Host genetic variation is likely to impact both adaptive and maladaptive host responses to HIV infection that play a role in neuropathogenesis, just as host genetics clearly impacts susceptibility to HIV infection and the rate of disease progression [
26‐
30]. Many candidate-gene studies and a single genome-wide association study spanning the pre-cART and cART eras, primarily focusing on HAD with or without HIV encephalitis, have identified variants in immune-regulatory genes and other gene classes as potential risk-modifiers or protective factors, but very few of these genes have been replicated in subsequent studies (Table
1) [
31•,
32•]. A number of factors that complicate the clinical definition, risk-stratification, and monitoring of HAND, have presented challenges to genetic studies aimed at better understanding susceptibility to these disorders: inherent fluctuations in individual neuropsychometric test scores over time, despite correction for practice effects, imprecision of diagnostic categories like ANI and MND, possible residual biases in testing protocols, and varying methods used to determine composite scores in prior published studies of NCI among HIV + persons. Distinguishing reversible NCI from the “legacy effect” of brain damage due to previously untreated HIV infection, and completely excluding confounding by comorbidities also remain difficult [
6•,
7]. In contrast to the pre-cART era, many converging pathogenic mechanisms are likely to contribute to HAND at present [
33,
34,
35•]. These challenges notwithstanding, recent genomic, transcriptomic, and epigenomic studies have highlighted metabolic pathways and physiologic processes that are disrupted in HAND (Table
2).
Table 1
Summary of published genetic associations (positive findings) in candidate-gene and genome-wide association studies (GWAS) of HIV-associated neurocognitive disorder (HAND) (See text for references). Only genes associated with a HAND-related phenotype in at least one published study, and studies that included HIV + subjects, are listed
Nuclear genes
| | | |
APOE (E4 allele) | AIDS with ADC/HAD ± HIVE; non-AIDS with HAND ± neuropathologic features | Autopsy (mostly case-control; one survival study with autopsy component; 2 uncontrolled); cross-sectional; longitudinal cohort | R |
TNFA
| HAD; HAD/ADC, or HIVE and/or HIV-LE | Autopsy case-control | NR |
MCP1/CCL2, CCR2
| HAD ± HIVE or AIDS/ADC, OR change in executive functioning and processing speed between 2 consecutive visits up to 15 yrs apart; or NCI (clinical rating score ≥ 5); HAE (children) | Retrospective case-control; longitudinal cohort ± cross-sectional analysis | R (MCP1) NA (CCR2) |
MIP1A/CCL3
| HAD; AIDS with HAD; OR change in executive functioning and processing speed between 2 consecutive visits up to 15 yrs apart; OR risk of NCI | Retrospective case-control; longitudinal cohort | R |
SDF1
| Decline in NC test scores and/or brain growth failure in children; OR change in executive functioning and processing speed between 2 consecutive visits up to 15 yrs apart; OR prevalent NCI (adults); change in GDS or cross-sectionaL GDS in co-HCV+ | Longitudinal cohort with cross-sectional component; retrospective case-control | NR |
MBL2
| Changes in GDS or cross-sectional GDS in co-HCV+; OR change in executive functioning and processing speed between 2 consecutive visits up to 15 yrs apart; OR prevalent NCI (adults) | Longitudinal cohort with cross-sectional component | NR |
CCR5 (δ32 del) | HAD/ADC; AIDS ± HAD; decline in NC test scores and/or brain growth failure in children; NCI in children; GDS (change and cross-sectional) | Longitudinal cohort ± cross-sectional component; case-control | R prior to 1991 only; NR in cART era |
COMT
| Executive functioning domain Deficit Scores ± stimulant abuse; HAND: standardized NP domain T-scores | Retrospective/Case-control | NR |
DRD2, DRD3
| GDS ≥ 0.5 (NCI); Global and cognitive domain T-scores in population with prevalent substance dependence | Cross-sectional/Case-control | R (DRD3 in substance users) |
HLA:DR, DQB1, A24, B27
| Time to CNS impairment (“deterioration in brain growth, psychological function and/or neurological status”) | Pre-cART cross-sectional study; cART era case-cohort study; longitudinal cohort | R (DR, B27) NA (DQB) NR (HLA A) |
APOBEC3G
| Brain growth failure, with NCI defined differently based on age | Pre-cART pediatric cohort study | NA |
PKNOX1/PREP1
| AIDS with dementia | Retrospective case-control | NA |
YWHAE
| HAND | Cross-sectional study with HIV+/HIV- controls | NA |
Mitochondrial & nuclear DNA structural changes
| | | |
8-oxoG modification | HAND “screen”, International HIV Dementia Score ≤ 10 | Autopsy case-control | NA |
Regulation of telomere length | Detailed NP test scores (global and ability domain scores) ± history of chronic psychological trauma (Childhood Trauma Questionnaire Short Form) | Cross-sectional with HIV+/HIV- controls | NA |
Table 2
Genes with significantly altered expression in transcriptomic and epigenetic studies and hence implicated in HAND pathogenesis
Monocytes
|
Pathways: | Brain metabolite neuroimaging traits, e.g., N-acetylaspartate concentration in FWM, anterior cingulate | Pulliam et al. 2011 [ 100]; Borjabad, 2012 [ 17•] |
IFN response/activation |
Example genes: IP-10
|
Pathways: | Neurocognitive impairment (GCR) | |
Mitotic cell cycle |
Translational elongation |
Oxidative stress |
Example genes: IL6R, casein kinase 1-alpha-1, hypoxia upregulated-1, LDL receptor-related protein 12, KEAP-1
|
Brain tissue/brain-derived cells
|
Pathways: | SIV ± SIVE, HIV ± HIVE brains; also primary neurons in vitro
| Eletto et al. 2008 [ 104]; Yelamanchili et al. 2010 [ 106] |
pre-synaptic proteins/general neuronal function |
Example genes: miR-128a, SNAP25, MEF2C, miR-142-3p, miR-142-5p, miR-21 (downregulation of target MEF2C in neurons) |
Pathways: | FWM from HIV-, HIV+/HIVE brains; immunohistochemistry; glutamate levels, brain neurotrophic factor levels | Noorbakhsh et al. 2010 [ 107] |
IFN response |
Neuroinflammation | Gene expression in primary astrocytes exposed to HIV Vpr protein in vitro vs. controls |
Nucleotide metabolism |
Cell cycle | Neurobehavioral abnormalities (locomotion) in transgenic mice with increased BG Vpr expression |
Mitochondrial function |
Apoptosis (astrocytes) |
Example genes: Caspase-6
|
Pathways: | Acute and chronic SIV model ± SIVE | Reviewed in Winkler et al. 2012 [ 92•] |
Inflammation |
Immune and acute-phase response |
Ion transport |
Example genes: B2M, STAT1, IFI44, IFIT3, MX1
|
Pathways: | HAND diagnosis (±cART), ±HIVE | Borjabad et al. 2012 [ 17•] |
Neurogenesis |
Synaptic transmission |
Antiviral/immune responses (including IFN responses, complement activation) |
Ion/Calcium transport |
Antigen presentation/processing |
Oxygen transport |
Signal transduction |
Cell cycle |
Oligodendrocytes/Myelination |
Microtubule-based movement |
Example genes (treated vs untreated HAND): CXCR2, CR1, HLA-DQB1, CXCL2, IFIT1, IFI44, STAT1, MOBP
|
Pathways: | HAND ± HIVE vs. HIV+/HAND- vs. HIV- controls; neurocognitive impairment (GCR) | Gelman et al. 2012 [ 93•]; Levine et al. 2013 [ 94•] |
Synaptic transmission |
Neuroimmune function |
Endothelial markers | | Gelman et al. 2012 [ 93•]; Levine et al. 2013 [ 94•] |
Neuronal function |
Glutamate signaling |
Axon guidance |
Clathrin-mediated endocytosis |
IFN response |
Antigen presentation/processing |
Inflammation/acute-phase response/toll-like receptors |
Oligodendrocyte function |
Mitochondrial function |
Cell signaling |
Protein ubiquitination |
Caveolin-mediated endocytosis |
Example genes: YWHAE/14-3-3 protein, GAD1, IFRGs, TFRC
|
Pathways common between HAND and AD: | HAND ± HIVE brains vs. HIV+/HAND-, HIV+/HAND-; neurocognitive impairment (GCR) in HIV + vs. Mini-Mental Status Exam data in AD | |
Cytoplasm |
Mitochondrial function |
Tricarboxylic acid cycle |
Transit peptide |
Synaptic |
Cell Differentiation |
Activator |
Repeat |
Cell communication |
Regulation of transcription |
Phosphorylation |
Pathways differentiating HAND + vs. – HIVE: | | |
Gliosis |
Dopaminergic tone Inflammation |
Example genes: GRK6, CCL2, ID2
|
Pathways: | HAND vs. HIV- controls | |
Neuronal RNA splicing/gene transcription |
Example genes: RbFOX3
|
Pathway: | Global and cognitive domain T-scores/GDS (all 7 domains) | Jacobs, 2013 [ 72•]; Gupta, 2011 [ 71] |
Dopaminergic response |
Example genes: DRD3
|
Pathways: | Subsyndromic HAND/ANI, MND or HAD ± HIVE, vs. HIV- controls | Desplats et al. 2013 [ 117•] |
Chromatin modification |
Inflammation |
Example genes: BCLB11B (targets IL-6, TNFα, CXCR4) |
Transcriptomic and Epigenetic Studies
The advent of microarray technology has led to numerous studies investigating the effects of HIV infection on host gene expression in phenotypes that contribute to but are relatively distant from HAND, including viral replication, HIV persistence, apoptosis, and immune dysregulation in general. Those studies performed prior to 2007, have been reviewed extensively elsewhere [
88] Subsequent functional genomics studies have yielded additional information on genes and pathways up- or down regulated in astrocytes, neurons, and glial cells, cell types intimately involved in HAND pathogenesis (Table
2) [
31•,
89‐
91]. A number of genes show consistently altered expression across studies of human astrocytes
in vitro, the human HIV-infected brain with or without HAD or HIVE, and the simian-immunodeficiency virus (SIV)-infected macaque model, pointing to their likely importance in HAND [
31•,
90,
92•]. While many studies to date have focused on patterns of RNA expression changes specific to HIVE in frontal gray matter [
92•], few have investigated differences in brain regions affected by HAND without HIVE. A microarray study by Gelman et al. [
93•] using brain tissues from HIV-infected and seronegative individuals enrolled in the National NeuroAIDS Tissue Consortium (NNTC) Brain Bank, revealed two different transcriptome patterns in HAND + HIVE and HAND alone. HAND combined with HIVE was associated with high viral load, global upregulation of genes involved in interferon responses, and general immune activation, while specific neuronal transcripts in frontal neocortex were down-modulated. HAND without HIVE, however, was associated with low viral load, upregulated endothelial-type transcripts, and the conspicuous absence of gene-expression changes noted in HIVE. Weighted-gene coexpression network analysis (WGCNA) of the same transcriptomic data, accounting for correlations amongst functionally related genes, also identified meta-networks of genes associated with global neurocognitive function; these included cancer-related genes and genes important in oligodendrocyte function [
94•] in frontal neocortex, frontal white matter, and the basal ganglia. Dysregulation of genes involved in mitochondrial function, cancer, the immune response, synaptic transmission and cell-cell signaling has also been suggested in other studies of HAND [
17•,
94•] (Table
2).
The role of antiretroviral drug toxicities in HAND remains controversial. The contribution of complex drug interactions, side effects when taking an increased number of drugs for advanced disease, and known mitochondrial effects of older dideoxynucleoside reverse-transcriptase-inhibitors such as stavudine and didanosine, to NCI is unknown [
8,
95]. Transcriptomic studies evaluating the impact of cART on gene expression patterns in HAND in brain tissue reveal alterations in expression of about 100 immune-regulatory, cell-cycle, and myelin-pathway genes that are not correlated either with brain viral burden or to antiretroviral drug CNS penetration effectiveness (CPE) score, suggesting a possible explanation for the difficulty to date in correlating CPE scores to neurocognitive outcomes despite their association with reduced CSF viral load [
8,
96•].
HIV-infected monocytes or monocytes from HIV-infected as compared to seronegative individuals have been the focus of many
in vitro microarray-based studies to identify upstream biological mechanisms relevant to HAND, due to their key role in BBB injury. Genes and pathways that have been found to be significantly upregulated in such studies include: a large number of chemotaxis- and inflammation-related genes [
97,
98] and genes involved in the interferon (IFN) response, as well as genes that promote antioxidant and anti-inflammatory responses [
31•,
99,
100]. Expression of subsets of these genes have also been associated in some, but not all [
99] studies, with mild NCI [
31•,
100], with HAND in hepatitis C/HIV-co-infected subjects on cART [
101•], and with metabolic neuroimaging traits such as
N-acetyl-aspartate in frontal white matter [
100]; however, monocyte transcriptome patterns have not always correlated with NCI in HIV + persons [
99,
102].
A recent postmortem study examined changes in expression of ephrin (
EPH) genes that mediate synapse formation and recruitment of glutamate receptors to synapses [
103•]. Postmortem brain tissues from cognitively characterized HIV-infected subjects and seronegative controls from the Manhattan HIV Brain Bank were examined for levels of expression of a variety of genes, including
EPHA4 and
EFNB2 (an ephrin ligand). Transcript levels of both of these genes in the caudate, and of
EPHB2 in the anterior cingulate were significantly lower in HIV-infected patients, and
EPHB2 mRNA levels in the cingulate correlated with premortem neurocognitive function. The authors hypothesized that decreased expression of
EPHB2 in the cingulate may represent a compensatory mechanism minimizing excitotoxic injury in the face of chronic inflammation.
The small number of published epigenetic studies of HAND have focused on expression of microRNA (miRNA), small non-coding RNA molecules that bind messenger RNA and regulate gene expression at the transcriptional or post-transcriptional levels. MicroRNA (miRNA) expression studies conducted in cortical neurons exposed to viral proteins such as Tat and Vpr [
104,
105•], or in tissue from individuals with HIVE or SIV-infected macaques with encephalitis (SIVE) have implicated upregulation of the following classes of host miRNAs in HIVE and SIVE: 1) immune response and inflammation, 2) nucleotide metabolism, 3) cell cycle, 4) oncogenesis (
e.g., miR-21, which targets a neuronal transcription factor), and 5) apoptosis (
e.g., caspase-6). Downregulated miRNAs included those involved in: 1) inflammation, 2) neuronal monoamine oxidase activity (possibly explaining the reduced dopaminergic activity in HAND), 3) apoptosis (
e.g., suppression of caspase-6 expression), 4) modulation of viral replication, 5) mitochondrial function, and 6) axonal guidance (Table
2) [
105•,
106‐
108,
109•,
110•,
111•]. These studies have provided some useful leads and validated several neuropathogenic mechanisms in HAND, but sample sizes have been extremely small (five individuals or less in human studies). In general, these studies have not evaluated associations with neurocognitive phenotypes or accounted for multiple statistical tests or potential confounders in the analyses. Findings in SIV models also require replication in humans.
Consolidation of short-term memories into long-term memory requires synaptic plasticity, which is characterized by structural changes and altered gene expression at neuronal synapses [
112•]. In keeping with the finding that synaptodendritic damage rather than neuronal loss is a neuropathological feature of milder forms of HAND [
113•], a recent study found downregulation of many synaptic plasticity genes in HIV-infected astrocytes, and increased expression of pro-apoptotic genes, compared to uninfected controls [
112•]. These findings translated into reduced dendritic spine density and altered dendritic morphology, which were most prominent in cells infected with clade B virus.
Finally, Lucas et al. [
113•] have reported a highly abnormal distribution of the RNA splicing factor NeuN/Rbfox3 in postmortem brain tissue from 22 HIV-infected individuals with MND/HAD as compared to seronegative controls. Very few targets have been identified for this splicing factor, which is usually confined to the nucleus, where RNA splicing occurs. The authors posit that altered localization of RbFox3 in HAND may reflect downregulation of expression of neuronal genes relevant to HAND pathogenesis. This finding requires further study.
Relatively few studies have evaluated the role of histone modification and DNA methylation in the context of HAND. Histone deacetylases (HDACs) function in epigenetic regulation by deacetylating histones and other proteins involved in transcription and chromatin remodeling; histone hypoacetylation has been linked to many neurodegenerative diseases. Saiyed et al. [
114•] showed that HIV-1 Tat protein upregulates HDAC2 expression in neuronal cells, leading to transcriptional repression of genes involved in synaptic plasticity and neuronal function and suggesting a potential therapeutic role for HDACs as a drug class in HAND [
115,
116]. More recently, Desplats et al. conducted a case-control study among 32 deceased HIV + individuals from the HIV Neurobehavioral Research Center and California NeuroAIDS Tissue Network, 72 % of whom underwent neurocognitive testing within 1 year of death [
117•]. The study compared epigenetic markers in postmortem brain tissue, such as B-cell CLL/lymphoma (BCL11B), a transcriptional silencer, among several patient groups: HIV + controls without detectable proviral DNA, RNA or p24 in the CNS, HIV + cases with high viral DNA but no HIV RNA or p24 (latent cases), and HIVE cases with high expression of viral DNA, RNA and p24. Up to half of HIV-infected subjects were on cART, and with the exception of HIVE cases, all HIV + subjects had mild to moderate NCI. Compared to HIV + controls, higher levels of BCL11B protein and other chromatin modifiers involved in transcriptional silencing of HIV-1 (including HDAC1) were observed in HIV + latent cases and were associated with dysregulation of pro-inflammatory genes like
IL6,
TNFA, and
CXCR4. Latent cases also displayed more cognitive impairment than HIV + controls. These results suggest that even in the absence of detectable viral replication, significant dysregulation of pro-inflammatory genes may still occur and that these changes are associated with increased levels of epigenetic factors such as BCL11B. These findings are highly relevant to strategies for eradicating viral reservoirs which might include modulation of BCL11B.
Narasipura et al. [
118•] examined the role of epigenetic regulation in maintaining latency of the virus in astrocytes
in vitro. DNA CpG methylation and histone modifications (methylation and deacetylation) at the HIV-1 promoter region are specific hallmarks of HIV-1 latency, and HDAC inhibitors reactivate the virus in cell culture models and in HIV-infected CD4+ T cells [
118•,
119•]. This study added to previous studies by demonstrating the role of epigenetic regulation in maintaining and reversing virus latency in astrocytes specifically, a process implicated in HAND [
90]. Inhibitors of class I HDACs and histone methyltransferases which demethylate DNA are able to activate the HIV-1 promoter in latently infected astrocytes, thereby confirming that these cells may be clinically important reservoirs for HIV in the brain. Other
in vitro studies have revealed epigenetic regulation of markers in T-regulatory cells, which normally maintain gut-mucosal immune tolerance via suppression of effector T-cell functions but are dysregulated in chronic HIV infection [
120•], as well as dysregulation of HDAC1 and DNA methyltransferases in oral epithelial cells, potentially contributing to HAND via increased oral microbial disease and peripheral immune activation [
121].
We know of only one unpublished study of DNA methylation in the context of HAND, the preliminary results of which were presented at the 19th Conference on Retroviruses and Opportunistic Infections in 2012 [
82]. This study of 17 HIV + participants in a longitudinal cohort study showed many strong positive and negative correlations of methylation profiles at autosomal sites with changes in neurocognitive test performance (scaled scores corrected for practice effects) measured at two consecutive time points.
Other very preliminary findings deserving of further exploration and replication in studies of HAND include: 1) interactions between opioid-related genes such as OPRM1, substance abuse, and HAND [
122•]; 2) mitochondrial DNA haplogroup effects on HAND risk within specific ethnic subpopulations [
123]; 3) the impact of iron metabolism, which is essential for mitochondrial function as well as dopaminergic metabolism [
124,
125•]; and 4) potential protection against HAND by promoter variants in the antioxidant response gene HMOX1 [
126].