Therapy with at least three ART medications has been standard treatment for HIV infected patients for since approximately 1995 . Indeed combination ART has dramatically reduced medical morbidity and mortality with HIV infection, but high rates of HAND continue to be reported . Heaton and colleagues sought to determine neurocognitive impairment in large groups of HIV + and HIV - participants from the pre-combination ART era (1988-1995; N = 857) and combination ART era (2000-2007; N = 937). The rates of impairment increased with successive disease stages in both eras: 25%, 42%, and 52% in pre-ART era and 36%, 40%, and 45% in combination ART era. In the medically asymptomatic stage, neurocognitive impairment was significantly more common in the ART era; indicating a possible iatrogenic mechanism whereby ART is salutary in the periphery, but possibly in some way deleterious in the CNS . Furthermore, the pattern of neurocognitive impairment also differed. Patients from the pre-ART era had more impairment in motor skills, cognitive speed, and verbal fluency. On the other hand, ART era patients suffered more memory (learning) and executive function deficits. Importantly, this study showed high rates of mild neurocognitive impairment persist at all stages of HIV infection, despite adequate viral suppression and immune reconstitution with combination ART .
In this context, we sought to identify the risk of adverse neuropathological side effects from various ART regimens in vitro. Here, we elucidate a potential mechanism whereby antiretroviral compounds may have neurotoxic effects, both alone and in combination. This may contribute to the neurological complications that are associated with advanced HIV infection and/or long-term ART seen clinically. Our study shows antiretroviral compounds may effectively increase Aβ generation while possessing the capability to inhibit its clearance by preventing microglial phagocytosis. By affecting both amyloidogenic fronts (generation and clearance), antiretroviral treatment may substantially enhance Aβ aggregation and deposition, which itself is neurotoxic.
We find the most significant amyloidogenic effects when the antiretroviral compounds 3TC, Indinavir, and Abacavir are used in combination (Figure 1). Also, we observe that particular antiretrovirals, Indinavir and Abacavir, may have detrimental additive effects on Aβ microglial clearance (Figure 2). Accordingly, it is likely that certain 3 drug regimens may present an even greater risk of neurological complications.
Though higher CNS-penetrating regimens have been associated with neurocognitive improvement, recent research demonstrates ART might also impart neurotoxic effects; adversely affecting cognition. Indeed in a recent clinical study discontinuation of ART in experienced subjects improved neurocognition and those results were not attributed to practice effects. Furthermore, subjects re-initiated on ART did not experience cognitive gains. Therefore, ART neurotoxicity might explain the unexpected results of this clinical study gains . Past reports have indicated the deleterious effects of ART on the brain were at least in part caused by damage to peripheral neural tissues (for review see ). NRTIs have been shown to induce toxicity in peripheral tissues by altering mitochondrial function, and PIs were shown to damage proteosome function [12, 18]. Also, subjects on didanosine and stavudine regimens had decreased N-acetylaspartate (NAA) concentrations in frontal white matter, a sign of neurotoxicity which positively correlated with treatment duration .
Recently, studies have addressed the influence of five NRTIs (2' 3'-dideoxyinosine, zidovudine, emtricitabine, and tenofovir), one NNRTI (efavirenz), and two PIs (ritonavir, atazanavir sulfate) on neuronal integrity and function. All of the antiretroviral medications tested except for 2' 3'-dideoxyinosine reduced mitochondrial membrane potential. Furthermore, several antiretroviral medications destabilized neuronal intracellular calcium homeostasis, showing a reduced acute response to glutamate . The ability of certain antiretroviral medications and combinations thereof to dysregulate neuronal calcium homeostasis and affect the mitochondrial membrane potential both promote the deposition of Aβ plaques and increased amyloidogenic processing of APP (for review see ). Furthermore, neurons treated with antiretroviral medications exhibited dendritic beading and pruning correlated over a range of doses, which has been linked to cognitive dysfunction .
In our experiments, the median toxic doses for several ARVs were well within the therapeutic concentration range in plasma of HIV-infected patients, and a few showed some signs of damage in the range of CSF concentrations. These initial observations highlight potential adverse effects of high concentrations of antiretroviral medications in the CNS and indicate that there may be some negative tradeoffs to traditionally delivering "therapeutic concentrations" of these compounds to the CNS.
As previous pharmacokinetic studies have confirmed the moderate to high oral bioavailabilities and low to moderate plasma protein binding properties of 3TC, Indinavir, and Abacavir, it is feasible that all of these antiretroviral compounds can reach systemic peak concentrations >100 uM following normal dosing regimens. Needless to say, only a fraction of these concentrations needs to be distributed in the brain to mediate the amyloidogenic effects we observed in our in vitro studies, which employed 10uM concentrations. Furthermore, ART effects in vivo are likely to occur over long-term exposures. Chronic, low dose, in vivo effects of any reagent are often very appropriately modeled in vitro, by proportionately higher doses of that same reagent, over more acute time frames . For these reasons we used 10uM ART dose for these experiments.
This brings us to a potential dilemma in ART concerning an important parameter, blood brain barrier (BBB) permeability. On one hand 3TC, Abacavir, and Indinavir have been reported to be moderately BBB permeable and consequently may be free to promote amyloidosis. On the other hand, these antiretroviral compounds may also be more capable of reducing HIV load in the brain, which may be essential to avoid general HIV encephalopathy. Furthermore, Liu and colleagues  demonstrated that the HIV-1 Tat protein competitively inhibits the LRP receptor, resulting in an inhibition of Aβ clearance. Therefore, by minimizing HIV replication and associated Tat protein expression in the brain, BBB permeable antiretroviral compounds may also prevent amyloidosis. In light of this complex situation, switching from an ART regimen that is associated with both amyloidosis and the potentially related lipodystrophy to one without these adverse effects may be the best course of clinical action to reduce the risk of neurological complications. On the other hand, consistent association of neurocognitive impairment with nadir CD4 across pre-and post-ART eras suggests that earlier treatment to prevent severe immunosuppression may also help prevent HAND. Clinical trials targeting HAND prevention should specifically examine timing of ART initiation .
Future studies are also warranted to investigate the dose-response effects of newer BBB permeable antiretroviral drugs including fusion inhibitors, alone and in combination, on microglial CNS Aβ levels in an in vivo murine model. Moreover, future studies will be required to determine the effect of antiretrovirals on APP proteolysis and microglial Aβ proteases including neprilysin, insulin-degrading enzyme, and endothelin-converting enzymes 1 and 2, as ART modulated proteolytic activity could affect the development of HAND and amyloidosis. This study was also limited to the effect of ART of microglia and neurons separately without other mitigating factors. In the future this work will need to be replicated in vivo in mouse models to not only account for astrocytes, but the entire brain milieu. Nevertheless, it is clear from the most recent update by the World Health Organization in 2010 that the cost of rapid ART scale-up is significant in terms of side-effects . There is a need from patients and health-care providers to phase in less toxic ART regiments while maintaining simplified fixed-dose combinations.