Yin and Yang of PDGF-mediated signaling pathway in the context of HIV infection and drug abuse

Abstract

The control and eradication of neurological complications associated with AIDS continues to be an important goal in efforts toward improving the well being of HIV-infected patients. Although combined antiretroviral therapies have contributed significantly to increasing the longevity of patients by suppressing the virus burden in the systemic compartments, the prevalence of HIV-associated neurological disorders continue to be on the rise. This in turn, leads to an impaired quality of life of the infected individuals who continue to suffer from mild to moderate cognitive decline and memory loss. Developing therapeutic interventions that reverse neuronal injury in the context of HIV infection, is thus of paramount importance in the field. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) has a neuroprotective potential against HIV envelope protein gp120 and Tat. Paradoxically, PDGF is also a cerebrovascular permeant with deleterious effects on the blood-brain barrier resulting in increased influx of monocytes in the CNS. Herein, we review the opposing roles of PDGF in the context of HIV-associated neurodegenerative disorder (HAND).

Introduction

Currently over 40 million people live with HIV worldwide. In the US, the aging population represents one of the fastest growing groups with HIV. The Center for Disease Control estimates that by the year 2015, half of all Americans living with HIV will be over the age of 50. Although combination antiretroviral therapy (cART) has improved the health of millions of those living with HIV, the penetration into the CNS of many of these drugs is limited, and the quality of life of the patients continues to be diminished by milder, residual neurocognitive impairment commonly referred to as HAND. Reversible neuronal loss, decreased neurogenesis and synaptodendritic injury are emerging as important mediators of cognitive deficits associated with HIV-associated neurodegenerative disorder (HAND).

Neurotrophic family of growth factors promote neurogenesis and thus play key roles in neuronal homeostasis (Almeida et al., 2005). Various neurotrophic factors have been implicated in the protection of neurons against cellular and viral neurotoxins including brain derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), nerve growth factor (NGF) & glia cell line-derived neurotrophic factor (GDNF) (Alzheimer and Werner, 2002; Almeida et al., 2005; Deierborg et al., 2008; Colafrancesco and Villoslada, 2011). Findings from our laboratories have identified a novel neurotrophic factor, platelet derived growth factor (PDGF) that plays a crucial role in reversing neuronal toxicity mediated by HIV proteins Tat & gp120 (Yao et al., 2009). Additionally, reports on the neuroprotective potential of PDGFs in various other pathologies are also extant.

Interestingly, in addition to its neuroprotective role, PDGF has also been suggested to have a paradoxical role as a vascular permeant that results in impairment of endothelial barrier during ischemic stroke (Su et al., 2008). Notably, HIV infection of the CNS during late stages of the disease process also results in endothelial barrier disruption and the ensuing neuroinflammation. It has been suggested that HIV-1 can enter the CNS through infected leukocytes likely via the Trojan horse mechanism. Findings from our lab have demonstrated up-regulation of PDGF in newly infiltrating macrophages lining the blood vessels (Potula et al., 2004). More recently we have also validated the role of PDGF as a vascular permeant in human brain microvascular cells exposed to cocaine (Yao et al., 2011a; Yao et al., 2011b). Effect of PDGF on endothelial cells has been the subject of many studies (Sato et al., 1993; Koyama et al., 1994; Cao et al., 2002). Herein we review the opposing roles of PDGF in the context of HIV infection with focus on cell signaling.

Neuroprotective role of PDGF

TRPC channels in neuroprotection mediated by PDGF-BB against HIV Tat in tyrosine hydroxylase (TH) neurons

HIV infection selectively targets the basal ganglia region of the brain leading to dopaminergic neuronal loss (Nath et al., 2000; Mocchetti et al., 2007). Importantly, evidence on the involvement of dopamine impairment observed in HAD comes from case studies on pathological specimens of HIV-infected individuals that demonstrate specific loss of these neurons (Nath et al., 2000). Experimental studies in animals have also demonstrated that nigrostriatal neurons in the region of basal ganglia are highly susceptible to toxicity mediated by HIV-1 proteins (Zauli et al., 2000). Since neurons are not infected by HIV-1, neuronal death is likely due to toxicity exerted by viral and cellular neurotoxins released from either virus-infected or activated cells (Eugenin et al., 2003). One of the key viral toxins implicated in neuronal injury/death is the virus transactivator protein, HIV-1 Tat that not only can be secreted from infected cells but is also taken up by neighboring non-infected cells, such as neurons (Liu et al., 2000). HIV-1 Tat is known to inhibit expression of tyrosine hydroxylase in dopaminergic neurons likely resulting in cognitive abnormalities observed in patients with HAD (Zauli et al., 2000; Everall et al., 2005).

Neuronal homeostasis is a result of the fine balance between neurotrophic and neurotoxic factors. Family of neurotrophic factors, such as BDNF, NGF, and GDNF have been known to play key roles in the protection of neurons against a variety of neurotoxins (Almeida et al., 2005; Arthur et al., 2006; Deierborg et al., 2008). Our findings have identified the role of yet another neurotrophic factor, PDGF that has been demonstrated as a critical factor in the postnatal brain development of rats (Smits et al., 1991).

PDGF is a family of growth factor comprised of five dimeric ligands (PDGF-AA, -AB, -BB, -CC and -DD) that are assembled from four gene products (PDGF-A-D) and bind to two classical tyrosine kinase receptors, PDGF-α (PDGF-αR) and PDGF-β receptor (PDGF-βR). PDGF families of factors play multiple roles during embryogenesis, brain development and in a variety of pathologies in the adult (Andrae et al., 2008). For the sake of clarity, PDGF chains A to D indicate the respective gene, while PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD refer to the protein isoforms. PDGF has been demonstrated to protect primary hippocampal neurons against glutamate-induced neuronal damage (Beazely et al., 2008). In our previous findings using SH-SY5Y cells we have reported that PDGF can mediate neuroprotection against the HIV envelope gp120-mediated toxicity (Peng et al., 2008b; Peng et al., 2008a).

Studies from our laboratories have demonstrated that exogenous PDGF exerted neuroprotective effects against HIV-1 Tat mediated neurotoxicity in primary midbrain neurons. This effect of PDGF in neuroprotection involved the role of transient receptor potential canonical (TRPC) channels. TRPC channels are Ca²⁺-permeable, nonselective cation channels that play key roles in various physiological functions including neuronal survival. Silencing of TRPC channels with either short interfering RNAs (specific for TRPC 5 and 6) or using pharmacological blockers resulted in suppression of both PDGF-mediated neuroprotection as well as elevation in intracellular Ca²⁺ in primary midbrain neurons. In these cells PDGF-mediated neuroprotection constituted parallel but distinct ERK/CREB and PI3K/Akt pathways. Blockage of TRPC channel also resulted in suppression of PDGF-induced Pyk2/ERK/CREB activation, but had no effect on Akt activation. Furthermore, administration of PDGF was able to restore susbtantia nigra dopaminergic neurons from Tat-mediated neurotoxicity. Pre-treatment of mice with the TRP blocker resulted in attenuation of neuroprotection, thus underscoring the novel role of TRPC channels in protection of neurons mediated by PDGF.

Role of synaptic plasticity gene Arc/Arg3.1 in neuronal plasticity mediated by PDGF-BB

Our previous findings had also dissected the role of PDGF in mediating long-term potentiation (LTP). Long-term forms of learning and memory are dependent on rapid synthesis of new RNA and proteins in the cells (Davis and Squire, 1984). Increasing evidence suggests that a set of immediate-early genes including Arc/Arg3.1 (Link et al., 1995), Homer (Brakeman et al., 1997), Narp (Tsui et al., 1996) and cpg-2 are critical for transcription-dependent plasticity. Arc/Arg3.1 gene regulation is very unique since it has been shown that in stimulated neurons Arc mRNA can translocate from the nucleus to the dendrites where it is translated and integrated into the postsynaptic zone along with other cytoskeletal proteins (Steward et al., 1998; Steward and Worley, 2001). Essential role of Arc in synaptic plasticity has also been shown in Arc/Arg3.1 knockdown studies, which leads to selective deficits in LTP (Guzowski et al., 1999). Arc expression in the neurite has thus been implicated as an index of synaptic activation.

We have demonstrated that in the rat hippocampal neurons PDGF-BB regulated the expression of Arc/Arg3.1 gene, which was confirmed in vivo in a model of PDGF injection. In these studies, PDGF-R tyrosine kinase inhibitor-STI-571 was also able to abolish PDGF-BB mediated LTP ex vivo in rat hippocampal slices. Molecular pathway involved in PDGF-mediated induction of Arc/Arg3.1 included activation of the MAPK/ERK (MEK) signaling. Upstream release of intracellular calcium stores was also critical in this process. Pharmacological blocking using inhibitors specific for either MAPK/ERK phosphorylation or calcium release pathway suggested the downstream convergence of the two pathways involving the activation of the immediate early gene (Egr-1).

TRPC1 channel is critical for PDGF-BB-mediated neurogenesis

It is well documented that not only is an increased neuronal damage/loss, but that there are also fewer adult neural stem/progenitor cells (NPCs) present in the dentate gyrus of the hippocampus of patients with HIV-associated CNS disease. A defect in reduced NPCs could in turn, account for increased prevalence of behavioral deficits observed in patients with HAND in the post-ART era. In the adult mammalian brain, new dentate granule cells are continuously generated from neural progenitor cells and are integrated into the existing hippocampal circuitry via a process termed as neurogenesis (Venkatesan et al., 2007). Adult hippocampal neurogenesis is regulated by a number of physiological as well as pathological stimuli. HIV Tat, an early viral protein,has been known to impair neurogenesis (Mishra et al., 2010). Additionally, psychostimulants such as cocaine can also negatively affect the self-renewing capacity of the hippocampus by down-regulating the proliferative capacity of NPCs (Yamaguchi et al., 2004; Yamaguchi et al., 2005; Hu et al., 2006). These findings thus raise the concern that cognitive dysfunction associated with HIV infection and drug abuse could, partially be attributable the impaired neurogenesis in the hippocampus.

Previous investigation from our laboratories has suggested that pre-treatment of rat hippocampal NPCs with PDGF-BB resulted in restoration of proliferation impaired by HIV Tat & cocaine and that this involved signaling via the cognate receptors. In this study essential role of TRPC was also implicated in PDGF-BB-mediated upregulation of proliferation. Signaling pathways involved in this process included parallel but distinct ERK/CREB, PI3K/Akt pathways with the downstream activation of mTOR/4E-BP & p70S6K and NF-kB signaling. Confirmation of this pathway by silencing of TRPC1 resulted in suppression of PDGF-mediated proliferation as well as PDGF-BB-induced ERK/CREB and mTOR/4E-BP & p70S6K pathways. Consistent with this in vitro finding injection of recombinant rAAV2-PDGF-B in the hippocampi of mice resulted in restoration of impaired NPC proliferation mediated by HIV Tat & cocaine. TRPC 1 channel can thus be envisioned as a novel target that regulates cell proliferation mediated by a neurotrophic factor such as PDGF-BB and has implications for development of therapeutic intervention strategies for restoration of impaired neurogenesis mediated by viral proteins and/or drug abuse.

Neuroprotection mediated by another isoform of PDGF, PDGF-CC: Involvement of TRPC-mediated inactivation of GSK 3β

PDGF isoforms are known to exert diverse effects in various cell types. In recent years identification of yet another PDGF isoform, PDGF-CC, which has neuroprotective potential in a variety of neurological disorders is being appreciated (Tang et al., 2010). In addition to testing the role of PDGF-BB, we have also explored the involvement of another PDGF subtype-PDGF-CC, which is the newly identified third membrane of family of five PDGF chains (PDGF A-D) that dimerizes prior to receptor binding.

Neuronal survival in response to neurotrophic factors has implicated the essential role of phosphatidylinositol-3 kinase (PI-3K) (Liot et al., 2004; Zheng and Quirion, 2004; Subramaniam et al., 2005). While most of the downstream effectors of PI-3K mediating neuronal survival have not been completely identified, Akt/glycogen synthase kinase-3β (GSK3β) is strongly implicated to play an essential role in this process (Wu et al., 2007; Wang et al., 2010). Role of TRPC channels in neuronal survival is being much appreciated in recent years (Jia et al., 2007; Sossin and Barker, 2007). It has been demonstrated that PDGF-CC mediated neuroprotection against Tat toxicity involves both TRPC and Akt signaling

In our efforts to understand the effect of viral protein Tat on modulation of intrinsic expression of PDGF-CC, we demonstrated downregulation of the growth factor at both the transcription as well as the translational level in human neuroblastoma SH-SY5Y cells. Reciprocally, pretreatment with PDGF-CC abrogated Tat-induced neurotoxicity by ameliorating apoptosis as well as neurite loss. Using both pharmacological and loss of function approaches the role of PI3K/Akt signaling was suggested as being crucial in PDGF-CC-mediated neuroprotection. Additionally, involvement of TRPC1 was also critical in modulating calcium transients in PDGF-CC-mediated neuroprotection. PDGF-CC via its binding to the cognate receptor mediated inactivation of the downstream mediator (GSK3β with its phosphorylation at Ser-9). Gain and loss of function studies using cells transfected with either the wild type or mutant GSK3β constructs further confirmed the findings. Pre-treatment of cells with either the PI3K inhibitor or TRPC blocker failed to inactivate GSK3β in response to PDGF-CC, thereby implying the intersection of PI3K and TRPC signaling at the common mediator -GSK3β.

MicroRNA-29 mediated regulation of PDGF-B in Opiate and HIV neurotoxicity

Opiate dependence is emerging as a co-morbidity of HIV infection owing to intravenous route of injection. In fact, there is a strong connection between opiate usage and HIV neuropathogenesis as evidenced by accelerated incidence and progression of HAND in HIV-infected opiate users. Various reports have indicated that opiates such as morphine can potentiate HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. Validation of these findings in SIV/macaque model of opiate dependence demonstrated reduced expression of the tropic factor PDGF-B with a concomitant increase in miR-29b in the basal ganglia compared with the SIV-infected controls. Molecular mechanisms involved in downregulation of PDGF-BB implied transport of miR29b via exosomal shuttling from HIV Tat and morphine treated astrocytes to neurons resulting in downregulation of the target gene- PDGF-B with a concomitant decrease in neuronal cell viability. These findings thus shed light on miRNA mediated regulation of the neurotropic genes such as PDGF and that downregulation of these vital tropic factors could in turn, lead to neuronal dysfunction/death manifesting as cognitive decline observed in HAND

Similar to most chemokines and growth factors, PDGF also exerts paradoxical effects in various cell types. The deleterious effects of PDGF in the context of HIV infection will be the discussed below.

Neuropathological role of PDGF

Role of PDGF as a vascular permeant

As described earlier HAND remains a common complication of HIV even in presence of virus suppression in the systemic compartment. In fact, HAND etiology involves a chronic low level activation leading to age-related morbidities (Nath et al., 2000). Inability of the medications to penetrate into the CNS further results in brain becoming a sanctuary for the virus and viral proteins. This in turn, leads to the ensuing neuroinflammation and endothelial breach commonly observed in HAND (Su et al., 2008). Our previous findings have demonstrated upregulation of PDGF in the brains of macaques with SIV encephalitis specifically in newly migrating macrophages surrounding the blood vessels (Potula et al., 2004). Intriguingly endothelial cells lining the blood vessels also demonstrated upregulation of PDGF in response to virus proteins. Since PDGF is a known vascular permeant as demonstrated in studies by Su et al on ischemic stroke(Su et al., 2008), it is likely that PDGF expressed by and around the blood vessels during SIVE, could be a factor contributing to endothelial barrier breach, adding validity to this hypothesis are various reports demonstrating deleterious effects of PDGF on endothelial cells (Sato et al., 1993; Koyama et al., 1994; Cao et al., 2002).

In addition to endothelial cells, astrocytes comprising the neovascular unit are also the source of PDGF. In fact, our previous study has demonstrated that exposure of rat and human astrocytes to the HIV-1 protein Tat resulted in the induction of PDGF at both the mRNA and protein levels via activation of ERK and JNK signaling pathways and the downstream transcription factor Egr-1. Exposure of astrocytes to PDGF-BB in turn, leads to increased proliferation and the release of proinflammatory mediators such as the chemokine monocyte chemotactic protein-1 (MCP-1). MCP-1 is a key chemokine that facilitates the recruitment of monocytes into the brain contributing to neuroinflammation and BBB leakiness (Fuentes et al., 1995; Weiss et al., 1999; Eugenin and Berman, 2007). This chemokine has been extensively studied and is expressed by a number of cell types including astrocytes, microglia and neurons (Lee et al., 1993; Rock et al., 2006). Elevated expression of MCP-1 has been demonstrated in various diseases including multiple sclerosis, amyloid lateral sclerosis, lupus nephritis, peripheral neuropathy, Alzheimer's disease and HIVE (Ransohoff et al., 1993; Berman et al., 1996; Van Der Voorn et al., 1999; Mahad and Ransohoff, 2003; Yamamoto et al., 2005; Tanuma et al., 2006; Groh et al., 2010; Marks et al., 2010). Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

Cocaine-mediated induction of PDGF: Implication for increased vascular permeability

Drug abuse has been implicated as a contributing risk factor for increased neuroinflammation associated with HIV-1 infection. Intriguingly, cocaine has been shown to disrupt the BBB (Fiala et al., 1998; Zhang et al., 1998; Fiala et al., 2005). Cocaine-mediated effects on the BBB are complex involving both direct pro-apoptotic effects on the endothelial cells as well as indirect paracrine effects that are manifested by pro-inflammatory modulators such as chemokines and cytokines (Zhang et al., 1998).

In keeping with the role of PDGF-CC as a vascular permeant that augmented barrier permeability during ischemic stroke (Su et al., 2008), our findings have also demonstrated a similar role of PDGF in disrupting the endothelial barrier. In our findings exposure of human brain microvascular endothelial cells (HBMECs) to cocaine resulted in induction of PDGF-BB via the binding of cocaine to its cognate sigma receptor, with subsequent activation of MAPKs and Egr-1 pathways. In vivo validation of the role of PDGF-BB in mediating brain endothelial permeability was validated in mice injected with cocaine in the presence or absence of pre-treatment with PDGF-BB neutralizing antibody.

PDGF-B chain is a novel target gene of cocaine-mediated Notch1 signaling

Notch signaling has emerged as an important regulator of neuronal homeostasis. Similar to the physiological effects of PDGF, Notch signaling also mediates intercellular signals that affect diverse physiological functions such as proliferation, survival and differentiation (MacKenzie et al., 2004; Noseda et al., 2004). Notch receptors (Notch1 to 4) are transmembrane proteins that bind to endogenous ligands such as Delta and Jagged-1 leading to proteolytic cleavage of the Notch receptors by the γ-secretase enzyme complex and a concomitant release of the Notch intracellular domain (NICD). NICD in turn, translocates to the nucleus and interacts with the DNA binding factor CSL (also known as RBP-Jk and CBF1), leading to transactivation of promoters for the Notch target genes HES and HEY. Additional immediate downstream genes of Notch signaling have also been characterized, suggesting the existence of a larger immediate Notch transcriptome. As an example, PDGF-βR has been shown to be a target of Notch signaling gene in vascular smooth muscle cells (Jin et al., 2008). More recently PDGF-B chain has also been identified as a new member of the Notch target gene in human HBMECs, thereby implicating Notch signaling as a key player in the maintenance of BBB integrity. Our previous study demonstrated that cocaine-mediated activation of Notch1 signaling leading to targeted expression of PDGF-B involved activation of the downstream effector CSL. These findings provided the first evidence of involvement of Notch1 activation in cocaine-mediated regulation of PDGF-B expression.

Conclusions

In the brains of individuals with HAND, upregulation of chemokines in the CNS is considered a correlate of neuroinflammation. However, recent evidence raises the possibility that, in addition to their role as chemoattractants, chemokines can also functions as neurotransmitters or neuromodulators (Rostene et al., 2007). An example of this is the chemokine fractalkine that functions as an inflammatory mediator by attracting peripheral macrophages into the brain and opposingly, can also serve as a neuroprotective factor (Tong et al., 2000; Eugenin et al., 2003). Moreover, it can also function to regulate neuronal survival through its antiapoptotic effects (Meucci et al., 1998).

In response to cellular damage, the host is also capable of generating trophic growth factors such as PDGF which, similar to fractalkine, can also exert somewhat paradoxically diverse roles. During the progression of HIV infection PDGF upregulation by macropahges and endothelial cells can results in increased expression of MCP-1 resulting in enhanced neuroinflammation and a concomitant breach of the blood brain barrier. On the other hand, PDGF can also provide tropic support for the neurons against a variety of neurotoxic mediators (cellular and viral products). Thus, depending on the cell type within the tissue, the same host factor can manifest diverse activation responses leading to diverse outcomes. The ultimate outcome of infection in the CNS (neuronal survival versus damage) is thus a result of the ensuing shift in balance between the neurotrophic versus neurotoxic products manifested over time following infection. Figure 1 summarizes the diverse functions of PDGF, both as a neuroprotective agent and also as a vascular permeant in the setting of HIV toxicity and drug abuse. Since this sort of paradoxical regulation is a common theme of the cytokines and growth factors, caution has to be exercised in development of therapeutic targets involving these mediators.

Figure 1. Schematic illustration demonstrating the paradoxical role of PDGF-BB in the context of HAND and drug abuse.

Yang role of PDGF-BB indicated that PDGF-BB-mediated engagement of the activation of TRPC 1 channels resulting in Ca²⁺ influx transients with subsequent activation of ERK/CREB, but not the Akt/NF-kB pathway, culminating in neuroprotection. Yin role of PDGF-BB indicated that exposure of cocaine leads to sigma receptor-mediated activation of ERK and JNK/Egr-1 or Notch pathways, result in PDGF-BB expression with concomitant BBB damage and neuroinflammation.

Abbreviation

BDNF

brain-derived neurotrophic factor

cART

combination antiretroviral therapy

FGF

fibroblast growth factor

GDNF

glia cell line-derived neurotrophic factor

GSK3β

glycogen synthase kinase-3β

HAND

HIV-associated neurodegenerative disorder

LTP

long-term potentiation

MCP-1

monocyte chemotactic protein-1

NGF

nerve growth factor

NICD

Notch intracellular domain

NPC

neural stem/progenitor cells

PDGF

platelet derived growth factor

PI-3K

phosphatidylinositol-3 kinase

TH

tyrosine hydroxylase

TRPC

transient receptor potential canonical