The National NeuroAIDS Tissue Consortium brain gene array: two types of HIV-associated neurocognitive impairment

Abstract

Background: The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders.

Methods: Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.

Results: With HIVE the HIV-1 RNA load in brain tissue was three log(10) units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.

Interpretation: Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).

Figure 1. Venn diagrams of regulated probes on the array.

The number of significantly regulated transcripts is shown in the three HIV-1 infected groups (Groups B, C, and D) as compared to uninfected controls (Group A) in three brain sectors. The criteria for significance employed a cutoff filter of ±2 fold change and p≤0.05 with the Benjamini-Hochberg correction for multiple testing comparisons using S+ArrayAnalyzer. Outer lobes represent probes regulated exclusively in one group. Regions within points of intersection represent probes regulated in more than one group. Group D (in green) contains NCI subjects with HIVE and had the most regulated genes in every brain sector. In neostriatum 91% of the probes with HIVE were upregulated. In neocortex 84% of the probes with HIVE were downregulated. Group C (yellow) contains NCI subjects without HIVE. Group B (purple) contains HIV-1-infected subjects without any NCI or HIVE. Groups B and C had few regulated probes that were delimited to neostriatum primarily. See Table 2 for the lists of regulated canonical pathways.

Figure 2. Interferon response genes (IFRGs) on the array.

Four array groups are designated A, B C and D on the abscissa. The top panels contain heat maps of normalized signal intensities for 242 unselected IFRG probes, and for 50 IFRG significantly regulated probes. The heat maps illustrate clustering of IFRGs in the four groups. The B panels are histograms of real time PCR confirmation for three IFRG transcripts and CD163 (mean ± SD, Student’s t test). The C panels illustrate confirmation at the protein level in Western blots of pooled extracts. Group D (NCI with HIVE) had significant regulation of IFRGs in all three brain sectors. Group C (NCI without HIVE) did not have significant IFRG regulation. Group B (HIV-1-infected subjects without any impairment or HIVE) had mild IFRG regulation. *p<0.05; **<0.01; ***<0.001.

Figure 3. Confirmation of downregulated neocortical GABA pathway mRNAs in HIVE.

GAD1 codes for the rate limiting enzyme in GABA synthesis in presynaptic neurons (glutamic acid decarboxylase, 67 kDa). GABRA1 codes for GABA receptor subunit A1 in postsynaptic neurons. Both of these mRNAs pertain to inhibitory neocortical neurons and were significantly decreased in frontal neocortex in subjects with NCI and HIV encephalitis (Group D). NCI subjects without HIVE did not have altered GABA transcripts (Group C). mRNA is expressed relative to GAPDH mRNA. Mean ± SD, Student’s t test.

Figure 4. Increased endothelial cell type transcripts on the array.

JAG1, PECAM1 and TFRC are gene symbols for mRNAs that are expressed by brain endothelial cells predominantly. All three were significantly increased in Group C on the array. Confirmation with RT-PCR is shown. A, uninfected subjects (n = 6); B/C/D, HIV-1 infected subjects (n = 18). The increased expression of these endothelial transcripts was found to be present in the HIV-1 infected groups generally. mRNA is expressed relative to GAPDH mRNA. Mean ± SD, Student’s t test.

Figure 5. Correlation of gene expression with brain viral load.

STAT1, S100A4, BTN3A1, ACP5, KLJL6, and NLRC5 are gene symbols for the top six mRNAs that show significant correlation with brain viral load. The Pearson correlation coefficient (r) and p value are shown, along with a linear regression line modeling the correlation. The different brain regions are shown by different colored and shaped symbols.