Vorinostat positively regulates synaptic plasticity genes expression and spine density in HIV infected neurons: role of nicotine in progression of HIV-associated neurocognitive disorder

HIV-1 infection, cigarette smoking and drug abuse are global public health concerns. Several studies report that nicotine, the psychoactive drug in tobacco products that induces dependence among smokers, is one of the major immunomodulatory agents [1‐5]. Nicotine dependence is the most common form of chemical dependence in the United States. Research suggests that nicotine may be as addictive as heroin, cocaine and alcohol [3, 5]. In the United States, the prevalence of cigarette smoking ranges from 35-70% in HIV-infected individuals compared to approximately 20% in general population [6‐9].

Infection with HIV is often associated with a wide range of neurological and neuropathological abnormalities, which are collectively known as HIV- associated neurocognitive disorder (HAND). HAND is characterized by development of cognitive, behavioral and motor abnormalities, and occurs in approximately 50% of HIV infected individuals [10, 11]. Previous studies indicate that HIV-1 clade B (predominant subtype found in the United States and in the western world) being more neuropathogenic than clade C (predominant in sub-Saharan Africa and Asia) [12‐14]. Experimental evidence and clinical observations suggest a correlation between smoking and increased risk for neurological disorders [15]. A great deal of research has focused on the pathophysiological and clinical effects of nicotine. In human studies, nicotine and other agonists of the nicotinic acetylcholine receptor (nAChRs) have been reported to improve cognitive performance in healthy adults and in different psychiatric populations [16, 17]. In studies with rodents, the results have been mixed with observations of improvement, impairment, or no effect on various measures of learning and memory following acute or chronic systemic treatment [16]. However, the effect of nicotine on HIV-1 pathogenesis has received limited research attention. Few studies have reported that nicotine exacerbates HIV-1 replication in in vitro-infected alveolar macrophages and primary human microglia [18, 19]. Further, limited examination of the neuropathogenic effects of nicotine on HIV-1 infection of CNS cells has shown that galantamine and nicotine exert synergistic effect on the inhibition of microglial cell activation induced by HIV-1 gp120 [20]. A recent study reported that exposure to tobacco smoke extract increases the blood–brain barrier permeability and plays a critical role in pathophysiology of ischemia [21].

Although few studies have reported deficits in learning, memory, and global cognitive performance among cigarette smokers living with HIV [22, 23], the underlying mechanisms associated with the use of nicotine and the development of HAND in these patients are unknown. New memory formation involves gene expression-dependent changes in synaptic structure and plasticity in the hippocampus [24]. Recently, we have reported dysregulated synaptic plasticity genes expression and decreased spine density in HIV infected CNS cells [13]. Although epigenetic modifications are known to regulate gene expression during HIV infection [25], molecular and biochemical evidence on epigenetic mechanisms in CNS cells in response to HIV and nicotine are lacking. Acetylation and deacetylation of histone proteins associated with chromatin plays a pivotal role in the epigenetic regulation of transcription and other functions in cells, including neurons.

HDACs often function as a component of the transcriptional repressor complex to silence gene expression and induce chromatin compaction through histone protein deacetylation. HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory [26]. HDAC inhibitors are emerging as an exciting new class of potential therapeutic agents for the treatment of solid and hematological malignancies. Vorinostat also known as suberanilohydroxamic acid (SAHA) was the first histone deacetylase inhibitor approved by the U.S. Food and Drug Administration for the treatment of Cutaneous T-Cell Lymphoma. Vorinostat binds to the catalytic domain of the histone deacetylases (HDACs). This allows the hydroxamic moiety to chelate zinc ion located in the catalytic pockets of HDAC, thereby inhibiting deacetylation and leading to an accumulation of both hyperacetylated histones and transcription factors. Vorinostat was also shown to cross the blood–brain barrier in a mouse model of Huntington's disease [27]. Therefore, use of vorinostat as a therapeutic agent in the CNS related diseases may be useful. In the down-stream, translation of eukaryotic mRNAs is regulated primarily at the level of initiation. Two main mechanisms by which translation is controlled are the formation of the ternary complex via eIF2 and the recruitment of the ribosome to the mRNA via 4E-BPs. Phosphorylation of an α subunit of eIF2 (at Ser51) converts the protein from a substrate to a competitive inhibitor of the GDP/GTP-exchange reaction by decreasing the rate of dissociation of eIF2 from eIF2B [28, 29]. This causes a decrease in general translational initiation [29, 30]. We hypothesize that by inducing the transcriptional repression, HDAC2 plays a major role in the neuropathogenesis caused by HIV and nicotine (Figure 1). In the down-stream, we hypothesize that translational process in HIV infected cells will be affected by eIF2α phosphorylation (Figure 2).

In the current study, we have analyzed the effect of nicotine on the differential expression of synaptic plasticity genes, spine density in an in vitro HIV infection model using neuronal cells. Along with the dysregulated synaptic plasticity genes and altered spine density, we have found increased levels of HDAC2 expression indicating the transcriptional repression of synaptic plasticity genes in these cells. We also found increased eIF2α subunit phosphorylation (at Ser51) which may associate with the translational repression of synaptic plasticity genes in HIV alone infected SK-N-MC cells and in the presence of nicotine. These negative regulatory effects of HIV infection and nicotine on the expression of synaptic plasticity genes and spine density were reversed by the HDAC inhibitor vorinostat. This study suggests that nicotine synergistically acts with HIV to down-regulate synaptic plasticity genes and alter spine density, potentially via upregulation of HDAC2. The use of vorinostat may help to reverse these negative regulatory effects (induced by HDAC2) on synaptic plasticity genes expression and spine density.

Nicotine is the single tobacco constituent demonstrated to have the potential to stimulate the production of HIV-1, as has been shown using in vitro-infected alveolar macrophages [13]. A growing body of evidence suggests chronic cigarette smoking alone is associated with abnormalities in brain morphology and neurophysiology [31, 32]. Reports on effect of nicotine on HIV associated neurocognitive disorders are scanty. We have used SK-N-MC cells to study the HIV infectivity and for the analysis of synaptic plasticity genes expression and spine density changes as these cells have been reported to be susceptible for the productive HIV-1 infection [33]. In this study, with the use of neuronal cells, we have observed that the presence of nicotine have significantly increased HIV infection which are in agreement with the previous reports observed in other types of cells [18, 19, 34]. Rock et al., reported that TGF-β1 may be involved in the effects of nicotine on enhanced HIV-1 expression in microglial cells [19]. Out of 84 synaptic plasticity genes analyzed, in this study, we have observed down-regulation of 28 synaptic plasticity genes (3–38 folds) and up-regulation (3–56 folds) of 8 genes in HIV infected cells. Our results indicate that HIV infection alters the expression of synaptic plasticity genes in neuronal cells which may contribute to the disrupted synaptic plasticity.

Nicotine treated SK-N-MC cells exhibit down-regulation in 23 synaptic plasticity genes (3–35 fold) and up-regulation in 16 genes (3–75 folds). Among these genes, 20 were commonly down-regulated in both nicotine-treated (alone) and HIV infected cells (alone). Recent studies have shown that nicotinic mechanisms influence forms of synaptic plasticity that are thought to underlie learning and memory [35, 36]. Although nicotine is known to have neuroprotective effects, reports indicate that nicotine do not provide any evidence of the amelioration of the learning deficit (and slightly worsened performance during acquisition) observed in the behavioral HIV-1 transgenic (HIV-1Tg) rat model [37]. In few studies with nicotine exposure, improved attention, learning and memory processes were reported in both experimental animals and humans [16, 17, 38]. But the positive effect of nicotine on memory and attention is still a matter of debate in case of chronic nicotine exposure. In this study, we observed both positive and negative regulatory effects of nicotine on synaptic plasticity genes expression in neuronal cells after 7 days of chronic nicotine exposure.

This is the first report to study the impact of nicotine on synaptic plasticity genes expression in HIV infected neuronal cells. HIV infected SK-N-MC cells in the presence of nicotine display a significant reduction in total 47 synaptic plasticity genes expression (3–84 folds) and significant up-regulation in only 4 genes expression (IGF1, NFKBIB, PPP2CA, RAB3A) (3–15 folds). All the genes down-regulated in nicotine/HIV infection alone are also down-regulated (few genes synergistically) in HIV infected cells co-treated with nicotine. The fold down-regulation ranges from 3–84 folds indicating increased neuropathogenicity in HIV infected cells treated with nicotine. Activation of immediate early transcription factors [immediate early genes (IEGs)] leads to the transcriptional activation of synaptic plasticity genes that presumably play an important role in the structural and functional neuronal changes involved in mammalian LTM [39, 40]. Out of 30 immediate early response genes we have analyzed, 18 genes were significantly down-regulated in HIV infected SK-N-MC neuronal cells in the presence of nicotine, indicating transcriptional repression of other synaptic plasticity genes, therefore decreased LTM. Synaptic plasticity gene-gene interactions/relationships were built using the GNCPro Gene Network Central for genes significantly down-regulated in SK-N-MC neuronal cells Infected with HIV (Figure 10a); Nicotine treated (Figure 10b) and HIV infected cells in the presence of nicotine (Figure 10c).

Transcription factor NF- kappa-B (NF-kB) regulates spatial memory formation, synaptic transmission, and plasticity. Inhibition of NF-κB leads to decrease in forskolin-induced CREB phosphorylation [41]. In this study, along with down-regulation of NF-κB1, we have also observed up-regulation of NFKBIB that belongs to the NF-kappa-B inhibitor family, which inhibit NF-κB1 by complexing with, and trapping it in the cytoplasm that may further disrupt the synaptic plasticity in HIV infected neurons in the presence of nicotine.

NCAM1 is another highly down-regulated synaptic plasticity gene in the HIV infected neuronal cells in the presence of nicotine. NCAM1 is a multifunction transmembrane protein expressed in both neurons and glial cells with cell recognition properties involved in cellular migration, synaptic plasticity [42, 43] and central nervous system development [42]. In this study, expression of NACAM1 was recovered with the use of vorinostat treatment.

Two other highly down-regulated synaptic plasticity genes in HIV infected neuronal cells and in combination with nicotine are the RHEB and TIMP1. While RHEB plays an important role in long term activity-dependent neuronal responses [44], TIMP1 is a candidate plasticity gene induced by seizures and by stimuli leading to long term potentiation. Down-regulation of these two genes may lead to the altered learning and memory.

We have also analyzed the dendritic spine density changes in HIV infected SK-N-MC cells co-treated with nicotine. We have got supporting results for the gene expression studies in SK-N-MC showing the significantly reduced spine density in the HIV infected cells co-treated with nicotine than the control, nicotine/HIV infected cells. We have also observed significantly decreased spine density in nicotine/HIV infected neuronal cells than the control cells indicating neurotoxicity in nicotine/HIV alone infected cells as well. Reduced cortical synaptic density has been reported in mild to moderate HAND patient’s autopsy samples [45]. Our study shows that nicotine severely affects spine density during HIV infection. This report provides evidence of increased neuropathogencity in HIV-infected neuronal cells exposed to nicotine and shows the induced synergistic decrease in synaptic plasticity genes expression and spine density than HIV/Nicotine alone infected cells. Among different nicotinic mechanisms influencing synaptic plasticity, role of overexpressed α7-nAChRs, which play a role in neurological disorders [46] was studied with nicotine/HIV. Chronic exposure to nicotine was reported to cause up-regulation of α7-nAChRs [47] and up-regulation of α7-nAChR has been reported as a contributor to the neurotoxicity associated with HIV infection as well [48]. Another important mechanism is the accumulation of excess glutamate in the extracellular space as a consequence of CNS trauma, neurodegenerative diseases, infection, or deregulation of glutamate clearance results in excitotoxicity. Most acute and chronic neuronal diseases, including HAND, have implicated this type of bystander pathology of excitotoxicity [49]. The presence of excess glutamate in the synaptic clefts activates glutamate gated ion channels and results in high levels of ion influx into neuronal cells allowing the over activation of downstream calcium ion-dependent effectors and signaling pathways, culminating in neuronal damage. Nicotine also reported to enhance the glutamate release [50, 51]. Therefore, it may be possible that HIV in combination of nicotine may increase the excitotoxicity mediated by glutamate release.

Further, we have investigated the mechanisms attributed for the transcriptional and translational repression of synaptic plasticity gens in HIV infected cells and in combination with nicotine. We analyzed one of the important mechanisms, the role of HDAC2 in the transcriptional repression of synaptic plasticity genes. Histone acetylation is an epigenetic mark in the chromatin that favors gene expression and is modulated by the balance between histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzyme activities, which add and remove, respectively, acetyl groups from histones. With the use of SKNMC, we showed significant up-regulation of HDAC2 in all the three study groups (1. HIV infected; 2. Nicotine treated; 3. HIV infected and co-treated with nicotine) indicating the possible role of HDAC2 in the transcriptional repression of synaptic plasticity genes in neurons. The evidence for HDAC2 in the negative regulation of memory and synaptic plasticity has been well documented [26, 52] and the HDAC inhibitors especially class-I were reported as cognitive enhancers [53, 54]. Loss of HDAC2 has been reported to improve the associative working memory and accelerated extinction learning [52]. Recently, Hanson et al., reported enhanced excitatory and reduced inhibitory synaptic transmission in HDAC2 knockdown single pyramidal neurons, whereas reduced excitatory and enhanced inhibitory synaptic transmission in HDAC2 overexpressed neurons [55]. Earlier, from our lab, we have reported the up-regulation of HDAC2 in human neurons in response to the HIV-1 Tat protein induced neurotoxicity [56]. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is known to regulate the transcription of many genes [57‐59]. Recently, the role of JAK/STAT pathway in synaptic plasticity has been reported [60].

Further we have used vorinostat, a potent, reversible HDAC inhibitor (both Class I and Class II) to inhibit HDAC2 expression in SK-N-MC cells infected with HIV and/or treated with nicotine. Interestingly, we observed increased HIV infection (latency disruption) in neuronal cells when treated with vorinostat at 1 μM concentration (no significant change in HIV infection in combined treatment with nicotine and vorinostat). Vorinostat reported to disrupt the HIV latency in resting CD4⁺ cells in vitro [61, 62] and in vivo as well [63]. Our results suggest that vorinostat can disrupt the HIV latency in HIV infected CNS cells as well. Therefore, reactivation of latent HIV in the neuronal cells (by using vorinostat) along with antiretrovirals may provide a precise approach to treat and eradicate latent HIV infection in the brain. Vorinostat could also able to inhibit the HDAC2 up-regulation in neuronal cells infected with HIV and/or treated with nicotine. This results in a more relaxed chromatin configuration that allows the transcription of synaptic plasticity genes. Figure 1 shows the schematic representation of role of vorinostat in recovery of synaptic plasticity genes expression in neuronal cells infected with HIV and/or treated with nicotine. We have found that in all the groups (Nicotine, HIV infected, HIV + Nicotine), synaptic plasticity genes expression and spine density were recovered and many synaptic plasticity genes were further up-regulated when treated with vorinostat compared to the control cells. In vorinostat alone treated cells, 19 synaptic plasticity genes were significantly up-regulated and 4 genes (CREM, NFKB1, RHEB, TIMP1) were significantly down-regulated than control cells. Effect of down-regulation of these 4 genes in synaptic plasticity needs to be further evaluated. These results indicate that vorinostat positively regulate synaptic plasticity genes expression and spine density in HIV infected neurons and may be useful as a therapeutic agent in the treatment of HAND. Zolinza (Merck, Whitehouse Station, NJ), a prescription drug contains vorinostat is currently being used for the treatment of cutaneous T-cell lymphoma [64], multiple myeloma [65], mesothelioma [66]. Therefore, further studies to use vorinostat as a therapeutic agent against HAND and in nicotine users need to be studied.

In the down-stream, we have also analyzed one of the important mechanisms i.e. eukaryotic initiation factor 2α phosphorylation (at Ser51), which is responsible for the translational repression of synaptic plasticity genes. Due to partial phosphoryation of eIF2α, general translation is reduced, and ATF4 mRNA translation is augmented. As a consequence, expression of synaptic plasticity and memory-related genes is depressed (Figure 2). Decreased eIF2α phosphorylation reduces ATF4 mRNA translation and enhances general mRNA translation, thus facilitating the induction of synaptic plasticity genes expression, which leads to L-LTP and long-term memory (LTM) consolidation [67]. In our study, we have found increased eIF2α phosphorylation in two-study group of neurons (1. HIV infected; and 2. HIV infected and co-treated with nicotine). It is indicating that translation of synaptic plasticity genes will also be effected in the neuronal cells infected with HIV and in combined treatment with nicotine. To the best of our knowledge, till now, there are no reports of translational repression in HIV infected cells mediated by eIF2α phosphorylation. Therefore, further regulation of eIF2α phosphorylation in HIV infected neurons should be studied to control the translation of synaptic plasticity genes.

There are few reports showing the cognitive impairment in heavy smokers [68‐71]. Effects of cigarette smoking on cognitive disorders in HIV infected patients are scanty [23, 72]. This is the first study in analyzing the molecular mechanisms behind the increased risk of HAND in HIV infected nicotine users. This study concludes that HIV infected nicotine users are more prone to develop HAND symptoms compared to HIV infected patients who are not nicotine addicts, due to the HDAC2 induced transcriptional repression. These negative regulatory effects on synaptic plasticity genes expression and spine density may be reversed by using the vorinostat (class I and II HDACs inhibitor). Vorinostat may be a useful therapeutic to inhibit the neurotoxic effects on synaptic plasticity in HIV infected nicotine abusers.