Introduction
Despite introduction of combination antiretroviral therapy (cART), HIV-associated neurocognitive disorder (HAND) continues to affect approximately 50 % of HIV(+) patients (Dore et al.
1999; Heaton et al.
2010). Furthermore, in the cART era, the underlying neuropathology has shifted from overt subcortical involvement to a more insidious cortical damage (Gannon et al.
2011). Various factors, such as poor adherence to drug regimen, emergence of resistant virus species, and residual viral DNA in the central nervous system (CNS), may contribute to these changes (Gannon et al.
2011). However, another likely contributor to HAND in the cART era is the virtually unstudied potential for antiretroviral (ARV)-related toxicity in the CNS. cART has decreased HIV-related morbidity and mortality by limiting T cell loss and controlling opportunistic infections. However, cART regimens are associated with potentially serious side effects, including dyslipidemia, lipohypertrophy, and increased risk of atherosclerosis (Vidal et al.
2010). Additionally, cART-associated toxicity in the peripheral nervous system is well documented, and it is likely that cART would trigger similar responses in the CNS. Pharmacokinetic studies suggest limited ARV penetrance into the CNS and indicate low cerebrospinal fluid (CSF) and parenchymal drug concentrations (Yilmaz et al.
2004; Yilmaz et al.
2009). However, direct blood–brain barrier (BBB) compromise by viral proteins and neuroinflammation and indirect BBB impairment due to concomitant factors, such as coexisting infections, can lead to increased CSF and parenchymal drug concentrations. Thus, the impact of ARVs in the CNS of HIV(+) patients is clinically relevant and must be examined.
Initial cART usually includes two nucleoside/nucleotide reverse-transcriptase inhibitors (NRTIs) in combination with a non-nucleoside reverse-transcriptase inhibitor (nNRTI) or with a protease inhibitor (PI) boosted with a low dose of a second PI, Ritonavir. NRTIs and nNRTIs bind to the HIV reverse-transcriptase enzyme and inhibit proviral DNA synthesis; PIs inhibit viral proteases needed for virus maturation and assembly. Despite some crossover, certain ARV classes are more highly associated with particular side effects and toxicities than are other classes. PIs alter lipid metabolism and induce the endoplasmic reticulum stress response in macrophages, linking PIs to increased risk of atherosclerosis (Touzet and Philips
2010). NRTIs inhibit DNA polymerase γ and lead to decreased mitochondrial DNA, loss of mitochondrial membrane potential, and oxidative phosphorylation, consequently precipitating oxidative stress (Nolan and Mallal
2004). Previous studies exploring possible side effects of ARVs in the CNS are scarce and mostly involve cell lines (Cui et al.
1997). Given the mutations and aberrations in immortalized cell lines, these studies may not reflect the ARV toxicity potentially occurring in biological settings. Peripheral dorsal root ganglia neurons are the only primary cell type of neural lineage previously studied for ARV toxicity (Werth et al.
1994). In this report, we examined effects of ARVs in primary CNS neurons both in vivo and in vitro. Our findings suggest that cART induces oxidative stress and neurotoxicity in the CNS, and that the patients on long-term cART regimens would benefit from adjuvant therapies that include antioxidant strategies to overcome deleterious effects of cART in the CNS.
Materials and methods
Chemicals and reagents
Chemicals and reagents comprise the following: (1) AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, antiretroviral reagents; (2) Abcam (Cambridge, MA), mouse monoclonal NAD(P)H/quinone oxidoreductase-1 (NQO-1) antibody (A180) and mouse monoclonal synaptophysin antibody (SY38); (3) BioRad (Hercules, CA), Biosafe Coomassie stain, immunoblot polyvinylidene fluoride (PVDF) membrane and prestained broad range molecular weight ladder; (4) Cell Signaling Technology (Danvers, MA), rabbit polyclonal antibody raised against cleaved caspase-3; (5) Citifluor, Ltd. (London, UK), citifluor AF1. Covance (Princeton, NJ), mouse monoclonal microtubule-associated protein 2 (MAP2) antibody (SMI-52); (6) Dako (Carpinteria, CA), rabbit polyclonal glial fibrillary acidic protein (GFAP) antibody (Z0334); (7) Enzo Life Sciences (Farmingdale, NY), rabbit polyclonal antibody to heme-oxygenase-1 (HO-1); (8) Frontier Scientific (Logan, UT), Sn(IV) mesophorphyrin IX dichloride (SnMP); (9) Jackson ImmunoResearch Labs (West Grove, PA), fluorescein isothiocyanate-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-rabbit IgG secondary antibodies; (10) Invitrogen (Carlsbad, CA), Dulbecco's modified Eagle's medium (DMEM), tetramethyl rhodamine methyl ester, goat anti-mouse beta-lactamase TEM-1 conjugate, fluorocillin green substrate, dihydroethidium (DHE), 4′,6-diamidino-2-phenylindole (DAPI), neurobasal media, and B27 supplement; (11) New England Biolabs (Ipswich, MA), tyramide amplification system; (12) Peptide International (Louisville, KY), poly-l-lysine; (13) Sigma Aldrich (St. Louis, MO), carbonyl cyanide m-chlorophenyl hydrazone, cytosine arabinoside (Ara-C), fetal bovine serum (FBS), monomethylfumarate (MMF), oligomycin, propidium iodide, and PI cocktail; (14) ScyTek Labs (Logan, UT), normal antibody diluent; (15) Thermo Scientific (Waltham, MA), goat anti-rabbit horse radish peroxidase (HRP) antibody and goat anti-mouse HRP antibody, SuperSignal West Dura extended duration substrate; and (16) Tocris Bioscience (Ellisville, MO), thapsigargin. The antibody against calpain-cleaved spectrin was a generous gift from Dr. Robert Siman (The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA).
Discussion
Virus-related factors, such as resistant virus species and persistent viral DNA in the CNS, may contribute to the persistence of HAND in the post-cART era. One recent focus in HIV neurovirology is the development of ARVs with greater CNS penetrance (Letendre et al.
2008; Marra et al.
2009; Tozzi et al.
2009; Edén et al.
2010; Heaton et al.
2010; Garvey et al.
2011; Smurzynski et al.
2011). However, potential ARV toxicity in the CNS remains largely unexplored. Our study supports a possible contribution of the ARVs themselves to neuronal and synaptic damage observed in patients with HAND.
In this study, we show cART-induced synaptophysin loss, indicative of synaptic injury in two animal models. In our first model, in an in vivo model of SIV-infected pig-tailed macaques, we report decreased synaptophysin and CaMKII levels in the SIV(+)/cART group compared with uninfected or SIV(+)/placebo groups, indicating synaptodendritic damage. Interestingly, MAP2 levels did not change significantly across groups, which may be because of the relatively short duration of infection in this retrospective study cohort. While these data demonstrate potential effects of cART drugs in the presence of viral infection, there are three variables to consider in interpretation of these findings: (1) the time to euthanasia from the start of the experiments is different between SIV(+)/placebo and SIV(+)/cART groups, (2) persistent viral DNA in the CNS of SIV(+)/cART group, and (3) the lack of SIV(-)/cART group. It is not yet known whether brain SIV DNA is replication competent. As we utilized post-mortem samples obtained from a cohort of macaques enrolled in a previous study addressing the efficacy of CNS penetrant cART in reducing viral loads in the CNS, further experiments that include an additional control group receiving cART but not inoculation with SIV will be instrumental to more clearly determine the contribution of viral DNA and cART to synaptic damage in this model.
In the second in vivo model presented here, adult rats received a therapeutically relevant combination of ARVs (NRTI+PI+Rit boost). In the small number of studies where pharmacokinetics and effects of ARVs in the CNS were examined, pathological read-outs of neuronal damage, such as MAP2 or synaptophysin loss, were not determined (Huisman et al.
2003; Anthonypillai et al.
2004; Anthonypillai et al.
2006). Synaptic injury is a known indicator of neuronal damage and dysfunction in various neurodegenerative diseases, including HAND (Gupta et al.
2010) and synaptodendritic injury persists in HIV-infected individuals in the post-cART era (Xu and Ikezu
2009). We observed decreases in synaptophysin and MAP2 protein levels in the hippocampus in response to ARV administration over 7 days. Thus, our model demonstrates that ARV-associated neurotoxicity warrants consideration in developing therapeutic regimens for HIV-infected patients.
We also show that the PIs, Rit and Saq, alone or in combinations with the NRTI, AZT, induce oxidative stress, and neuronal damage/death in primary cultures at clinically relevant doses. Previous studies examined ARV-induced toxicity in cell lines, and Robertson et al. have provided the first evidence for ARV-induced neurotoxicity in primary rat neurons (Robertson et al.
2012). Here, we provide further evidence that PI-induced oxidative stress and neuronal death in primary neurons can be blocked by the activation of the endogenous antioxidant response. Interestingly, in our experimental paradigm, the NRTI, AZT, neither induced neuronal damage/death by itself, nor augmented PI-induced damage/death when used in combination. We observed similar effects from a combination using another NRTI, stavudine (d4T; not shown). In agreement with our observations, a previous study presented similar findings, specifically that neither AZT nor d4T inhibited cell growth or neurite regeneration in PC-12 cells after long-term drug exposure (Cui et al.
1997). It should be noted that NRTIs are unequivocally tied to peripheral neuropathy, where the underlying pathology is mitochondrial toxicity and oxidative stress. However, NRTIs affect only certain cell populations, as they are formulated as pro-drugs and, to become active, need to be phosphorylated by two kinases, thymidine kinase 1 and 2 (TK1 and TK2), and the expression of the cytoplasmic TK1 is cell cycle dependent (Bazzoli et al.
2010). Thus, in our model utilized to study post-mitotic neurons, AZT is most likely not converted to its active form, and thus does not contribute to neuronal damage/death. It is of note that our in vitro model of ARV-induced neurotoxicity utilized primary neuroglial cultures rather than cell lines. Primary cells are untransformed, and therefore more accurately reflect and predict ARV-associated effects occurring in the brains of patients on cART than would immortalized cell lines. The molecular pathways we investigated in this study are highly conserved from yeast to human cells; thus, the results obtained here in cells of rodent origin are likely conserved in human cells as well.
The drug concentrations used in this study are based on the plasma and CSF levels reported by various in vitro and in vivo studies(Huisman et al.
2003). As reported in such studies, AZT can be detected in the CSF at concentrations that are similar to those measured in the plasma (Wynn et al.
2002). Contrarily, as backed by various in vitro and in vivo studies (Wynn et al.
2002), both Rit and Saq are predicted to have limited CNS penetrance due to the strong tendency of these drugs to bind plasma protein because of their lipophilic nature and pharmacokinetic properties. However, a comprehensive study conducted in an in situ guinea pig model suggests that Rit can achieve high concentrations in choroid plexus and parenchyma through diffusion via the choroid plexus; in fact, the levels of Rit were comparable to levels measured in plasma (Anthonypillai et al.
2004). Furthermore, the study showed that, surprisingly, the CSF levels of Rit were lower than the levels measured in the choroid plexus and the parenchymal compartments. Thus, the authors concluded that CSF concentrations of Rit may not necessarily reflect the parenchymal levels, which are indeed a better indicator of effective drug levels in the CNS. Rit and Saq concentrations in our experiments fall well within plasma ranges reported in patients receiving cART.
Evidence of oxidative stress has long been associated with the most severe forms of HAND (Ngondi et al.
2006; Mielke et al.
2010). Interestingly, despite systemic control of viral replication in cART-treated patients, markers of oxidative stress, such as elevated levels of lipid and protein oxidation, are still detectable in the brains of these individuals (Ances et al.
2008). Of note, oxidative stress is one of the underlying mechanisms involved in NRTI and PI-induced toxicity in the periphery. Our data suggest that the sustained ROS accumulation in neurons due to prolonged exposure to ARVs might induce the oxidative stress associated with cART-induced toxicity in the CNS, leading to the observed changes in synaptophysin levels and the subsequent neuronal death.
Our data also suggest that astrocytes do not show neither ROS accumulation nor the endogenous antioxidant response in vitro. We also observe that astrogliosis in SIV-infected animals is resolved in cART-treated animals. While these findings suggest that astrocytes may not be highly impacted by ARVs in the short term, it is possible that prolonged exposure to ARVs might overwhelm astrocytes, which help buffer ROS accumulation in neurons under normal conditions, precipitating further neuronal damage.
We further show that the ARV drug-induced effects observed in this study can be blocked by MMF via activation of an endogenous antioxidant response. Cells respond to oxidative stress by activating the transcription of a subset of genes in an effort to clear excess ROS within the cell. Among these genes are NQO-1 and HO-1 (Chen and Kong
2004). Here, we show increased levels of NQO-1 and HO-1 mRNA and protein in ARV-treated neuronal cultures. Additionally, pretreatment of cultures with MMF led to further increases in HO-1 protein levels in ARV-treated cultures and provided protection against ARV-induced toxicity. Finally, our finding that the protection provided by MMF is reversed with chemical inhibition of HO-1 further provides evidence that ARV toxicity is mediated via induction of oxidative stress. The neuronal damage and death that occurs despite the cellular initiation of the endogenous antioxidant response following exposure to ARVs suggests that this response may be insufficient or too delayed to protect cultures from ARV toxicity. In support of this explanation, we observed that MMF-mediated augmentation of antioxidant responses is strongly protective against the neurotoxic effects of ARVs. Further studies using this in vitro model are warranted to determine whether other clinically relevant ARV drug combinations induce neurotoxicity and, if so, to establish whether similar pathways are involved.
DMF, a psoriasis treatment used in Europe since 1994, is currently being tested as a disease-modifying agent for multiple sclerosis (MS) (Anonymous
2011; Krieger
2011; Linker et al.
2011). MMF is the active DMF metabolite in vivo. Our report is the first to show MMF as a neuroprotectant against ARV-induced damage. Data from studies in MS patients show that DMF/MMF has good tolerability, can cross the BBB efficiently, and has relatively few and minor side effects. Furthermore, we have recently shown that DMF suppresses HIV replication, induces the antioxidant response in macrophaghes, and blocks neurotoxin release from macrophages (Cross et al.
2011). Overall, our findings make this immunomodulatory and antioxidant agent a good potential adjunctive therapeutic for use in HAND.
One of the paradoxical outcomes of cART is the persistence of HAND, despite successful viral control. Moreover, there is a shift from overt dementia to subtler neurocognitive impairments. Based on data presented here, we propose that different mechanisms by which HIV infection and cART induce neuronal damage underlie the changing neuropathology of HAND. Extensive studies have shown that during lentiviral infection, viral proteins and soluble factors secreted by infected cells lead to neuronal and synaptic damage via several direct as well as indirect mechanisms involving several cell types, including astrocytes. However, our data suggest that cART-mediated synaptic damage may involve direct mechanisms occurring specifically in neurons, such as oxidative stress, and that neurons, and not astrocytes, are the primary targets of cART-mediated damage in the CNS.
It is most likely that chronic exposure to cART regimens including neurotoxic ARVs over many years is associated with a slower, nonetheless insidious changes including synaptic damage in the absence of neuronal loss, and these neuronal perturbations may contribute to, and may even precipitate, some of the clinical and pathological changes observed in the chronic course of HAND in the cART era. While detrimental, such slow damage also suggests that alterations in cART regimens to include ARVs with low neurotoxicity profiles, such as NRTIs or nNRTIs may halt synaptic damage, and provide a point where previous damage may be reversed, either due to the withdrawal of the toxic drug, or with the help of an adjuvant, such as fumaric acid esters. Future studies, first in primates, then in humans, will be crucial to explore the specific impact of treatment interruption on recovery from cART-mediated neuronal damage and to determine the eddicacy of potential adjunctive therapies necessary to mitigate the side effects of cART in the CNS.