected drug abusers failed to show differences in cognitive function compared to the non-drug abusers (Concha et

Acknowledgments

This work was supported by NIH grants P01MH070056, P20RR015592, R01NS043990, and R01NS039253.

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