Background
Despite the availability of combination antiretroviral therapy (CART), which has successfully controlled HIV viremia and improved immune function in many treated HIV-infected patients, HIV-associated neurocognitive disorder (HAND) remains highly prevalent [
1]. The pathogenesis of HAND is still unclear, and is very often associated with nonviral neurobiological factors [
2,
3]. Numerous studies suggest that HAND is primarily the result of neuronal loss/dysfunction from direct or indirect viral effects, inclusive of inflammation driven by chronic low-level infection, loss of trophic factors, and elaboration of excitotoxic molecules (for example, [
4]). Morphologically, HIV-associated cognitive impairment has been linked to alterations in the synaptodendritic network in HIV-infected brain [
5,
6]. Currently, it is commonly accepted that cytokines and chemokines secreted by activated microglia and astrocytes in inflammatory conditions lead to alterations in synapse and dendritic spine structures in HIV-infected subjects, with a major role ascribed to glutamate neurotoxicity.
Despite numerous reports of effects of exogenous opiates, particularly drugs of abuse, on replication of HIV, HIV-associated neurotoxicity and modulation of immune responses in cell culture, animal models, and AIDS pathology in humans, (for example, [
7,
8]) much less is known of the impact of the endogenous opioid system on HIV neuropathogenesis and HIV-associated neurocognitive impairment. The opioid system comprises the mu (MOR), delta (DOR) and kappa (KOR) opioid receptors, which are activated by the endogenous opioid peptides beta-endorphin, enkephalin and dynorphin, respectively (for example, [
9] for review).
In vivo and
in vitro studies showed that stimulation of opioid receptors by exogenous MOP agonists like morphine leads to suppression of multiple components of the immune response including phagocytosis, natural killer cell activity, chemokine-induced chemotaxis, antibody response and cell-mediated immunity ( for review [
10]). Several reports demonstrate that Dynorphin A modulates the capacity of immunocytes to enhance or suppress chemotaxis through direct or indirect stimulation of KOR. Ruff
et al. [
11] have shown that Dynorphin 1-13 is a potent stimulator of human mononuclear cell chemotaxis. In recent studies of bi-directional heterologous desensitization between the chemokine receptor CXCR4 and KOR, Finley
et al. [
12] showed that treatment of the Jurkat T cell expressing KOR and CXCR4 with the KOR agonist U50,488H diminished the chemotaxis response to chemokine CXCL12. In the context of HIV, chronic opiate exposure has been associated with decreased expression of macrophage activation markers in brain [
13]. In contrast to mu opioid receptor ligands, dynorphin peptides (primarily endogenous KOR agonists) decrease basal and drug-induced dopamine levels in several areas of the dopaminergic nigrostriatal and mesolimbic-mesocortical systems as well as in tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus [
14]. In animal models, dynorphin/KOR system activation is also implicated in depression and anxiety, which may be secondary to the dopaminergic modulation [
15]. In humans and nonhuman primates, exogenous high-efficacy κ-opioid-receptor agonists have dose-dependent central nervous system (CNS)-mediated effects that include sedation (for example, unresponsiveness to environmental stimuli), dysphoria, anhedonia, depressive symptoms and psychotomimesis [
16‐
18].
In different experimental models of neurodegeneration and traumatic brain injury (TBI), dynorphin was shown to be either neuroprotective [
19‐
21] or neurotoxic [
22]. In the context of HIV infection, kappa opioid receptor ligands have demonstrated potential anti-inflammatory and neuroprotective properties in several
in vitro models of HIV neuropathogenesis. The synthetic KOR agonist (for example, U50,488) suppresses HIV-1 production in human microglial cells [
23] and CD4 T lymphocytes [
24], and dampens chemokine production in astrocytes [
25]. However, it has been shown that dynorphin stimulates TNF-a and IL-6 expression in human brain cell cultures, and the stimulatory effect of dynorphin resulted in upregulation of HIV-1 expression when human brain cells were co-cultured with human promonocytic cells U1 [
26].
In the present study we have examined expression of opioid genes
OPRM1,
OPRK1 and
PDYN in two brain regions, the caudate (a terminal field of the dopaminergic nigrostriatal system) and anterior cingulate (a terminal field in the mesocortical dopaminergic system) of postmortem brain of HIV-infected and control subjects; these areas are known to contain opioid receptors in humans [
27]. Several studies suggest that HIV-mediated neuropathogenesis includes the loss of dopaminergic terminals in the basal ganglia, including the caudate and putamen, either through degeneration of dopaminergic neurons in the substantia nigra or via local HIV-induced striatal pathology. This is postulated to lead to deficits in central dopaminergic activity, resulting in progressive impairment of diverse neurocognitive and motor functions [
28,
29]. The anterior cingulate cortex (ACC) is a heterogeneous subregion of the prefrontal cortex. Functions of the ACC include cognitive and attentional processing, autonomic regulation, motor control, and emotional control. [
30]. Studies on the distribution of cortical dopamine neurons in primates showed that the dopamine innervation is most dense in the motor and anterior cingulate cortex [
31]. Recently, decreased levels of the preproenkephalin mRNA (
PENK) and dopamine receptor D2 (
DRD2) in the dorsolateral prefrontal cortex (DLFPC) in postmortem brain of subjects with HIV/AIDS has been reported [
32].
In order to investigate whether there is an impact of the KOR/PDYN system and OPRM1 on HIV-related neuropsychological impairment, we examined the postmortem brains of HIV-infected and control subjects to identify any changes in quantitatively measured levels of PDYN, OPRK1 and OPRM1 as well levels of macrophage markers CD68 and CD163 in the caudate and anterior cingulate. We have found lower PDYN and greater OPRK1 mRNA levels in the anterior cingulate in HIV+ subjects. There was a positive correlation PDYN and OPRK1 levels with expression of macrophage/microglia marker CD163 in the anterior cingulate of HIV+ subjects. Furthermore, there was a positive correlation between better T-scores in motor domain scale and PDYN mRNA levels in this region.
Discussion
The major findings of the present study are that:
1.
There were opposite directions of change in the levels of PDYN and OPRK1 mRNA in the anterior cingulate in postmortem brain of HIV+ subjects: lower levels of PDYN with greater levels of OPRK1.
2.
There were positive correlations of PDYN and OPRK1 levels with expression of the anti-inflammatory microglial/macrophage marker CD163. In contrast, within HIV+ subjects, OPRK1 and PDYN were not correlated with levels of proinflammatory CD68.
3.
There was a positive correlation between better T-scores in the motor domain scale and PDYN mRNA levels in the anterior cingulate.
To our knowledge this is the first report of region-specific alterations in expression of these two opioid system genes in postmortem brain of HIV-infected individuals. One interesting result of the present study is the finding of lower levels of
PDYN mRNA in the anterior cingulate in HIV-infected subjects. Other studies have reported an increase or no change in
PDYN expression in postmortem brain in subjects with schizophrenia and other psychiatric disorders [
36].
The prodynorphin gene contains several calcium-responsive enhancer elements in its promoter region, including calcium/cAMP responsive element, phorbol ester-responsive element, and downstream regulatory element (DREAM) and is highly responsive to calcium levels ([
37] for review). Although dynorphins preferentially bind KOR and are potent and efficacious KOR agonists, several studies in cell culture and in the rodent CNS suggest that dynorphin peptides may potentiate glutamatergic receptor function and neurotoxicity, possibly through non-KOR sites of action [
38‐
40]. However, in human neurodegenerative diseases there is currently no direct evidence in support of this proposed alternative dynorphin-mediated mechanism of neurotoxicity.
Studies of a rodent model of viral encephalitis based on Borna disease virus (BDV) showed dynorphin depletion in the hippocampus due to depopulation of the granule layer and loss of competence of surviving granule cells to express dynorphin [
41]. Lower
PDYN mRNA levels and dynorphin peptides were observed in several experimental animal models of neuropathological conditions [
42‐
44]. Cell culture studies showed that activation of the human macrophage cells U-937 with lipopolysaccharide (LPS) led to a decrease in
PDYN mRNA levels through transcriptional inhibition of gene expression [
45].
Recently, significantly lower levels of another opioid neuropeptide mRNA, preproenkephalin (
PENK) were found in the dorsolateral prefrontal cortex in subjects with HIV encephalitis (HIVE) compared to seronegative controls [
32]. The authors did not find differences in
PENK mRNA levels in HIV-infected subjects with and without neurocognitive impairment, and concluded that the lower PENK levels were related neuropathologically to HIVE.
Mechanistic
in vitro studies support the hypothesis that the release of numerous factors by activated macrophages, glial cells and astrocytes could be a cause of elevated levels of
OPRK1 mRNA in the anterior cingulate of HIV+ subjects observed in our study. For example, incubation of the murine macrophage cell line J774 with the proinflammatory cytokine IFN gamma for 24 h led to upregulation of
Oprk1 expression at both transcriptional and protein levels [
46]. Functionality of KOR in macrophages was demonstrated by Dynorphin-A (1-17)-induced phosphorylation of ERK1/2. Also, in adjuvant-induced inflammation in rats, the proinflammatory cytokine interleukin-1 beta (IL-1 beta) induced upregulation of
Oprk1 in dorsal root ganglia [
47].
Clinical and experimental central nervous system injuries elicit an inflammatory response that comprises mostly activated macrophages [
48]. These cells exist in a state of dynamic equilibrium within the lesion microenvironment. Thus, depending on the inflammatory conditions in the lesion microenvironment, they may differentiate into proinflammatory cells that aggravate tissue injury, or anti-inflammatory cells that promote CNS repair [
49]. In our study, there was elevation of proinflammatory CD68 and anti-inflammatory CD163 mRNA in both the caudate and anterior cingulate of HIV+ subjects. Also, levels of
PDYN and
OPRK1 mRNA were positively correlated with CD163 mRNA in the anterior cingulate, but not in the caudate. In contrast, within HIV+ subjects mRNA levels of
OPRK1 and
PDYN were not correlated with levels of CD68. In a rodent model of Parkinson’s disease, treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine led to higher levels of proinflammatory macrophages (CD16, CD32 and CD86) in dynorphin knockout mice (Dyn
-/-) than the wild-type, suggesting anti-inflammatory and neuroprotective properties of
PDYN gene products (dynorphin peptides) [
50]. MPTP-induced more severe motor deficits in Dyn-/- than in wild-types, and Dyn-/- mice also exhibited greater dopaminergic depletion. This suggests that endogenous dynorphins play an important role in protection of nigrostriatal DAergic neurons from chemical insults.
One of our findings is a positive correlation between levels of anterior cingulate
PDYN mRNA levels and better T-scores in motor domain scale in HIV+ subjects. Animal models of experimental traumatic brain injury (TBI) provide some clues to a role of dynorphin and kappa opioid receptors in spatial memory and motor tasks. For example, TBI in rat resulted in increased
Pdyn mRNA and dynorphin peptide levels in hippocampus, and intracerebroventricular administration of the KOR antagonist nor-BNI exacerbated motor and vestibulomotor deficits [
19]. Of the cognitive domains we assessed, the association with motor performance likely reflects both a neurochemical association as well as a neuroanatomic specificity. The lack of association with other cognitive domains may be, in part, a function of the brain regions we examined and those we did not. For example, we did not assess the hippocampus, which is critical to learning and memory, nor did we examine orbitofrontal and dorsolateral prefrontal regions, implicated in executive functioning. On the other hand, the association of anterior cingulate with initiation of motor activity has been documented in humans, and is in keeping with the anatomic localization of our findings [
51]. Thus, further study of other brain regions may be necessary to fully understand the extent of association between DYN/KOR and cognitive processes in humans.
The present study has several limitations that could be the focus of larger follow-up studies. The alterations in levels of
PDYN/OPRK1 system and macrophage markers in brain of HIV-infected subjects were measured only at the mRNA level, and not the peptide/protein gene products. Cell heterogeneity in the samples may also be a consideration, since
OPRK1 is expressed in diverse cell types and phenotypes (for example, neurons, microglia, macrophages). This may potentially ‘mask’ a more specific relationship of
OPRK1 or
PDYN with HIV-associated neurocognitive impairment. Of interest, the anterior cingulate cortex (ACC) can be divided anatomically and functionally into distinct subregions, dorsal and ventral ACC. The dorsal ACC is connected with the prefrontal cortex, parietal cortex and the motor system. The ventral part of the ACC is connected with the amygdala, nucleus accumbens, hypothalamus, and anterior insula. Moreover, a study of the distribution of binding sites of 15 neurotransmitter receptors showed a differential pattern of expression of glutamate, GABA, acetylcholine, serotonin, and dopamine receptors among ACC subregions and neurons in humans [
52]. It would be of great interest to study an interaction of the DYN/KOR system with other receptors in specific ACC regions. In addition to cell heterogeneity, postmortem tissues cannot be rigorously controlled for terminal events and certain medical factors; this variability may mask associations in relatively small ‘n’ study. Thus, our findings need replication in larger groups of individuals, both with and without HIV infection.
Competing interests
The authors declare that they have no competing of interests.
Authors’ contributions
VY performed extraction of RNA, quantification of mRNA levels using RT-PCR, and drafted the manuscript. SM provided postmortem brain material, dissected brain regions and was involved in data analyses and writing the manuscript. AH performed the statistical analyses. ERB was involved in writing and editing the manuscript. MJK participated in study design, interpretation of data and editing the manuscript. All authors read and approved the final manuscript.