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* Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, Kentucky, USA
1Correspondence: 600 North Wolfe St., Pathology 509, Baltimore, MD 21287, USA. E-mail: anath1{at}jhmi.edu
SPECIFIC AIMS
The possibility that human matrix metalloproteinases (MMPs) interact with viral proteins has not been explored. Hence, we coincubated the HIV transactivating protein, Tat, with select MMPs to determine if they would cause synergistic or additive neurotoxicity.
PRINCIPAL FINDINGS
Neurotoxic properties of Tat and MMP-1
Previous work has shown that both Tat and MMP-1 independently can cause neurotoxicity. Furthermore, Tat can synergize with gp120, glutamate, and drugs of abuse such as morphine, cocaine, and methamphetamine to cause increased neurotoxicity. Hence, we coincubated Tat and MMP-1 to determine whether they too would similarly cause synergistic neurotoxicity. Surprisingly, we found that when mixed neuronal cultures were exposed to Tat and MMP-1 together, there was a decreased amount of neurotoxicity as measured by changes in mitochondrial membrane potential (Fig. 1
A). At lower MMP-1 concentrations, the neurotoxic effect of Tat was predominant, but with increasing amounts of MMP-1 a reverse dose response curve was noted, leading to a progressively decreasing amount of neurotoxicity. At higher concentrations of MMP-1, the neuroprotective effect was lost presumably because the neurotoxic effect of MMP-1 alone became predominant. Similar effects were seen when MMP-1 and Tat were preincubated together prior to adding them to the cell culture (Fig. 1)
and when they were added simultaneously to the cultures without preincubation (not shown).
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The combination of Tat and MMP-1 was also protective against cell death as measured by trypan blue exclusion (Fig. 1B
). Furthermore, MMP inhibitors block the neuroprotective effect of MMP-1. We recognized that mitochondrial membrane potential is an intermediate measurement of neuronal viability, and so tested MMP-1’s ability to protect against Tat-induced cell death. We incubated Tat and MMP-1 alone or together in mixed neuronal cultures with or without the MMP inhibitor, FN-439. Results demonstrate that coincubation of Tat and MMP-1 prevents the cell death observed with either Tat or MMP alone. Furthermore, addition of FN-439 reversed this protective effect.
Cleavage of HIV-Tat by MMP-1
We hypothesized that the observed protective effect could occur if Tat and MMP-1 bind to one another and interfere with each other’s neurotoxic potential or if MMP-1’s endopeptidase activity cleaves Tat and thus decreases its neurotoxicity. To determine whether MMP-1 could directly interact with Tat, we coincubated Tat and MMP-1 in an MMP reaction buffer and analyzed them by colloidal Coomassie blue staining, silver staining, and Western blot (not shown). We found that MMP-1 could cleave Tat, as demonstrated by disappearance of the Tat band from the gel, utilizing all three techniques.
Furthermore, when either batimastat or FN-439, both broad spectrum MMP inhibitors, were added to the reaction mixture, the Tat band reappeared, strongly suggesting that Tat was indeed cleaved by MMP-1’s endopeptidase activity.
To test the kinetics of the Tat-MMP cleavage reaction, Tat and MMP-1 were coincubated for various periods, resolved by SDS-PAGE, and the gels were silver stained (not shown). Degradation of Tat was complete within 60 min. There is a higher running band present with the Tat, which is either a contaminant or a multimer of Tat. It was not degraded and therefore serves as an internal control, showing that our MMP-1 preparation did not nonspecifically degrade all proteins.
Interaction of MMP-1 with Tat inhibits HIV-long terminal repeat transactivation
Based on these results, we concluded that the observed neuroprotective effect, as measured by mitochondrial membrane potential and cell death, was due to cleavage of Tat by MMP’s endopeptidase activity. Also, both cleavage and the neuroprotective effect are reversible by addition of an MMP inhibitor. To further confirm this observation, we hypothesized that Tat incubated with MMP-1 would not efficiently transactivate long terminal repeat (LTR) because it would be cleaved. MMP-1, Tat, and/or FN-439 were coincubated, as appropriate, with SVGA-long terminal repeat-GFP cells in culture and transactivation activity was measured by assessing the change in green fluorescence under each experimental condition (Fig. 2
). Tat alone produced significant transactivation compared to untreated controls, but transactivation returned to untreated levels when MMP-1 was added. Further addition of FN-439, thus inhibiting MMP endopeptidase activity, returned transactivation to levels seen with Tat alone. Neither FN-439 alone nor MMP-1 alone caused significant transactivation.
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MMP-2 and -9 also have protective properties against Tat neurotoxicity
We next determined whether MMP-1’s cleavage and neuroprotective properties were specific to MMP-1 or more generalizable to other MMPs. MMPs-2 and -9 were unable to cleave Tat (not shown). However, we further determined whether MMP-2 and-9 could produce synergistic neurotoxicity, neuroprotection, or have no effect on Tat activity. MMP-2 and Tat were coincubated in mixed neuronal cultures and neurotoxicity was measured by a change in mitochondrial potential and by neuronal cell death (not shown). Results demonstrate that MMP-2 and Tat were independently toxic when compared to untreated controls and that coincubation of Tat and MMP-2 prevents the neurotoxicity and cell death observed with Tat alone. Addition of FN-439 did not reverse the protective effect, consistent with the observation that MMP-2 does not cleave Tat. Similar results were seen for MMP-9. MMP-9 and Tat were toxic vs. untreated controls; the combination was protective vs. either protein alone and the MMP inhibitor failed to reverse this protection.
CONCLUSIONS AND SIGNIFICANCE
Neuronal loss of up to 90% has been observed in certain brain regions of patients with HIV dementia, yet actual infection of neurons by HIV is rare. The perivascular macrophages and microglia are the cell types productively infected in brain, suggesting that the observed neurotoxicity is an indirect effect of HIV-infected cells produced by factors that are released into the extracellular matrix of the central nervous system and trigger inflammatory cascades and oxidative stress. These mediators likely include two main groups—viral proteins, like Tat, and host proteins, such as matrix metalloproteinases (Fig. 3
).
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Tat has synergistic neurotoxic effects with multiple other toxins, and Tat’s neurotoxic mechanisms seem to overlap with those of MMPs. We thus expected to find synergistic, or at least additive, effects. Our work demonstrates that MMP-1 can cleave Tat protein and protect against its neurotoxic and HIV-long terminal repeat transactivation properties (Fig. 3)
. Cleavage of Tat by another human protease, furin, was recently shown as well. However, this is the first demonstration of interaction between MMPs and viral proteins, and, surprisingly, our data imply that MMP-1 cleavage of Tat may actually reduce overall neurotoxicity by reducing Tat levels.
Unlike MMP-1, neither MMP-2 nor -9 cleaves Tat. However, like MMP-1, we show that MMP-2 and -9 have neuroprotective effects against Tat-induced neurotoxicity. This neuroprotective effect must be mediated by a mechanism other than cleavage. The data generated from this research will guide future studies of other host-viral protein interactions. We now hypothesize that MMP production is an innate host response to HIV infection that plays a dual role in HIV neuropathogenesis. MMPs alone are well documented to have neurotoxic potential. Alternatively, our work now shows that MMPs may bind, cleave, or antagonize viral proteins, neutralizing their effects on brain cells.
Though MMPs clearly have many other roles, it is possible they have evolved from a primitive host defense mechanism. Plants without a specific adaptive immune system may use MMPs, along with other innate defense mechanisms, to combat infection. For example, the MMP2 gene of the soybean, Glycine max, is up-regulated in response to a variety of infections.
It is thus clear that the complex interactions between MMPs, infectious proteins, and host defense mechanisms need to be fully understood to develop a better rationale for MMP-directed therapeutic approaches. Since neurons themselves are not typically infected by HIV, but rather die due to secondary mediators, there is an excellent opportunity to prevent HIV dementia by blocking these mediator pathways. MMPs are prime candidates as therapeutic targets. However, it may not be possible to identify a single target pathway or molecule; combination therapy may be necessary. In addition to HIV-associated dementia, MMPs have been implicated in several other disease conditions, including tumor metastasis, atherosclerosis, emphysema, arthritis, and neurodegenerative disorders such as Alzheimer’s, multiple sclerosis, and even stroke. Optimal treatment of these conditions may require the knowledge and ability to choose appropriate MMP selective inhibitors, or even MMP selective augmentors, while broad spectrum MMP modulation may actually be harmful.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5619fje
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