Catechins protect neurons against mitochondrial toxins and HIV proteins via activation of the BDNF pathway
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- Nath, S., Bachani, M., Harshavardhana, D. et al. J. Neurovirol. (2012) 18: 445. doi:10.1007/s13365-012-0122-1
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Currently, there is no effective treatment for neurological complications of infection with the human immunodeficiency virus that persists despite the use of combination antiretroviral therapy. A medium throughput assay was developed for screening neuroprotective compounds using primary mixed neuronal cells and mitochondrial toxin 3-nitropropionic acid. Using this assay, a library of 2,000 compounds was screened. Out of 256 compounds that showed variable degrees of neuroprotection, nine were related to epicatechin, a monomeric flavonoid found in cocoa and green tea leaves that readily crosses the blood–brain barrier. Hence, catechin, epicatechin, and the related compound, epigallocatechin gallate (EGCG) were further screened for their neuroprotective properties against HIV proteins Tat and gp120, and compared to those of resveratrol. Epicatechin and EGCG targets the brain-derived neurotrophic factor (BDNF) and its precursor proBDNF signaling pathways, normalizing both Tat-mediated increases in proapoptotic proBDNF and concomitant Tat-mediated decreases in the mature BDNF protein in hippocampal neurons. Epicatechin and epigallocatechin gallate were more potent than catechin or resveratrol as neuroprotectants. Due to its simpler structure and more efficient blood–brain barrier penetration properties, epicatechin might be the best therapeutic candidate for neurodegenerative diseases including HIV-associated neurocognitive disorders where oxidative stress is an important pathophysiological mechanism.
KeywordsCatechinsNeuronsMitochondrial toxinsBDNF pathway
Infection with the human immunodeficiency virus (HIV) can result in severe complications affecting the nervous system, such as HIV sensory distal neuropathy (HIV-SN) and HIV-associated neurocognitive disorders (HAND). These complications occur and persist despite the advent and implementation of combined active antiretroviral therapy (CART) (McArthur et al. 2010). As effective CART allows HIV-infected patients to live longer, a growing proportion of patients experience some form of neuropathy and cognitive deficits. These complications are a result of oxidative stress-induced neurotoxicity that results directly from HIV infection in tissue reservoirs, where effective levels of antiretroviral drugs may be hard to achieve and low level HIV replication may continue. The coat protein of HIV, gp120, can cause neurotoxicity by multiple mechanisms. Exposure of neurons and glial cells to toxic viral proteins such as Tat, which can be produced by HIV-infected cells despite the use of CART, can set up positive feedback loops resulting in widespread neuronal dysfunction. Oxidative stress is the final common pathway for the neurotoxicity induced by both viral proteins (Mattson et al. 2005). Development of therapeutic strategies to protect against this neuronal damage and the ensuing clinical deficits remains a major unmet medical need. Neuroprotective compounds, if they can be identified and developed preclinically, may provide a treatment for HIV-SN and HAND.
One approach to identify potential neuroprotective agents would be to develop a medium throughput screening assay to assess the protective efficacy of a wide array of diverse chemical compounds. We have developed assays that utilize mixed primary cultures of neurons and glial cells to replicate the cellular conditions in situ. These cultures have been adapted to microtiter plates and exposed to oxidative stressors, such as 3-nitropropionic acid (3-NP), to mimic oxidative damage which results from HIV infection of these tissues. Confirmatory assays are then performed using HIV toxic proteins gp120 and Tat. Thus, we screened a library of 2,000 compounds comprised of natural substances, FDA-approved drugs, and compounds with prior human exposure (Spectrum Collection, Microsource Discovery). We identified epicatechin, epigallocatechin gallate (EGCG) and other catechin flavonoids as potential neuroprotective agents. These compounds are found in high concentrations in green tea leaves, purple grapes (Dani et al. 2009), and in seeds of the cacao tree which is used to make chocolate. While these compounds have reported antioxidant activities, these compounds have not been evaluated against mitochondrial toxins or HIV toxic proteins.
Materials and methods
Neuronal cultures from rodent cerebral cortex and from hippocampus were prepared from embryonic day 18 Sprague–Dawley rats using methods similar to those described previously (Haughey et al. 2004). Tissues were dissociated by gentle trituration with a firepolished glass pipette in calcium-free Hank’s balanced salt solution. The single cell suspension was centrifuged at 1,000×g and re-suspended in minimal essential medium containing 10 % heat-inactivated fetal bovine serum and 1 % (v/v) antibiotic and antimycotic solution (penicillin G 104 U/mL, streptomycin 10 mg/mL, and amphotericin B 25 mg/mL; Sigma, St Louis, MO, USA). Cells were allowed to attach for 3 h before the media was replaced with serum-free neurobasal medium containing 2 % (v/v) B-27 supplement (Gibco, Rockville, MD, USA) and 1 % (v/v) antibiotic and antimycotic mix (Sigma). Rat mixed hippocampal neurons were generated from freshly cultured rat hippocampi in neurobasal media containing 5 % (v/v) fetal bovine serum and 2 % (v/v) B27 supplement. Hippocampal neurons were plated in 96-well plates at a density of 4 × 105 cells/mL for neurotoxicity studies.
Human neuronal cultures were prepared as described previously (Magnuson et al. 1995). Briefly, the cells were mechanically dissociated, suspended in Opti-MEM with 5 % (v/v) heat-inactivated fetal bovine serum, 0.2 % (v/v) N2 supplement (Gibco), and 1 % (v/v) antibiotic and antimycotic solution and plated in flat-bottomed 96-well plates. The cells were maintained in culture for at least 1 month before use. Mixed neuronal cultures were grown in low serum conditions with N-2 supplement to encourage neuronal growth and inhibit glial proliferation. These cultures contain 70 % neurons, 30 % astrocytes, and <1 % microglia. These cultures can be maintained for several months. Previous experiments in our laboratory and those of others have shown that prolonged cultures of neurons ensures the expression of excitatory amino acid receptors and growth of processes (Magnuson et al. 1995; Nath et al. 1996). The original screening assays were done with three to four replicates per plate, including an internal positive control of 10 μM resveratrol as a neuroprotectant.
Neurons were plated on polyethyleneimine-coated 15-mm diameter glass coverslips at a density of 2 × 105 cells/mL for calcium imaging and in 35-mm diameter plastic plates at a density of 4 × 105 cells/mL for neurotoxicity studies.
Cell survival assay
Neuronal cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay (Mosmann 1983) is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals. These crystals are largely impermeable to cell membranes, and thus, accumulate within healthy cells. The resultant formazan precipitates are solubilized with dimethyl sulfoxide (DMSO) and read on a multiwell scanning spectrophotometer (SpectraMAX M5e, Molecular Devices). The number of surviving cells is directly proportional to the level of the formazan product created.
The Spectrum Collection of compounds by Microsource Discovery contains 2,000 compounds, of which about half are FDA-approved drugs and the remaining compounds are natural products or other compounds with some prior human exposure and safety testing data. The compound collection is dispensed and maintained on 96-well plates at a concentration of 10 mM in 100 % DMSO and stored at −80 °C. The compound mother plates were thawed one time in order to make four sets of daughter plates, and one set of daughter plates was used for these screening assays.
Rat mixed hippocampal cultures were treated with test compounds for 1 h at 37 °C prior to addition of 3-NP, Tat, or gp120. Cells were then exposed to these toxins for 18 h, after which time cell viability was assessed by MTT assay (Mosmann 1983). For Tat toxicity assays, purified recombinant Tat1-72 was utilized at 500 nM final concentration. After 18 h of exposure to Tat, the MTT assay was utilized to assess cytotoxicity. Hydrogen peroxide neurotoxicity was achieved with 2 h exposure to 100 μM H2O2, which is applied to the cultures following a 1-h preincubation with the neuroprotectant. Cell viability was quantified by MTT assay. Moderate throughput functional screening assays were done in triplicate or quadruplicate wells, while confirmatory dose–response assays were done with N = 8 per treatment group and replicated three times.
Fluorescence axodendritic degeneration assays
Rat mixed hippocampal or cortical neurons were transfected with a plasmid containing the neuron-specific CaMKII promoter, with a fusion construct of tau protein and td-Tomato genes or βIII tubulin–tdTomato genes (Nucleofector 2S, Lonza, MD), according to the manufacturer’s instructions. Transfection efficiency was about 60 %. After 7–10 days in culture, cells were utilized in neurotoxicity assays to assess the effects of catechins on Tat-mediated axodendritic degeneration. Following catechin treatment and Tat exposure, images were captured via live imaging on a Zeiss AxioObserver Z1 inverted microscope and the relative fluorescence of Tau-tdTomato was determined with a SpectraMax M5e multimode plate reader (Molecular Devices, CA).
HIV-1 proteins and reagents
The preparation of recombinant Tat1-72 protein has been described previously (Ma and Nath 1997). Tat protein varies in length from 86 to 101 amino acids and is made from two exons. The first exon form the first 72 amino acids and the neurotoxic epitope resides in this region (Nath et al. 1996), hence we have utilized recombinant Tat1-72 for these studies. The toxic effects of Tat1-72 can be blocked by anti-Tat Ig and likewise Tat∆31-61 has been used as a control (Turchan et al. 2001). Gp120 derived from HIVSF was acquired from the NIH-AIDS repository (Bethesda, MD, USA).
All data are represented as mean ± SEM and analyzed by one-way analysis of variance (ANOVA). Group-wise post hoc comparisons were assessed by Newman–Keuls multiple comparison tests, with statistical significance at p < 0.05.
Development of a neurotoxicity assay for screening neuroprotectants
Neuroprotection by the catechin class of compounds
Neuroprotective properties of the catechin family of compounds
Spectrum collection compound
% Protection vs 3-NP
Epicatechin, tetramethyl ether
Epigallocatechin octamethyl ether
Dose–response curves of neuroprotection by epicatechin and catechin
Epicatechin and catechin protect against HIV proteins
Neuroprotective properties against hydrogen-peroxide-induced toxicity
Protection from Tat-mediated neuritic damage by catechins
Potential neuroprotective mechanism(s) of catechin-mediated protection from HIV
High-throughput assays have been the mainstay of pharmaceutical companies for drug discoveries. Recently, the National Institutes of Health and other academic institutions have put in major effort towards the development of such assays for drug discovery that would particularly target rare disorders or other diseases that pharmaceutical companies have traditionally ignored. The neurological disorders associated with HIV infection fall into this category. Most throughput assays are either enzyme based or binding assays. However, cellular functions are very complex and involve multiple pathways, hence we developed a moderate throughput assay using primary mixed neuronal cultures and used rigorous experimental conditions to minimize variability and maximize reproducibility. We used 3-NP for causing neurotoxicity since its acts on the mitochondria and mitochondrial dysfunction have been implicated in most neurodegenerative diseases. Further, 3-NP is a stable compound with a wide and reproducible dose curve. Using this assay, we have identified several groups of compounds that have variable neuroprotective properties. We focused our initial efforts on catechins since nine related compounds had neuroprotective properties and have been shown to cross the blood–brain barrier (Faria et al. 2011). Further these compounds are natural substances present in green tea and other plants although the gallate group is predominant in green tea (Henning et al. 2003; Fraga and Oteiza 2011) Dark chocolate from the plant Theobroma cocao is also a rich source of epicatechin (Engler et al. 2004). Hence, humans have been regularly exposed to these compounds in food products for hundreds or thousands of years.
Several studies have investigated the neuroprotective properties of epigallocatechin gallate (reviewed in (Weinreb et al. 2009; Mandel et al. 2008)), the main catechin polyphenol constituent in green tea, and found that it protects neurons against 3-NP (Kumar and Kumar 2009) amyloid beta peptide (He et al. 2011), traumatic injury (Wei et al. 2011), cerebral ischemia (Park et al. 2009), cisplatin-induced neurotoxicity (Lee et al. 2010), and rotenone-induced neurotoxicity (Moldzio et al. 2010). However, one study failed to show any neuroprotection against 6-hydroxydopamine (Leaver et al. 2009). Although the exact mechanism of action of EGCG remains unknown, prevention of generation of reactive oxygen species, and nitric oxide synthesis has been implicated (Kim et al. 2009). These compounds have also been shown to inhibit ERK and NF-κB pathways (Fraga and Oteiza 2011; Lee et al. 2010). It can also cause proliferation of neural progenitor cells (Yoo et al. 2010). Our data in this study implicates the BDNF pathway in the neuroprotective actions of the catechins. The proapoptotic proBDNF (Teng et al. 2005) induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. proBDNF is converted to the mature and neuroprotective BDNF by extracellular proteases (Lu et al. 2005). Perhaps exposure of neurons to Tat caused increases in proBDNF (along with decreases in BDNF) by blocking the proteolytic processing of the proBDNF, which was reversible by catechin treatment. Interestingly, gp120 treatment also caused decreased BDNF levels in vitro and in vivo after 24 h of exposure (Nosheny et al. 2004), preceding caspase-3 activation and neuronal cell death. It is possible that catechin-mediated protection from gp120 (Fig. 5) may also utilize this mechanism. Recent studies in the CSF of HIV-infected patients demonstrate a strong correlation with increasing severity of HIV-mediated neurologic disease and a decline in growth factors, particularly BDNF and NT-3 (Meeker et al. 2011). Thus, a potential mechanism of catechin-mediated neuroprotection which promoted BDNF availability and signaling may be an effective strategy against HIV-mediated neurotoxicity and HAND. While the potential role of BDNF as a neurotherapeutic agent has long been considered (Rothman et al. 2012), its use has been limited due to the need to directly administer it to the brain as a recombinant protein or via viral vectors (Kells et al. 2008). Catechins thus represent a novel set of compounds by their ability to act via the BDNF pathway and may thus open a new area in neurotheraputics research.
Importantly, epigallocatechin has been shown to be neuroprotective following oral administration in an animal model of acute neurotoxicity (Kang et al. 2010) and aging-related oxidative injury in the brain (Li et al. 2010). Other compounds related to the catechins have not been well studied. We found that epigallocatechin gallate was only moderately neuroprotective against 3-NP neurotoxicity. Catechin, epicatechin, and epicatechin monogallate were much more potent than epigallocatechin in our screening assay. Of these compounds epicatechin had the best potency against 3-NP-induced neurotoxicity although they seemed to be similarly protective against HIV proteins with apparent EC50 of 1 μM compared to an EC50 of 10 μM for 3-NP neurotoxicity. Epicatechin gallate has been shown to have cytoprotective effects in a glial cell line at much lower concentrations of 0.1 to 1 μM (Abib et al. 2010). However, catechin and epicatechin have a smaller molecular mass and are efficient in crossing the blood–brain barrier (Faria et al. 2011). Catechin has also been shown to improve blood flow by causing cerebral vasodilatation (Drouin et al. 2011). The bioavailability of the compounds improves with repeated dosing. In one study, catechin and epicatechin were undetectable in the brain of rodents after administration of a single dose; however, after daily administration for 10 days, it reached 576.7 ± 227.7 and 290.7 ± 45.9 pg/g of brain tissue, respectively (Ferruzzi et al. 2009). Defining the therapeutic window of these compounds will be critical since at very high dosages, they may have proapoptotic properties and can cause neurotoxicity (Yin et al. 2009). Our observations also suggested a similar trend. We found that resveratrol was also neuroprotective against HIV-Tat protein-induced neurotoxicity; however, due to its hydrophilic properties, it does not cross the blood–brain barrier as efficiently.
Oxidative and nitrosative stress play an important role in mediating the neuropathogenesis of HIV infection and protein carbonyls, nitrosylated and hydroxynonenol-modified proteins can be found in brain and CSF of these patients, where they correlate with the severity of neurocognitive impairment (Turchan et al. 2003; Li et al. 2008). HIV proteins Tat and gp120 have been implicated in causing excitotoxic neurotoxicity by directly acting on neurons or via indirect mechanisms leading to activation of macrophages and glial cells. Oxidative stress is the final common pathway in all these mechanisms (Mattson et al. 2005). Currently available antiretroviral medications are unable to control complete viral replication in the brain and they do not have any effect of Tat production once proviral DNA has been formed. In this study, we found that the catechins were able to block the neurotoxic effects of the HIV proteins and thus may be of therapeutic potential in HAND. However, conducting clinical trials in this patient population carries many challenges and to date all trials with potential neurotherapeutic agents have failed in phase 2 studies. The availability of good surrogate markers is critical for monitoring such clinical trials so that early decisions on the continuation of these trials can be made. Thus, measurement of oxidized or nitrosylated proteins in CSF represents an excellent marker for monitoring clinical trials (Schifitto et al. 2009). It is important to note that even though catechins have been shown to be protective in a variety of models of neurodegenerative diseases, to date no clinical trials have been conducted with these compounds. This is in part because monitoring clinical end points in these diseases requires large sample sizes and monitoring of patients for long periods of time. CSF is not routinely obtained from these patients for either diagnostic purposes or for monitoring clinical progression. In patients with HAND, CSF monitoring of viral load and inflammatory markers is common practice, hence this population might represent an excellent opportunity for monitoring the therapeutic efficacy of these compounds.
In conclusion, we have developed a novel screening assay for identification of neuroprotective compounds and identified the family of catechins as a class of compounds with potent neuroprotective properties. Of these compounds, epicatechin is the most promising therapeutic candidate because of its high potency, simple structure, excellent bioavailability following oral administration, and efficient ability to cross the blood–brain barrier.
We thank Tanya Malpica-Llanos for technical assistance, Norman Haughey for providing rat neuronal cultures, and Avindra Nath at the National Institute of Neurological Diseases and Stroke, National Institutes of Health for carefully reading the manuscript and for helpful discussion/comments.