Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.     

VEGF inhibition alters neurotrophin signalling pathways and induces caspase-3 activation and autophagy in rabbit retina

This study sought to evaluate the prospective role exerted by vascular endothelial growth factor (VEGF) in the modulation of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) signalling pathways in the rabbit retina. To reach this aim, the anti-VEGF agents aflibercept and ranibizumab were used as a pharmacological approach to evaluate the putative consequences elicited by VEGF inhibition on neurotrophin signalling. VEGF inhibition determined a marked imbalance in proneurotrophin expression, a significant reduction in TrkA and TrkB phosphorylation states and a decrease in the pan-neurotrophin receptor p75. Importantly, VEGF blockade also caused a strong increase in cleaved caspase-3, beclin-1 and lipidated LC3. The effects were more pronounced in the aflibercept group when compared with ranibizumab-treated rabbits, particularly 1 week after injection. This study demonstrates that VEGF exerts pivotal physiological roles in regulating NGF and BDNF pathways in the retina, as its inhibition by anti-VEGF agents deeply impacts neurotrophin homeostasis. These events are accompanied by a sustained induction of apoptotic and autophagic markers, suggesting that anti-VEGF-dependent impairments in neurotrophin signalling could be responsible for the activation of retinal cell death pathways.     

Molecular and Structural Insight into proNGF Engagement of p75NTR and Sortilin

Nerve growth factor (NGF) is initially synthesized as a precursor, proNGF, that is cleaved to release its C-terminal mature form. Recent studies suggested that proNGF is not an inactive precursor but acts as a signaling ligand distinct from its mature counterpart. proNGF and mature NGF initiate opposing biological responses by utilizing both distinct and shared receptor components. In this study, we carried out structural and biochemical characterization of proNGF interactions with p75NTR and sortilin. We crystallized proNGF complexed to p75NTR and present the structure at 3.75-Å resolution. The structure reveals a 2:2 symmetric binding mode, as compared with the asymmetric structure of a previously reported crystal structure of mature NGF complexed to p75NTR and the 2:2 symmetric complex of neurotrophin-3 (NT-3) and p75NTR. Here, we discuss the possible origins and implications of the different stoichiometries. In the proNGF-p75NTR complex, the pro regions of proNGF are mostly disordered and two hairpin loops (loop 2) at the top of the NGF dimer have undergone conformational changes in comparison with mature NT structures, suggesting possible interactions with the propeptide. We further explored the binding characteristics of proNGF to sortilin using surface plasmon resonance and cell-based assays and determined that calcium ions promote the formation of a stable ternary complex of proNGF-sortilin-p75NTR. These results, together with those of previous structural and mechanistic studies of NT-receptor interactions, suggest the potential for distinct signaling activities through p75NTR mediated by different NT-induced conformational changes.     

Nerve growth factor promotes formation of lumen-like structures in vitro through inducing apoptosis in human umbilical vein endothelial cells

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.     

Mature pig oligodendrocytes rapidly process human recombinant pro-nerve growth factor and do not undergo cell death

The neurotrophin family with its first member, nerve growth factor (NGF), binds two classes of receptors, more specifically to Trk receptors and to a shared p75NTR receptor. It has been shown that proNGF rather than NGF is predominant in the mature central nervous system. A recent finding indicated that a furin-resistant proNGF preferentially binds to p75NTR, initiating a pro-apoptotic cascade even in the presence of TrkA. In this context, rodent oligodendrocytes were reported to undergo cell death when exposed to proNGF. We have investigated the effect of a non-mutated 32 kDa human recombinant proNGF (rhproNGF) on cultured pig oligodendrocytes which express TrkA, p75NTR and sortilin. Pig oligodendrocytes respond to rhproNGF (50 ng/mL) with an enhanced regeneration of their processes as already observed for NGF. Activity of mitogen-activated protein kinase (MAPK), which plays an important role in oligodendroglial process formation, was increased even when rhproNGF processing was inhibited by the furin inhibitor Decanoyl-RVKR-CMK. Similarly, a cleavage-resistant proNGF (R-1G) activated MAPK and promoted oligodendroglial process regeneration. High concentrations of rhproNGF (300 ng/mL) did not induce cell death. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blotting revealed that oligodendrocytes process rhproNGF to NGF. NGF was detected in Western blots of oligodendroglial lysates already 10 min after rhproNGF exposure, followed by a release of NGF into the culture medium. Indirect evidence indicates that rhproNGF processing occurs via an endocytotic route.     

Pro-domain in precursor nerve growth factor mediates cell death

Nerve growth factor (NGF) is synthesized as a precursor, proNGF that undergoes post-translational processing to generate the biologically active mature NGF. While the neurotrophic function of NGF is well established, the activity of the proNGF precursor is still unclear. In this study, we have cloned the pro-domain of the precursor NGF molecule and have elucidated its function. We have used both mature and the furin resistant pro((R/G))NGF as controls in our experiments. Both pro((R/G))NGF and mature NGF (NGF) exhibited neurotrophic activity on PC12 cells while the pro-domain itself promoted cell death. The pro-domain, has been found to mediate apoptosis possibly by promoting the formation of a signaling complex comprising of endogenous p75(NTR) receptor, Bim/Bcl2 group of proteins and JNK and MEK1/2 signaling pathways.     

Effects of a Single Intravitreal Injection of Aflibercept and Ranibizumab on Glomeruli of Monkeys

Purpose

It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

Methods

The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm.

Results

Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased.

Conclusion

Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli''s VEGF level. Whether this is caused by aflibercept''s higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.     

Bevacizumab and ranibizumab on microvascular endothelial cells: A comparative study

Given its broad effects in endothelium, vascular endothelial growth factor (VEGF) represents the primary rate‐limiting step of angiogenesis. Therefore, VEGF targeting therapies were soon developed. Bevacizumab and ranibizumab are two of these therapeutic agents already in clinical use. Bevacizumab was first used for cancer treatment, whereas ranibizumab was designed to target choroidal neovascularization, the main cause of blindness in age‐related macular degeneration. The present study aims to compare the multiple effects of bevacizumab and ranibizumab in human microvascular endothelial cells (HMECs). HMEC cultures were established and treated during 24 h with the anti‐VEGF agents within the intravitreal‐established concentration range or excipients. Analyses of VEGF content in cell media and VEGF receptor‐2 (VEGFR‐2) expression in cell lysates were performed. No cell cytotoxicity (MTS assay) was found in anti‐VEGF‐treated cultures at any concentration. Apoptosis (TUNEL assay) was significantly increased and cell proliferation (BrdU assay), migration (transwell assay) and assembly into vascular structures were significantly reduced by incubation with both agents at the two doses used. These findings were accompanied by a strong decrease in VEGF release, and in phosphorylated VEGFR‐2 and Akt expression for both agents at the clinical concentration. Interestingly, phosphorylated Erk was only significantly reduced upon bevacizumab treatment. In addition, proliferation was more affected by ranibizumab, whereas migration, capillary formation, and phosphorylated VEGFR2 expression were significantly reduced by bevacizumab as compared to ranibizumab. Therefore, although both agents presented anti‐angiogenic actions, distinct effects were exerted by the two molecules in HMEC. These findings suggest that a careful confirmation of these effects in clinical settings is mandatory. J. Cell. Biochem. 108: 1410–1417, 2009. © 2009 Wiley‐Liss, Inc.     

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

不同anti-VEGF药物联合玻璃体切除术治疗增殖型糖尿病性视网膜病变效果及对视力水平的影响

摘要 目的：研究不同anti-VEGF药物联合玻璃体切除术治疗增殖型糖尿病性视网膜病变（PDR）效果及对视力水平的影响。方法：随机将2016年4月～2022年6月甘肃省人民医院收治的268例PDR患者分为雷珠单抗组（88例）、康柏西普组（90例）和阿柏西普组（90例）,雷珠单抗组行雷珠单抗注射联合玻璃体切除术,康柏西普组行康柏西普注射联合玻璃体切除术,阿柏西普组行阿柏西普联合玻璃体切除术,观察三组患者的临床治疗效果,并检测其术前、术后7 d和28 d后的血管内皮细胞生长因子（VEGF）和促红细胞生长素（EPO）、最佳矫正视力（BCVA）、黄斑中心凹厚度（CMT）和眼压。结果：三组治疗总有效率比较,差异无统计学意义（P＞0.05）。康柏西普组、阿柏西普组治疗7 d后VEGF、EPO水平明显低于雷珠单抗组（P＜0.05）。康柏西普组、雷珠单抗组治疗7 d后BCVA水平明显低于阿柏西普组;康柏西普组、阿柏西普组治疗7 d后CMT水平低于雷珠单抗组（P＜0.05）。三组治疗前、治疗7 d后、治疗28 d后眼压比较,差异无统计学意义（P＞0.05）。结论：雷珠单抗、康柏西普、阿柏西普联合玻璃体切除术在PDR治疗中均具有较为显著的治疗效果,但康柏西普和阿柏西普在减少黄斑厚度方面效果更为显著,而雷珠单抗与康柏西普在改善视力方面效果更明显。     

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGF^Loxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75^NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75^NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.     

Sortilin participates in light-dependent photoreceptor degeneration in vivo

Both proNGF and the neurotrophin receptor p75 (p75(NTR)) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75(NTR) co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75(NTR), and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75(NTR) and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the "pro" domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75(NTR)/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies.     

2,5-Hexanedione induced apoptosis in rat spinal cord neurons and VSC4.1 cells via the proNGF/p75NTR and JNK pathways

Increasing evidence suggests that n-hexane induces nerve injury via neuronal apoptosis induced by its active metabolite 2,5-hexanedione (HD). However, the underlying mechanism remains unknown. Studies have confirmed that pro-nerve growth factor (proNGF), a precursor of mature nerve growth factor (mNGF), might activate apoptotic signaling by binding to p75 neurotrophin receptor (p75NTR) in neurons. Therefore, we studied the mechanism of the proNGF/p75NTR pathway in HD-induced neuronal apoptosis. Sprague–Dawley (SD) rats were injected with 400 mg/kg HD once a day for 5 weeks, and VSC4.1 cells were treated with 10, 20, and 40 mM HD in vitro. Results showed that HD effectively induced neuronal apoptosis. Moreover, it up-regulated proNGF and p75NTR levels, activated c-Jun N-terminal kinase (JNK) and c-Jun, and disrupted the balance between B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Our findings revealed that the proNGF/p75NTR signaling pathway was involved in HD-induced neuronal apoptosis; it can serve as a theoretical basis for further exploration of the neurotoxic mechanisms of HD.     

Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.     

Nerve growth factor and its precursor differentially regulate hair cycle progression in mice.

Nerve growth factor (NGF) promotes proliferation via its high affinity receptor (TrkA). Its precursor proNGF promotes apoptosis via the pan-neurotrophin-receptor p75. Recently, we have identified NGF and p75 as important hair growth terminators. However, if proNGF is involved or if NGF can also promote hair growth via TrkA is unclear. By RT-PCR we found that NGF/proNGF mRNA levels peak during early anagen in murine back skin, whereas NGF/proNGF protein levels peak during catagen, indicating high turnover in early anagen and protein accumulation in catagen. By immunohistochemistry, NGF and TrkA are found in the proliferating compartments of the epidermis and hair follicle throughout the cycle. In contrast, strong proNGF is found in the highly differentiated inner root sheath and adjacent to the p75+ regressing epithelial strand in catagen. Commercial 7S NGF, which contains both NGF and proNGF, promotes anagen development in organ-cultured early anagen mouse skin, whereas it promotes catagen development in late anagen skin. Together, our findings suggest an anagen-promoting or anagen-supporting role for NGF/TrkA, and a catagen-promoting role for proNGF/p75 interactions. This has important implications for the future design of specific neurotrophin receptor ligands as novel pharmaceuticals in the modification of tissue remodeling processes such as hair growth or wound healing.     

ProNGF inhibits NGF-mediated TrkA activation in PC12 cells

Degeneration of cholinergic basal forebrain neurons (CBFN) is a hallmark in the pathology of Alzheimer's disease (AD). Critically depending upon the neurotrophic support through nerve growth factor (NGF), CBFN in the AD brain face elevated concentrations of the pro-form of NGF (proNGF) and suffer from an imbalance between TrkA and p75(NTR) expression. Research for the underlying mechanisms of CBFN death suggested a pro-apoptotic activity of proNGF. However, this finding could not be confirmed by all investigators and other studies even observed a neurotrophic function of proNGF. In the presence of these controversial findings we investigated the activity of proNGF in PC12 cells with specific emphasis on its neurotoxic versus neurotrophic action. In this study, we show that proNGF can mediate TrkA receptor signaling directly, yet in the manner of a partial agonist with a lower maximum activity than NGF. A pro-apoptotic activity of proNGF could not be confirmed in our cellular system. Interestingly and surprisingly, pre-incubation with proNGF at low, sub-active concentrations inhibited TrkA-mediated neurotrophic NGF signaling in PC12 cells. Our data support a novel hypothesis for the role of elevated proNGF levels in CBFN pathology in AD. Thus, proNGF can indirectly contribute to the slow neurodegeneration in AD by reducing NGF-mediated trophic support.     

A novel hypothesis for Alzheimer disease based on neuronal tetraploidy induced by p75NTR

Cumulative evidence indicates that neuronal cell cycle re-entry represents an early and critical event in AD, recapitulating known hallmarks of the disease including tau hyperphosphorylation and production of Aβ peptide-containing plaques. Neurons that duplicate their DNA are rarely observed to undergo mitosis, and they remain for long time as tetraploid cells, in accordance with the chronic course of the disease. We have recently shown that cell cycle re-entry and somatic tetraploidization occurs during normal development in a subpopulation of RGCs, giving rise to enlarged neurons with extensive dendritic trees. Tetraploization in these neurons occurs in response to the activation of the neurotrophin receptor p75NTR by an endogenous source of NGF. In contrast, BDNF inhibits G2/M transition in tetraploid RGCs, preventing their death by apoptosis. In AD both proNGF and p75NTR are overexpressed, and AD-associated oxidative conditions have been shown to enhance proNGF function. This suggests that p75NTR could be a trigger for neuronal tetraploidization in AD, being the p75NTR-mediated pathway a putative target for therapeutical intervention. Functional changes in affected neurons, derived from tetraploidy-associated hypertrophy, could compromise neuronal viability. The known decline of BDNF/TrkB expression in AD could facilitate G2/M transition and apoptosis in tetraploid neurons.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Intrinsic structural disorder of mouse proNGF

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm‐proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X‐ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti‐NGF antibodies and the soluble forms of TrkA and p75^NTR receptors. SAXS measurements revealed the rm‐proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular “crab‐like” and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the “crab‐like” to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75^NTR receptors and a panel of neutralizing anti‐NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners. Proteins 2009. © 2008 Wiley‐Liss, Inc.