|Home | About | Journals | Submit | Contact Us | Français|
Work from our laboratories has validated mixed lineage kinase type 3 (MLK3) as an enzyme pathologically activated in the central nervous system (CNS) by HIV-1 neurotoxins. In this review, we discuss MLK3 activation in the context of the neuropathogenesis of HIV-1 associated neurocognitive deficits (HAND). We use findings from the literature to substantiate the neuropathologic relevance of MLK3 to neurodegenerative disease, with an emphasis on Parkinson’s disease (PD) that shares a number of important phenotypic and neuropathologic characteristics with HAND. We discuss signal transduction pathways downstream from MLK3 activation, with an emphasis on their involvement in microglia and neurons in preclinical models of HAND. Finally, we make a case for pharmacologic intervention targeted at inhibition of MLK3 as a strategy to reverse HAND in light of the fact that combination antiretroviral therapy, despite successfully managing systemic infection of HIV-1, has been largely unsuccessful in eradicating HAND.
Combination antiretroviral therapy (cART) has transformed HIV-1 infection, once almost uniformly fatal to adults and children, into a chronic, manageable disease. Prior to the advent of cART, HIV-1 infection frequently resulted in a devastating subcortical dementia (HIV-1 associated dementia or HAD) in adults and a progressive encephalopathy (PE) in children. After the widespread availability of cART, HAD and PE all but disappeared, but a more indolent phenotype, HIV-associated neurocognitive disorder (HAND) remains a frequent, insidious complication of HIV-1 infection that pervasively diminishes quality of life and overall functionality for the activities of daily living. However, ongoing longitudinal and cross-sectional studies of adult and pediatric patients with HIV-1 have shown that efficacious control of viral load with cART, has not eradicated the constellation of cognitive neurologic disease that comprises HAND. Current interim estimates of aggregate data from the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) consortium suggest that 52% of their cohort (Total N = 1562) have some form of HAND. Thus, despite widespread availability and adherence to cART, the prevalence of HAND has actually increased.[1, 2] Moreover, neuropathologic retrospective analyses from patients in the post-cART era have shown a 17.5% incidence of parenchymal brain pathology and cART with increased CNS penetration has been associated with poorer neurocognitive performance - raising the possibility that chronic exposure to cART may have long-term neurotoxic consequences independent of HAND.
More effective forms of cART may be possible through the use of agents capable of reactivating and flushing otherwise stable viral reservoirs.[4–6] However, the development and clinical testing of new drugs capable of reversing HIV-1 latency in the CNS will present unique hurdles and may require considerable time to bring to fruition. As a result, effective adjunctive therapies for HAND will remain a crucially important unmet medical need for the foreseeable future. This is especially true in light of the lack of efficacy of other candidate adjunctive therapies for HAND - as summarized in a recent meta-analysis of ten trials of adjunctive therapies involving some 711 subjects. It is worth adding that most of the agents studied in these trials had not been evaluated in an in vivo model to directly test whether the candidate adjunctive therapy was efficacious in reversing either neuroinflammation or restoring synaptic function.
A 2003 study of HIV-1 infected subjects; previously naïve to antiretroviral therapy, who received 3 months of cART and subsequently underwent neuropsychologic tests and magnetic resonance spectroscopic (MRS) studies, demonstrated significant improvement in CD4 counts, suppression of plasma and CSF viral loads. However, the study cohort had persistent elevations of MRS parameters creatine (Cr) and choline (Cho), which served as markers of inflammation in the frontal lobes. Additionally, neuropsychologic impairments persisted and Cr and Cho increased in the basal ganglia after CART regardless of whether one or two cART agents effectively penetrated the BBB.
A year prior to this study, Ernst et al. showed that there was an increased magnitude and volume of activation measured by blood oxygen level dependent (BOLD) functional neuroimaging studies in the lateral prefrontal cortex of cognitively normal HIV-1 subjects while performing neuropsychologic tasks of increasing difficulty compared to matched HIV-1 seronegative controls. Seven years later, the authors conducted an equally intriguing BOLD imaging study during which HIV-1 positive and negative subjects performed tasks of increasing attentional load at an initial trial and a one year follow up trial. While both sets of subjects had normal neuropsychologic indices at the initial and follow-up visits, during the follow-up visit the HIV-1 positive subjects had increased magnitudes of brain activation in the prefrontal and posterior parietal cortex with high load attentional tasks while the HIV-1 seronegative subjects had decreased magnitudes of brain activation.
Collectively, these studies suggest that HIV-1 infection in the CNS is a chronic neuroinflammatory condition that affects the brain’s ability to achieve increased synaptic efficiency, the basis for long term potentiation (LTP) that is associated with learning and memory, and that to compensate, the brain must activate more poly-synaptic networks to achieve normal processing of information. This places increased metabolic demands on the synapse, increasing the potential for permanent damage to synaptic architecture. It also forecasts an increasing neurologic burden for the aging population with HIV-1, who may also be susceptible to developing Alzheimer’s (AD) and Parkinson’s disease (PD), because they will sustain an additive or even synergistic type of neurodegeneration. Thus we posit that for therapies to be effective at reversing HAND, they must reverse inflammation at the synapse before neuronal function can be ameliorated.
Development of neuroprotective therapies for neurodegenerative disease face extraordinary challenges, related to both the complexity of neuropathogenesis, bioavailability of drugs in vulnerable brain regions and timing of treatment. For example, AD and PD likely begin many decades before a clinically significant phenotype is noticeable; thus therapeutic strategies face an uphill battle because damage to vulnerable neuronal pathways may have passed a critical point for repair and return to pre-morbid homeostasis. In contrast, HAND provides some unique opportunities for therapeutic intervention not least because (i) the onset of HIV-1 infection is usually a definable event that can be routinely tested and (ii) early events in HAND likely involve reversible synaptic injury (see above).
Despite this, previous trials of adjunctive therapy for HAND have been unsuccessful. As noted above, a recent meta-analysis of the published outcomes of ten trials of adjunctive therapies involving 711 people with HIV-1 and neurologic disease, concluded that there was no evidence for efficacy in terms of cognitive improvement. While many of these agents had not been evaluated in an appropriate in vivo model of HAND, a notable exception was memantine. Memantine blocks the NMDA receptor-associated ion channel only when it is excessively (i.e. pathologically) open, and as such, does not remain in the channel long enough to block normal excitatory neurotransmission. Despite success in preclinical small animal models, memantine failed to significantly improve any neuropsychologic indices in a phase II double-blind, randomized, placebo-controlled, multi-center trial (plus best anti-retroviral therapy) in patients with HAND. Interestingly, this was the first clinical study of HIV-associated dementia in which surrogate markers (magnetic resonance spectroscopy, i.e. MRS) were validated with clinical presentation, as quantified by neurocognitive testing, and in which the surrogate markers appeared to be even more sensitive than the cognitive testing or neurologic examination. MRS was used to quantify N-acetylaspartate (NAA), a metabolite that is chiefly found in neurons in the adult brain, and the ratio of NAA to creatine (Cr) was used as an index of neuronal function and viability. Significant increases in NAA/Cr were observed in the multivariate analysis among individuals receiving memantine compared to placebo in the frontal white matter and the parietal cortex. Thus, memantine’s failure to elicit clinical improvements in neuropsychologic parameters despite an increase in the MRS parameter NAA that was presumably indicative of preserved neuronal integrity in corresponding brain regions, may reflect the fact that neuroinflammation can prevent activity-dependent (i.e. LTP) processes both upstream and downstream of the NMDA-R channel.[13–15]
This emphasizes the need to design adjunctive therapies that take into account both neuroinflammation caused by HIV-1 viral products and pro-inflammatory mediators, and the activity-dependent nature of neuronal networks vulnerable to these neurotoxins, rather than simply quantifying neuronal apoptosis or even synaptic transmission as a therapeutic endpoint.
Mixed-lineage kinases (MLKs) are MAPK kinase kinases (MKKKs) with features of both serine-threonine and tyrosine kinases (hence the nomenclature “mixed lineage”) that regulate the c-Jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPK) signaling cascade, and also regulate the other two major MAPK pathways, p38 and extracellular signal-regulated kinase (ERK).[16–18]
MLK-3 (aka MAP3K11) is the most widely expressed MLK family member,[16–18] and is expressed in neurons, dendritic cells,[20, 21] and many other cell types. At the cellular level, MLK3 is activated by cellular/metabolic stress, including reactive oxygen species, ceramide and TNFα.[22, 23] At the molecular level, MLK3 is activated by the small GTPases Cdc42 and Rac, which bind to the interactive binding region of MLK3, and can cause it homodimerize via a leucine zipper interface, resulting in autophosphorylation at Thr277 and Ser281 within the protein activation loop, ultimately resulting in MLK3 activation.[24, 25] HIV-1 Tat also leads to phosphorylation at these same residues, with accompanying activation of MLK3 in primary rat neurons.
MLK3 has been implicated in neuronal apoptosis leading to neurodegenerative disease.[26–29] In the context of Parkinson’s disease (PD), the first-generation MLK3 inhibitor, CEP-1347, has been shown to prevent the induction of neuronal cell death, motor deficits and neuronal degeneration in the MPTP model of Parkinsonism,[30–33] and CEP1347-mediated neuroprotection has also been demonstrated in an in vitro model for PD, using methamphetamine-exposed human mesencephalic-derived neurons. In addition, MLK3 has been implicated as playing a causal role in peripheral neuronal degeneration, including the development of HIV-associated peripheral neuropathy, which can be induced both by soluble HIV-1 gene products and also by the antiviral drugs used to treat HIV-1.[34, 35]
The results of a Phase II trial of CEP1347 for treatment of PD were reported in 2007. This trial, the Parkinson Research Examination CEP-1347 Trial (PRECEPT), enrolled PD patients in the early stages of disease and not yet requiring dopaminergic therapy (N = 806). The primary endpoint was time to disability requiring dopaminergic replacement therapy with L-dopa. Unfortunately, the study was concluded early due to futility, raising questions about the appropriateness of the PD models used in preclinical studies with CEP-1347, as well as about the CNS pharmacokinetics, target selectivity and mechanism of action of CEP-1347 itself. Wang and Johnson  surmised that failure of the PRECEPT trial was due to the fact that CEP-1347 inhibition of MLK3 in dopaminergic neurons was insufficient to ensure survival without additional trophic support from brain-derived neurotrophic factor (BDNF) signaling via TrkB receptors.
In vitro studies of Cephalon’s “first generation” MLK inhibitor, CEP1347, showed that this agent can protect primary rat hippocampal neurons as well as dorsal root ganglion neurons from the otherwise lethal effects of exposure to HIV-1 gp120.[34, 35] Studies from our laboratories subsequently examined the effect of HIV-1 Tat and gp120 on MLK3. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons and this was abolished by the addition of CEP1347. CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, over-expression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat.
We further confirmed and extended these in vitro findings – by showing that CEP1347 is neuroprotective in an in vivo model of HIV-1 infection, reversing microglial activation and restoring normal synaptic architecture, as well as restoring macrophage secretory profiles to a trophic vs. toxic phenotype in response to HIV-1 infection. Collectively, these studies suggest that MLK3 activity is increased by HIV-1 neurotoxins, resulting in downstream signaling events that trigger neuronal death and damage, along with monocyte activation (accompanied by release of inflammatory cytokines). We have delineated some of the key features of MLK3 activation in the cartoon (Figure 1A and B) below.
Monocyte recruitment to the CNS has long been recognized as an important contributor to viral neuroinvasion, and macrophage burden in the CNS represents a strong correlate of HIV-associated neurologic disease. Many of these macrophage express activation markers and are thought to originate from peripheral monocytes, including subsets of monocytes with inflammatory characteristics that have been associated with the clinical presentation of HIV-1 or SIV encephalitis.[42–48] Importantly, infiltration of peripheral monocytes and their persistent activation in certain brain regions such as hippocampus and adjacent parts of entorhinal and temporal cortex persists in cART-treated HIV-1 infected patients.[49–52] Thus, monocyte chemotaxis into the CNS is thought to play an important role in the neuropathogenesis of HAND.
Expression of the potent monocyte chemokine, CCL2 (aka MCP1) is upregulated by HIV-1 Tat,[53–65] and is elevated in brains from subjects with HIV-1 dementia. Interestingly, CCL2-mediated monocyte chemotaxis is synergistically enhanced by CXCL8 (aka IL-8),[65–67] which is also upregulated by HIV-1 Tat.[55, 65] These observations are relevant to MLK3 signaling events because astrocytic expression of CCL2 and CXCL8 is regulated by JNK,[37, 65, 68–72] and JNK lies downstream of MLK3 (see Figure 1B). Moreover, CCL2 engagement of its cognate receptor, CCR2, results in activation of downstream MLK3 signaling events – including activation of the JNK and p38 MAPK pathways.[62, 73, 74] Thus, MLK3 regulated signaling cascades likely play an important role in monocyte chemotaxis to the CNS, via increased CCL2 and CXCL8 production from astrocytes (Figure 1B).
MLK3 also modulates neuroinflammation through effects on cytokine production by microglia and brain macrophages. In particular, CEP1347-mediated inhibition of MLK3 results in a significant decrease in Tat-stimulated release of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 by macrophage and microglia. This is important because TNFα mediates a significant part of Tat-mediated toxicity in models of HAND, and inhibition of this pathway by CEP-1347 may account for the reduction in neuroinflammation in an in vivo model for HAND.
There is another, equally important aspect to the role of MLK3 inhibition as a potential therapeutic strategy for HAND: its role in promoting neurotrophin signaling and synaptic homeostasis (Figure 1B). These processes may be coupled since trophic factor production by microglia (e.g., release of BDNF) may be reduced in response to the presence of infiltrating leukocytes within the CNS – leading to a reduction in trophic support for TrkB bearing neurons.[77–80] Moreover, changes in neurotrophin signaling may affect microglial activation, but the mechanisms for this are complex since microglia express truncated TrkB receptors (Maggirwar et al., unpublished data) and respond to neurotrophins (including BDNF), but are likely to have different downstream signaling repertoires than neurons that express full-length TrkB receptors. Indeed, BDNF inhibits nitric oxide (NO) release by activated microglia, suggesting that it may have a direct anti-neuroinflammatory effect.[81, 82]
MLK regulates Trk receptor expression at the transcriptional level, and MLK3 inhibition results in a robust up-regulation of TrkB expression.[83, 84] Moreover, ongoing studies in our laboratories demonstrate that pharmacologic inhibition of MLK3 is also able to increase TrkB expression in primary neurons (data not shown) – suggesting that this inhibition may potentiate neutrophin signaling.
Finally, there is evidence that the HIV-1 envelope protein gp120 reduces levels of BDNF in vivo, presumably decreasing its ability to activate TrkB receptors and ERK1/2-mediated neuroprotective pathways. Thus, gp120 induces neurotoxicity in part by its ability to interfere with BDNF signaling via TrkB and activates apoptotic pathways by a caspase-3-dependent mechanism. The overall contribution of gp120 vs. Tat to the pathogenesis of HAND, as well as the relative overlap in MLK3 and BDNF-associated signaling pathways, still remain active areas of investigation.
In summary, our studies underscore the importance of MLK3 as a therapeutic target in HIV-associated neurocognitive disorder. In particular, our data implicate microglia and inflammatory leukocytes infiltrating into the CNS as key targets for MLK3 inhibition to prevent neuroinflammation and destruction of vulnerable synaptic architecture. This type of therapeutic strategy is necessary to address the ongoing neuroinflammation that persists in the face of successful resolution of systemic HIV-1 infection by cART. Our data also suggest a compelling need for next generation pharmacologic approaches to inhibit MLK3 and to elaborate the identification of MLK3-based neuroprotective mechanisms.
We are grateful for the support of NIH awards PO1 MH64570 (HAG, SD, SBM, HEG), RO1 MH56838 (HAG), RO1 MH078989 (HAG), R21 MH03851 (HAG), RO1 DA026325 (SD), RO1 NS054578 (SBM), T32 AI49105 (MK) and the generous support of the Geoffrey Waasdorp Pediatric Neurology Fund without which this work would not have been possible.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.