Epicatechin blocks pro-nerve growth factor (proNGF)-mediated retinal neurodegeneration via inhibition of p75 neurotrophin receptor proNGF expression in a rat model of diabetes
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- Al-Gayyar, M.M.H., Matragoon, S., Pillai, B.A. et al. Diabetologia (2011) 54: 669. doi:10.1007/s00125-010-1994-3
Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes.
Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot.
Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75NTR), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg−1 day−1, orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75NTR by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures.
Diabetes-induced peroxynitrite stimulates p75NTR and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75NTR apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.
KeywordsDiabetes Epicatechin Neuroprotection Peroxynitrite p75NTR proNGF
Glial fibrillary acidic protein
Mitogen-activated protein kinase
Nerve growth factor
p75 neurotrophin receptor
Retinal ganglion cell
Retinal Müller glial cell line
Relative optical density
Small interfering RNA
Tyrosine kinase A
Diabetic retinopathy, a progressive and potentially devastating vascular-neurodegenerative disease, is the leading cause of blindness in working-age adults in the USA . Current therapeutic options, which include photocoagulation and vitrectomy, are invasive and limited by considerable side effects, as reviewed by members of this author team . Therefore, there is a great need for new non-invasive therapies to prevent diabetic retinopathy. Retinal neurodegeneration is a critical component of diabetic retinopathy and has been linked to impairment of visual function due to cell death of the inner retinal and ganglion cells [3, 4, 5, 6, 7].
A growing body of evidence supports the role of oxidative stress and in particular peroxynitrite in activating glia and secreting growth factors as part of a defence mechanism. We and others have demonstrated increases in expression of nerve growth factor (NGF) in experimental models of diabetes and in clinical diabetes [7, 8, 9]. Recent findings by our group showed that diabetes-induced peroxynitrite formation impairs cleavage and maturation of NGF, leading to accumulation of its precursor ‘proNGF’ at the expense of the mature NGF levels, both in experimental models and in clinical diabetes . Accumulation of proNGF after injury has been detected in several neurodegenerative diseases, such as Alzheimer’s, [11, 12]. However the role of proNGF in the diabetic retina and the specific molecular mechanism regulating proNGF production and its subsequent effects on retinal neurodegeneration are not fully understood. While mature NGF mediates neuronal cell survival through binding of tyrosine kinase A (TrkA) and p75 neurotrophin receptors (p75NTR), reviews by others have established that proNGF can promote neuronal apoptosis because of its high affinity to p75NTR [13, 14]. A recent study demonstrated that the outcome of proNGF signalling, i.e. neurotrophic or apoptotic, can be dependent upon relative levels of its receptors [15, 16]. Therefore, we examined the expression of proNGF and its receptors TrkA and p75NTR in the diabetic retina and isolated retinal cells cultured in high glucose.
Our previous studies had shown that excessive peroxynitrite formation, as indicated by increases in nitrotyrosine formation, positively correlated with accelerated vascular cell death, blood–retina barrier breakdown and neuronal cell death in models of diabetes, and with ischaemic retinopathy and retinal neurotoxicity [6, 7, 10, 17, 18, 19, 20, 21, 22]. Of particular note, our recent study demonstrated a specific role of peroxynitrite in impairing the survival receptor TrkA via tyrosine nitration and upregulation of the death receptor p75NTR, leading to neuronal death in experimental models and in clinical diabetes . These findings prompted us to examine the neuroprotective effects of epicatechin, a green tea constituent and selective inhibitor of tyrosine nitration, in a streptozotocin-induced animal model of diabetes. The current study also elucidates the role of p75NTR in inducing proNGF expression in retinal Müller cells and in mediating retinal ganglion cell (RGC) death in response to proNGF and high glucose.
All procedures with animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the Charlie Norwood VA Medical Center Animal Care and Use Committee. Male Sprague–Dawley rats (n = 66; ~250 g body weight; Harlan Laboratories, Indianapolis, IN, USA) were randomly assigned to control, treated control, diabetic or treated diabetic groups. Diabetes was induced by intravenous injection of streptozotocin (60 mg/kg) dissolved in 0.01 mol/l sodium citrate buffer, pH 4.5. Detection of glucose in the urine of injected animals and blood glucose levels >13.9 mmol/l were used as markers of diabetes. One week later, treated groups received oral gavages of 100 mg kg−1 day−1 of epicatechin in PBS for the entire study. Control and diabetic animals received oral gavages of PBS only. After 4 weeks of diabetes, animals were killed and eyes enucleated for analyses.
Determination of nitrotyrosine
Slot-blot analysis was used as described previously [7, 21], with 30 μg of retinal homogenate from rat samples being immobilised on to a nitrocellulose membrane. After blocking, membranes were reacted with antibodies against nitrotyrosine (Calbiochem, San Diego, CA, USA) and the optical density of various samples compared with that of controls.
Evaluation of neural cell death in rat retina
TUNEL assay was performed to detect retinal cell death by immunoperoxidase staining (ApopTag-peroxidase) in whole-mount retina as described previously by our group . Formalin-fixed retinas were flat-mounted, dehydrated in ethanol, defatted by xylenes and rehydrated. After permeabilisation, TUNEL-horseradish peroxidise staining with 3-amino-9-ethylcarbazole was performed following the manufacturer’s instructions. The total number of TUNEL-horseradish peroxidise-positive cells was counted in each retina using light microscopy. TUNEL was also performed in 10 μm optical coherence tomography-frozen eye sections using the ApopTAG in situ cell death detection kit (TUNEL-FITC) as described previously [7, 20].
Determination of glial activation and immune-localisation studies
Optical coherence tomography-frozen sections (10 μm) of eyes were fixed using 2% (vol./vol.) paraformaldehyde in PBS and reacted with monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (for glial cell activation; Affinity BioReagents, Rockford, IL, USA), polyclonal anti-proNGF (Alomone Labs, Jerusalem, Israel) or polyclonal anti-p75NTR (Millipore, Billerica, MA, USA), followed by Texas red or Oregon green-conjugated goat anti-mouse or goat anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). Data (three fields/retina, n = 6 in each group) were analysed using a microscope (AxioObserver.Z1; Zeiss, Germany) and Axio-software to quantify the density of immunostaining.
Retinal protein extraction and western blot analysis
Retinas were isolated and homogenised in RIPA buffer as described previously . Samples (50 μg protein) were separated by SDS-PAGE and electroblotted to nitrocellulose membrane. Antibodies for proNGF (Alomone), p75NTR (Millipore), phospho-p38 mitogen-activated protein kinase (p38MAPK; Cell Signaling Technology, Danvers, MA, USA), nitrotyrosine (Calbiochem), cleaved poly-(ADP-ribose) polymerase (PARP; BD Bioscience Pharmingen, San Diego, CA, USA), TrkA (Chemicon International, CA, USA) and phospho-TrkA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Membranes were re-probed with β-actin (Sigma-Aldrich, St Louis, MO, USA) to confirm equal loading. The primary antibody was detected using a horseradish peroxidase-conjugated sheep anti-rabbit antibody (GE-Healthcare) and enhanced chemiluminescence. The films were scanned and the band intensity was quantified using densitometry software (alphEaseFC) and expressed as relative optical density (ROD).
Tissue culture studies
Retinal ganglion cells
We used RGC-5, a cell line kindly donated by N. Agarwal (Department of Cell Biology, UT Health Science Center, Fort Worth, TX, USA) and previously characterised . Cells were grown to confluence in complete medium (DMEM with 6% [vol./vol.] FBS and 10% [vol./vol.] penicillin/streptomycin) then switched to normal-glucose medium (5 mmol/l) or high-glucose medium (25 mmol/l) in the presence or absence of epicatechin (100 μmol/l) for 3 days.
Cultures of a transformed retinal Müller glial cell line (rMC-1), which has been previously characterised , were obtained from V. Sarthy (Department of Ophthalmology, Chicago, IL, USA) and grown to 80% confluence. Cells were maintained in high glucose and normal glucose for 72 h in the presence or absence of epicatechin (100 μmol/l). Condition media of rMC-1 were concentrated using filter devices (Amicon-Ultracentrifugal 10K MWCO; Millipore) and used to determine proNGF and p75NTR levels using western blot; they were also used for co-culture studies with RGCs.
Viability of RGC-5 cells was determined by incubating cells for 4 h at 37°C with 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS. MTT, a yellow dye, is reduced to purple formazan in living cells, which was dissolved in acid isopropanol (1:9 of 1 N HCl/isopropanol). Optical density was measured at 540 and 690 nm using microplate reader (Bio-Tek Instruments, VT, USA).
Silencing of p75NTR expression using small interfering RNA
RGCs (2 × 106) were transfected using electroporation and a cell line kit (basic Nucleofector; Lonza, Switzerland) according to the manufacturer’s protocol. Both the SMARTpool of small interfering RNA (siRNA) specific for p75NTR (gene also known as Ngfr) and its scrambled form were obtained commercially from Dharmacon (Lafayette, CO, USA). Pilot experiments were performed to optimise levels of p75NTR siRNA. Results showed that 200 nmol/l p75NTR siRNA inhibited its mRNA expression by 76%. Transfection of RGCs with siRNA or a scrambled form (200 nmol/l) was performed together with 2 μg pmax green fluorescent protein (in Lonza kit) to assess the transfection efficiency after electroporation by Nucleofector. The transfected cells were cultured for 16 h in six-well plates with complete medium containing 10% (vol./vol.) FBS. Transfection efficiency was monitored by fluorescence microscope 16 h after transfection by calculating the percentage of green fluorescent protein-producing cells per total number of cells (Electronic supplementary material [ESM] Fig. 1). Three independent cultures were used for each condition. The medium was then changed and cells switched to high glucose or normal glucose for 3 days, followed by treatment with proNGF (50 ng/ml) for 24 h.
Determination of TUNEL-positive cells in RGCs
RGC death was determined using TUNEL fluorescence (ApopTag-Fluorescein) counterstained with DAPI. The total number of TUNEL-positive cells was counted and expressed as percentage of TUNEL-positive cells per total number of cells in various groups.
The results are expressed as mean ± SEM. Differences between experimental groups were evaluated by ANOVA and the significance of differences between groups was assessed by the post-hoc test (Fisher’s protected least significant difference) when indicated. Significance was defined as p < 0.05.
Epicatechin treatment does not alter body weight or blood glucose levels
Effects of streptozotocin-induced diabetes on body weight and blood glucose levels in rat groups as indicated
Start weight (g)
End weight (g)
Blood glucose (mmol/l)
250.2 ± 4.26
273.4 ± 10.72
10.9 ± 1.1
249.8 ± 5.58
234.6 ± 7.38
27 ± 1.05*
Control + epicatechin
242.2 ± 6.26
281.4 ± 9.51
10.5 ± 1.27
Diabetes + epicatechin
251.5 ± 5.17
226.5 ± 4.8
30.4 ± 0.69*
Epicatechin prevents diabetes-induced neuronal cell death
Epicatechin blocks diabetes-induced glial activation and proNGF expression in rat retina
Epicatechin blocks diabetes-induced tyrosine nitration and restores TrkA phosphorylation
Epicatechin blocks diabetes-induced p75NTR expression in rat retina
Epicatechin blocks high glucose-induced expression of p75NTR and proNGF in Müller cells
Epicatechin blocks diabetes-induced p75NTR apoptotic pathway in rat retina and RGC culture
ProNGF mediates RGC death in a p75NTR-dependent way
The main findings of the current study are that diabetes-induced peroxynitrite causes retinal neurodegeneration via multiple mechanisms including: (1) glial activation and upregulation of p75NTR expression leading to increasing proNGF levels; (2) activation of the proapoptotic pathway p75NTR and p38MAPK pathway in RGCs and (3) impairment of phosphorylation of the survival receptor TrkA-Y490 via tyrosine nitration. These effects were blunted by treatment of epicatechin, a safe and dietary supplement. To the best of our knowledge, our study demonstrates for the first time a dual role of p75NTR in inducing proNGF expression and in mediating RGC death.
Epicatechin or polyphenolic flavonoid is one of several green tea constituents including epigallocatechin gallate, epigallocatechin, epicatechin gallate and epicatechin. The limited bioavailability of green tea extracts usually restricts their use as effective therapeutics. We used a repeated dosing of epicatechin by oral gavage (100 mg kg−1 day−1), as it has proven successful in improving epicatechin bioavailability to cross blood–brain barrier (282%) in Sprague–Dawley rats compared with a single acute dose . While the glucose-lowering effects of epigallocatechin gallate are well-documented in experimental models of diabetes at lower doses (25 mg kg−1 day−1) via its antioxidant properties [31, 32], little is known about the neuroprotective effects of epicatechin. Interestingly, our results demonstrated that treatment of diabetic animals with epicatechin did not affect blood glucose level or systemic antioxidant defence. In agreement with the above, we and others have previously shown that epicatechin is a selective inhibitor of tyrosine nitration, which does not affect the antioxidant defence [17, 21, 26, 33]. Our results here show that treatment with epicatechin (100 mg kg−1 day−1, orally) significantly reduced TUNEL-positive cells in retina flat mount and RGC cultures. Therefore, the protective effects of epicatechin in diabetic animals cannot be attributed to its ability to reduce blood glucose level or to the traditional antioxidant properties of green tea constituents. A growing body of evidence supports the role of oxidative stress and in particular peroxynitrite in activating glia to secret growth factors such as proNGF. Our results showed significant increases in nitrotyrosine formation in diabetic rat retinas compared with controls. These results lend further support to previous findings showing enhanced peroxynitrite formation [6, 7, 10, 18, 34] and increases in NGF levels to compensate for neuronal injury [2, 8, 9]. Treatment of diabetic animals with epicatechin (100 mg kg−1 day−1, p.o.) blocked nitrotyrosine formation and proNGF expression.
We next investigated the possible mechanisms by which diabetes enhances proNGF levels, and the protective action of epicatechin. Our recent study demonstrated a critical role of peroxynitrite in inhibiting the protease matrix metalloproteinase-7, which cleaves proNGF into NGF leading to accumulation of proNGF . Interestingly, epicatechin had minimal effects on cleavage of proNGF into NGF, suggesting its potential role in reducing proNGF expression. Previous studies have identified Müller cells as the main glia to secret proNGF in the retina [10, 25]. In agreement with this, our results showed prominent glial Müller cell activation, which was evident from immunostaining of GFAP and accumulation of proNGF in the diabetic retina and in high glucose-cultured Müller cells. In light of the fact that p75NTR is expressed in Müller cells and RGCs [27, 28, 29], our finding that the neuroprotective effect of epicatechin in vivo was associated with blocking the increases in expression of proNGF and p75NTR suggests a paracrine effect of glial cells on neurons as target tissue. A co-culture model of RGCs with condition media from high glucose-treated glial rMC-1 showed an approximately 2.2-fold increase in proNGF levels and a 56% reduction in RGC viability compared with RGCs treated with normal glucose treatment conditioned medium. Treatment of Müller cultures with epicatechin blunted p75NTR expression and proNGF levels, and did not significantly alter RGC viability (85% ~ of basal level). Together, these results suggest a dual role of enhanced p75NTR expression in the retina, i.e. activation of proNGF expression in Müller cells and activation of neuronal death in RGCs. In support of this, several previous studies have shown that inhibiting p75NTR on glial cells prevents apoptosis of photoreceptors and RGCs, and retinal degeneration via inhibition of proNGF or TNF-α [25, 35, 36]. However, we believe that our study is the first report demonstrating these effects in diabetes.
NGF and its precursor, proNGF, exert distinctive biological functions because of their different affinity to transmembrane receptors TrkA and p75NTR, and the outcome, neurotrophic or apoptotic, will depend on expression and activity level of these receptors [15, 16]. Our previous study demonstrated that retinas from diabetic humans and rats showed no alteration in TrkA expression levels among various groups . Therefore, we evaluated the post-translational modification of TrkA by determining its tyrosine nitration and phosphorylation. Our results showed a 2.2-fold increase in tyrosine nitration of TrkA and a 45% decrease in its phosphorylation site (Y490) in diabetic rat retinas compared with non-diabetic controls. Interestingly, tyrosine 490 is responsible for activating the phosphoinositide-3 kinase–Akt survival signal. These effects were associated with a fivefold increase in neuronal cell death. Protein tyrosine nitration and subsequent loss of protein function have been well documented in response to peroxynitrite [17, 19, 21, 37, 38, 39]. The finding that blocking tyrosine nitration using epicatechin protects retinal neurons and inhibits the expression of p75NTR supports a specific role of peroxynitrite in inducing neuronal death via upregulation of p75NTR expression, which has been demonstrated previously in vitro and in vivo [40, 41, 42]. However, it is not completely understood how peroxynitrite increases the expression of p75NTR. TrkA and p75NTR are co-expressed throughout the nervous system and cross-talk between the two receptors showed a bidirectional relationship. Previous studies, in neuronal cells, showed that blocking TrkA expression or abrogating phosphorylation of TrkA (Y490) results in significant upregulation of p75NTR expression [43, 44]. This effect was attributed to the fact that Y490 phosphorylation is necessary for the binding of an adaptor protein, Src homologous and collagen protein, and for downstream activation of Ras, leading to p75NTR expression. In agreement with the above, our results show that diabetes-induced peroxynitrite formation abrogated Y490 phosphorylation at a site on TrkA via tyrosine nitration, and that this was reversed by epicatechin treatment. Other possible activators of p75NTR expression include activation of protein kinase C , accumulation of glutamate, and activation of N-methyl-d-aspartate receptors , as well as diabetes-induced ischaemia . Further studies are warranted to explore whether these upstream events share a common signalling cascade.
A critical role of p75NTR, a member of the TNF superfamily, in mediating neuronal death has been documented in models of neurodegenerative diseases, including diabetes [47, 48, 49]. In agreement, our results showed that diabetes stimulates p38MAPK phosphorylation and cleaved PARP in vivo, and that high glucose stimulates p75NTR and p38MAPK, and induces RGC death in vitro, all of which were blocked by epicatechin treatment, supporting a causal role of p75NTR in mediation of RGC death. There is no commercial and reliable p75NTR inhibitor; therefore, we used a molecular approach to confirm the causal role of p75NTR in mediating RGC death. Silencing the expression of p75NTR with siRNA completely blocked proNGF-induced or high glucose-induced RGC death compared with the scrambled form. Interestingly, our finding that exogenous proNGF exacerbated p75NTR-mediated RGC death in basal and high glucose-treated RGC cultures suggest a vicious circle, where diabetes-induced initial neuronal injury will stimulate proNGF production, which will further damage RGCs. The notion that proNGF exacerbates RGC damage by enhancing p75NTR expression is supported by a previous report showing a positive loop between increased level of neurotrophin and stimulation of p75NTR expression in the target tissue . Further assessment of retinal neuroglial dysfunction using electroretinogram in diabetic animals should provide valuable information that could be translated to humans.
In summary, diabetes-induced retinal neurodegeneration is an early and critical event that can be prevented using epicatechin via multiple mechanisms. Epicatechin inhibits tyrosine nitration and restores the TrkA survival signal, which is impaired in diabetic retina. As such, epicatechin inhibits upregulation of p75NTR and hence proNGF levels in Müller cells, as well as activation of p75NTR and p38MAPK pro-apoptotic signals in RGCs. The fact that epicatechin is a safe dietary supplement offers an additional advantage for its potential use as add-on therapy in diabetic patients.
This work was supported by an American Heart Association Scientist Development Grant (0530170N to A. B. El-Remessy), the Juvenile Diabetes Research Foundation (grant 2-2008-149 to A. B. El-Remessy) and the University of Georgia Research Foundation (A. B. El-Remessy).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.