Dysregulation of Neuronal Cholesterol Homeostasis upon Exposure to HIV-1 Tat and Cocaine Revealed by RNA-Sequencing

Abstract

HIV-1 Tat protein is released from HIV-1-infected cells and can enter non-permissive cells including neurons. Tat disrupts neuronal homeostasis and may contribute to the neuropathogenesis in people living with HIV (PLWH). The use of cocaine by PLWH exacerbates neuronal dysfunction. Here, we examined the mechanisms by which Tat and cocaine facilitate alterations in neuronal homeostatic processes. Bioinformatic interrogation of the results from RNA deep sequencing of rat hippocampal neurons exposed to Tat alone indicated the dysregulation of several genes involved in lipid and cholesterol metabolism. Following exposure to Tat and cocaine, the activation of cholesterol biosynthesis genes led to increased levels of free cholesterol and cholesteryl esters in rat neurons. Results from lipid metabolism arrays validated upregulation of several processes implicated in the biogenesis of β-amyloid and Alzheimer's disease (AD), including sterol o-acyltransferase 1/acetyl-coenzyme A acyltransferase 1 (SOAT1/ACAT1), sortilin-related receptor L1 (SORL1) and low-density lipoprotein receptor-related protein 12 (LRP12). Further studies in Tat-treated primary neuronal cultures and brain tissues from HIV-1 transgenic mice as well as SIV-infected macaques confirmed elevated levels of SOAT1/ACAT 1 proteins. Our results offer novel insights into the molecular events involved in HIV and cocaine-mediated neuronal dysfunction that may also contribute to neuropathogenic events associated with the development of AD.

Figure 1

Expression profiling of the cellular mRNAs for control, cocaine, Tat and Tat/cocaine. (A) Box plot showing the statistical features of the expression profiles under different experimental conditions. MA plots of Tat vs. control, (B) cocaine vs. control, (C). Tat/cocaine vs. Tat, and (D) Tat/cocaine vs. cocaine, (E) Hierarchical clustering of (F) Cellular mRNAs and (G) Mitochondrial mRNAs are illustrated. RPKM, Reads per Kilobase Million; MA, log ratio vs. mean average.

Figure 2

Tat and cocaine dysregulate cholesterol metabolism by altering key genes involved in APP trafficking (SORL1) and cholesterol esterification (SOAT1/ACAT1). (A) Functional annotation of k-means clustering identified a group of genes involved in the dysregulation of lipoprotein metabolism pathways. (B) Lipoprotein signaling and cholesterol metabolism pathway PCR array validated dysregulation of key genes in this pathway. (C) qRT-PCR confirmed the upregulation of SOAT1/ACAT1, SORL1 and LRP12 in HIV-1 Tat protein treated rat neurons. (D) Adeno-Tat/cocaine treatment of rat neurons showed that cocaine has an inhibitory effect on Tat-mediated overexpression of SOAT1/ACAT1. (E) Relative SOAT1/ACAT1 mRNA expression in rat primary neurons in the presence of Tat protein. The statistical analyses were conducted via t-test with n = 3.

Figure 3

Expression of SOAT1/ACAT1 protein in the presence of HIV-1 Tat. (A) Immunocytochemical images of SOAT1/ACAT1 upregulation in rat hippocampal neurons exposed to 50 ng/ml rTat labeled with MAP2 (green) and SOAT1/ACAT1 (red) antibodies. (B) Immunocytochemistry images of SOAT1/ACAT1 upregulation in rat cortical neurons exposed to 50 ng/ml rTat labeled with MAP2 (green) and SOAT1/ACAT1 (red) antibodies. (C) Representative western blot and quantification of rat cortical neurons immunoblotted with anti-SOAT1/ACAT1 antibodies shows upregulation in response to Tat expression. GAPDH was used as a loading control. (D) Quantification of Western blot, n = 3, ^*p < 0.0001 (via t-test) (E) Immunohistochemistry on formalin fixed paraffin embedded slides for Tat-transgenic mouse (TG26) and wild type mice brain tissue confirmed in vivo upregulation of SOAT1/ACAT1 in TG26 (negative control for each panel have been shown in this figure).

Figure 4

Neuronal cholesterol synthesis is induced by cocaine, Tat and Tat/cocaine. (A) Normalized free cholesterol levels following cocaine, Tat and Tat/cocaine treatments exhibit an average increase greater than 50% in rat neurons. (B) Normalized total cholesterol levels as increased by Tat and cocaine. The total cholesterol includes free cholesterol and cholesteryl esters. (C) Normalized cholesteryl esters in neurons exposed to cocaine, Tat and Tat/cocaine indicate an additive effect of Tat plus cocaine on the cholesteryl ester amount. The statistical analysis was conducted via t-test, n = 4.

Figure 5

Schematic showing the upregulation of cholesterol biosynthesis genes as a result of HIV-1 Tat according to RNA-Seq data in neurons. Tat activates mevalonic acid and cholesterol esterification genes including SOAT1/ACAT1 leading to the accumulation of cholesteryl ester in neurons. Other gene symbols are as following: Hmgcs: hydroxymethylglutaryl-CoA synthase; Mvk: mevalonate kinase; Mvd: mevalonate diphosphate decarboxylase; Fdt1: farnesyl-diphosphate farnesyltransferase 1; Lrp1: Low density lipoprotein receptor-related protein 1.